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Anaïs Nin
Anaïs Nin

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Henri Matisse
Henri Matisse

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Guy Ritchie
Guy Ritchie

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Claude Bernard
Claude Bernard

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The ins and outs of glutamate receptor
trafficking during synaptic plasticity
Water and ion channels in kidney function
Nicoll, Roger A. (Department of Cellular and Molecular
Tokyo, Japan)
Pharmacology, University of California at San Francisco, San
Francisco, California, USA)
Glutamate, the major excitatory neurotransmitter in the brain, acts primarily on two types of ionotropic receptors, AMPA receptors and
NMDA receptors. Work over the past decade indicates that the number
of synaptic AMPA receptors is tightly regulated and may serve as a
mechanism for information storage. Recent studies show that stargazin,
the mutated protein in the ataxic and epileptic mouse stargazer, is necessary for the expression of surface AMPA receptors in cerebellar granule
cells. Stargazin is a small tetraspanning membrae protein and is a member of a family of proteins referred to as transmembrane AMPAR regulatory proteins (TARPs). These proteins are differentially expressed
throughout the brain. TARPs control AMPA receptor trafficking through
the endoplasmic reticulum and are important for the maturation and
proper folding of AMPARs. They are necessary for the delivery of
AMPA receptors to the cell surface, as well as to the synapse. Finally
TARPs control the gating of synaptic receptors. The role of TARPs is
analogous to non-pore forming auxiliary subunits of voltage gated ion
channels. Thus TARPs provide the first example of auxiliary subunits of
ionotropic receptors. In this talk I will review the pivotal role that TARPs
play in the life history of an AMPA receptor.
Kidney is the main organ in the maintenance of water and electrolyte homeostasis of extracellular fluid of the body. Extensive physiological research has been performed to understand this important kidney function.
The research has developed from the organ level to molecular level and
the development was accompanied by advancement of experimental
technology, i.e., from the clearance method to molecular biology. Molecular biological studies have identified a wide variety of channel and
transporter proteins that exist in renal epithelial cells. Coupled with human genetic studies it has been shown that mutations of the genes encoding renal membrane transport proteins (channels and transporters) cause
human hereditary diseases such as water and solute loosing or retaining
diseases. At present nearly all genes responsible for relatively popular
human hereditary solute and water disorders have been determined and
the current research interest is focusing on the mechanisms of regulation
of the membrane transport proteins at molecular level. For this aim the
disease-causing mutations found in hereditary diseases have provided
valuable information, for example, possible phosphorylation sites and
protein-protein interaction domains. In this lecture, I will present our
data on water channels (aquaporin) and chloride channels and their diseases of the kidney, and will show how molecular and genetic studies
work together to open a new field of the transport protein research.
The Actin Cytoskeleton and Synaptic
Sasaki, Sei (Dept. Nephrology. Grad. Sch.Tokyo Med Dent Univ.
Matus, Andrew (Friedrich Miescher Institute, Basel, Swiss
Epegenetics and Regulation of Gene
Expression through Nuclear Hormone
Kurokawa, Riki (Division of Gene Structure and Function,
Dendritic spines form the postsynaptic contact elements for most excitatory synapses in the central nervous system. Using time-lapse imaging
of living neurons expressing proteins tagged with green fluorescent protein (GFP) we discovered that dendritic spines undergo rapid changes in
shape thus identifying them as the major sites of morphological plasticity in neuronal circuits of the brain. This motility is driven by dynamic
actin filaments and is differentially regulated by various subtypes of
postsynaptic glutamate receptors. Activation of AMPA receptors produces an immediate blockade of spine motility which is reversed as soon
as the stimulus is withdrawn. By contrast, blockade of spine motility via
NMDA receptors requires 30 min to develop and persists for hours after
the stimulus is withdrawn. To understand the cellular mechanisms underlying these effects we examined the influence of actin binding proteins in dendritic spines. Profilin shows activity-dependent targeting to
spine heads which depends on activation of NMDA receptors and is induced by electrical stimulation patterns associated with changes in synaptic strength such as LTP. Simulataneously actin dynamics are
suppressed and spine motility is blocked for several hours. Conversely,
blocking profilin targeting to spines by expressing a peptide that inhibits
its binding to VASP family proteins destabilizes spine morphology. Together with data for other actin binding proteins to be presented, this suggests that several distinct receptor dependent mechanisms regulate the
dynamic state of the spine actin cytoskeleton and hence morphological
plasticity at excitatory synapses.
Research Center for Genomic Medicine, Saitama Medical School)
Coactivator and corepressor (cofactors) are essential for the transcriptional regulation by nuclear receptor (NR). Coactivator possesses intrinsic histone acetylase (HAT) activity and corepressor is associated with
histone deacetylase (HDAC). These enzyme activities are indispensable
for the function of these cofactors, and also for modulation of chromatin
structures, or epigenetic regulation. Recently, we have shown that dysregulation of the HAT activity causes human diseases such as Rubinstein-Taybi Syndrome (RTS) and Huntington's disease.Our present
efforts are to identify the roles of HAT activity in eukaryotic transcriptional regulation. Recently we have found an RNA-binding protein, TLS
(translocated in liposarcoma)/FUS, which has potent inhibitory effect on
the HAT activity of CREB-binding protein (CBP). This inhibitory molecule has a regulatory role in a CREB-dependent transcription system.
Unexpectedly, specific RNA sequences stimulate the inhibitory activity
of TLS. Such RNA sequence exists at 94% of coding regions of human
genome. The specific RNAs form guanine (G) quartet structures in a K+dependent manner. This structure enhanced binding of the RNAs to TLS.
Addition of the RNAs in HAT assay of CBP with TLS resulted in more
efficient inhibition to the HAT activity. These results have demonstrated
that the RNAs with G-quartet structure function as ligands for TLS and
that the TLS-RNA complex is a potent repressor for the CREB-dependent transcriptional systems, suggesting that TLS mediates RNA-dependent transcriptional repression.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Genetically engineered rodents in brain
Yanagawa, Yuchio1,2 (1Grad. Sch. Med. Gunma Univ.,
Clonal identification of mulltipotent stem/
progenitor cells in the developing liver and
pancreas using flowcytometric cell sorting
Maebashi, Japan; 2SORST, JST, Kawaguchi, Japan)
Taniguchi, Hideki1,2 (1Department of Regenerative Medicin,
Certain types of neurons in the brain are difficult to study because they
cannot be easily identified by location or morphological criteria alone.
One approach to identify such neurons is to label them with a reporter
protein. GABAergic inhibitory neurons play an important role in the regulation and stabilization of network activities, but they are primarily
scattered throughout mammalian central nervous systems and thus can
be hardly identified in live brain preparations. GABA is synthesized
from glutamic acid by glutamate decarboxylase (GAD) and is accumulated into synaptic vesicles by vesicular GABA transporter (VGAT).
Two isozymes of GAD, GAD65 and GAD67 and VGAT are primarily
expressed in GABAergic neurons. To facilitate the study of GABAergic
neurons, we generated the GAD67-GFP mice using a gene targeting
method via homologous recombination in ES cells. EGFP fluorescence
was specifically observed in the GABAergic neurons in the GAD67GFP mice. The GAD67-GFP mice have helped to elucidate the anatomical profile of GABA neuronal network and its electrophysiological activity as well as the development of GABAergic neurons. In addition, we
generated bacterial artificial chromosome transgenic rats, which specifically express a modified YFP, Venus protein under the control of the
VGAT promoter. In VGAT-Venus rats, Venus fluorescence was sufficiently bright to visualize GABAergic neurons and thus allowed wholecell patch-clamp recordings from visually identified GABAergic neurons. The detailed morphological and functional analyses of GABAergic
neurons in brain slices will greatly benefit from these genetically engineered rodents.
Graduate School of Medicine, Yokohama City University; 2Research
Unit for Organ Regeneration, Center for Divelopmental Biology,
Using flowcytometry combined with single-cell-based assays, we prospectively identified hepatic stem cells with multilineage differentiation
potential and self-renewing capability in the developing mouse liver. cMet+ CD49f+/low CD29+ c-Kit- CD45- TER119- cells in fetal liver
could be clonally propagated in culture, where they continuously produced hepatocytes and cholangiocytes as descendants while maintaining
primitive stem cells. When cells that expanded in vitro were transplanted
into recipient animals, they morphologically and functionally differentiated into hepatocytes and cholangiocytes, with reconstitution of hepatic
cord and bile duct structures. These data indicate that self-renewing multipotent stem cells are retained in midgestational developing liver. The
pancreas also contains a population of pancreatic stem cells that generate
endocrine, exocrine, and ductal cells during development, neogenesis,
and regeneration. By combining flowcytometry and clonal analysis, we
show here that stem/progenitor cells of pancreatic endocrine and exocrine cells that reside in the neonatal mouse pancreas. Clonally isolated
stem/progenitor cells could be used to reveal the mechanism of cell differentiation in digestive organs, and also could provide new insight into
therapies for liver diseases, diabetes mellitus and cancer.
Visualizing Columnar Architectures with
High-Resolution fMRI
Development of a comprehensive cardiac
cell model
Cheng, Kang (RIKEN Brain Science Institute, 2-1 Hirosawa,
Wako-shi, Saitama 351-0198, Japan)
Noma, Akinori (Grad. Sch. Med. Kyoto Univ. Japan)
Among the many neuroimaging tools available for studying human brain
functions, functional magnetic resonance imaging (fMRI) is the most
widely used today. One advantage of fMRI over other imaging techniques is its relatively high spatial resolution. High-field fMRI, with its
superb signal-to-noise ratio, has strengthened the capability of fMRI and
allowed mapping of fine cortical architectures in human primary visual
cortex (V1). In this presentation, I will explain the factors limiting the
spatial precision and resolution of fMRI, describe the benefits that highfield fMRI offers in dealing with these issues, and introduce several
high-resolution studies that have been conducted in our laboratory on the
functional organization of human primary V1, including mapping of ocular dominance columns, temporal frequency dominance domains and
orientation selectivity.
To understand mechanisms of various functions of cardiac cells, the
whole cell model was developed. The model is composed of ion channels, ion transporters, membrane receptors, coupling between the sarcolemmal Ca channel and the ryanodine receptor channels, sarcoplasmic
reticulum with Ca, SERCA, calsequestrin, the contraction machinery, intracellular ion concentrations, mitochondria model, and gap junction
channels. The function of these functional units are mostly described
with experimenatal equations in literatures, otherwise model adjusted
refering to experimental observations on macroscopic levels. The whole
cell model, we call 'Kyoto Model', well reconstracts the pacemaker activity in the sinoatrial node cell, the ventricular action potential, the contraction, and homeostasis of the intracellular ion concentrations, energy
metabolism and the cell volume regulation, classic regulation by the autonomic nervous transmitters. Responses to various experimental interventions are reversible. Model based new hypotheses were obtained for
the pacemaker mechanisms, cell volume regulation via ion fluxes
through sarcolemma, and the Ca mediated upregulation of the mitochondrial ATP production on increasing the work load. The model still needs
to be revised and implemented with new mechanisms, thereby the integrations of the experimental knowledge will be most efficiently and systematicall achieved through developing the whole cell model. All these
model constructions were conducted on simBio, which is newly developed Java package by us for constructing cell models on a large scale (http://www.sim-bio.org/).
J. Physiol. Sci., Vol. 56, Suppl., 2006
Integrative Physiology of Cardiac Function
Suga, Hiroyuki (Nat'l Cardiovasc Ctr, Suita, Osaka, Japan)
I would like to review the four eurekas I have experienced in my cardiac
function research over these 40 years. In 1966, the major concepts of cardiac pump function were Frank's ventricular pressure (P)-volume (V) relation, Starling's law of the heart, Sarnoff's ventricular function curve,
and Sonnenblick's myocardial force-velocity relation. Since these could
not persuade me, I started my own research, using canine in situ beating
hearts. I obtained P-V loops of the left ventricle (LV) and their relations
with LV contractility. My first eureka suggested in 1967 that the contracting LV could be modeled as a time-varying elastance E(t). I then
found that its end-systolic peak (Emax) could serve as a reliable contractility index, later adopted as a core concept in cardiac physiology. My
second eureka suggested in 1974 that the E(t) model could provide a specific P-V area (PVA) as a measure of the total mechanical energy generated by an LV contraction. Both Emax and PVA could reliably predict
LV oxygen consumption (Suga: Ventricular energetics. Physiol Rev,
1990). My third eureka suggested in 1994 that the total amount of calcium recruited in the excitation-contraction coupling could be calculated
from a set of Emax, PVA, and a decay time constant of the post-extrasystolic transient alternans. My forth eureka suggested in 2003 that the sliding length of a crossbridge per ATP could be calculated from a set of
PVA and its oxygen cost to be variable up to more than 20 times unit
step. These integrative physiological findings seem to have advanced a
better integrative understanding of the pump function of a beating heart.
signaling and regulation of cell cycle
in mammalian fertilization
Miyazaki, Shunichi (Dept. Physiol., Tokyo Women's Med. Univ.
Sch. Med., Tokyo, Japan)
Late Professor Susumu Hagiwara (1922-89) is a great physiologist who
discovered Ca2+-dependent action potentials in barnacle muscle fibers in
1964, distinct from Hodgkin/Huxley-type Na+ spikes. Thereafter, he
characterized Ca2+ and other ion channels in a wide variety of cells in relation to functions. He was interested in Ca2+-regulated cell functions.
Fertilization is one of such phenomena. A dramatic increase in intracellular Ca2+ occurs at fertilization in eggs of all species examined to date,
and it is a pivotal signal for egg activation seen in resumption of meiosis
and cell cycle progression. Ca2+ signaling at fertilization was first investigated in sea urchin and fish eggs in mid-70s. We began to address the
mechanism in mammals in 1980. Mammalian eggs exhibit repetitive
Ca2+ increase mainly due to Ca2+ release from the ER via type 1 IP3 receptor/Ca2+ channel. Accumulated evidence indicates that a cytosolic
sperm factor is driven into the ooplasm upon sperm-egg fusion and induces repetitive Ca2+ release. Recent studies have shown that a novel
isozyme of phospholipase C, PLCζ, is a strong candidate of the sperm
factor. Fertilization-like Ca2+ oscillations are induced by injection of
sperm extract, PLCζ RNA, or recombinant PLCζ into mouse eggs.
PLCζ has extremely high Ca2+-sensitivity in PLC activity and nuclear
translocation ability. These properties qualify PLCζ as the sperm factor
that initiates and drives cell cycle-dependent Ca2+ oscillations. Ca2+
activates CaMK II and thereby ubiquitin/proteasome system, leading to
degradation of cyclin, inactivation of metaphase promoting factor, and
resumption of meiosis.
Exploring the Logic for Olfactory
Mori, Kensaku (Grad. Sch. Med. Univ. Tokyo, Tokyo, Japan)
Exploring the Logic for Olfactory Perception Mori, Kensaku (Dept.
Physiol., Grad. Sch. Med. Univ. of Tokyo, Japan) The olfactory perception plays a key role in the daily life of human and animals. Since the discovery of odorant receptors in 1991, we have witnessed a rapid progress
in the understanding of the olfactory system. However, the recent studies
focused on the early olfactory processing at the levels of odorant receptors, sensory neurons and olfactory bulb (OB). The central processing of
olfactory information in the mammalian brain is still not well understood. Late Professor Sadayuki Takagi and his colleagues are the pioneers who explored the odorant-response specificity of neurons from the
OB through the olfactory cortex to the orbitofrontal cortex in the monkey
brain. In the OB, individual glomeruli represent a single odorant receptor, and the glomerular sheet of the OB forms odorant receptor maps.
Studies of OB mapping show that (1) individual glomeruli respond to a
range of odorants that share a specific combination of molecular-features, that (2) each glomerulus appears to be unique in its molecular receptive range property, and that (3) glomeruli with similar molecular
receptive range properties are located in proximity and form molecularfeature clusters. The olfactory cortex reads the molecular-feature maps
in the OB and is thought to integrate information from different molecular-feature detecting glomeruli to form the olfactory image of objects.
We discuss also behavioral-state-dependent gating of olfactory information flow at the level of the olfactory cortex.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Temporal organization of physiology and behaviors—Functions of multi-oscillatory hierarchical system [IUPS Symposium in
the 83rd Annual Meeting of the Physiological Society of Japan]
Luminescence reporter techniques have
uncovered complexity among oscillatory
structures in the mammalian circadian
Yamazaki, Shin1; Davidson, Alec J.2 (1Department of
Biological Sciences, Vanderbilt University, Nashville TN, USA;
2Departmenf of Biology, University of Virginia)
Temporal organization of
physiology and behaviors—
Functions of multi-oscillatory
hierarchical system [IUPS
Symposium in the 83rd Annual
Meeting of the Physiological Society
of Japan]
Luminescence reporters have been used successfully in studies of circadian rhythms in many organisms. Ever since luciferase was introduced
for real-time monitoring of gene expression rhythms in plants and cyanobacteria, luminescence reporter techniques have become a powerful
tool for noninvasive assays of circadian oscillations. Using a real-time
light detection system from cultured rodent tissues, we were able to
record a circadian oscillation from the cultured suprachiasmatic nucleus
(master pacemaker in the brain) for more than 16 month. We also discovered that most peripheral tissues were rhythmic in culture with distinct
phases relative to the light dark cycle to which the animal had been exposed. Daytime restricted-feeding uncoupled the rhythms in digestive
tissue from the environmental light cycle. Therefore the mammalian circadian system consists of at least two oscillatory systems; one is coupled
with environmental light and another is coupled with food. This might
have significant meaning for the adaptation of the circadian system to the
natural environment. The study of multiple oscillatory systems also has
had significant impact on medicine. We have begun to understand the
circadian relationship between tumor cells and normal cells. Management of circadian rhythms may provide a new therapeutic approach for
combating human disease.
SCN vs Non-SCN clocks
Honma, Ken-ichi; Abe, Hiroshi; Honma, Sato (Department
Reconfiguring Cellular Ensembles Within
the Suprachiasmatic Nucleus
of Physiology, Hokkaido University Graduate School of Medicine)
Schwartz, William J. (Department of Neurology, University of
The circadian system in mammals entrains to several oscillating factors
in the environment. Among them, a light-dark cycle is the common as
well as most potent factor so far examined. Entraining light signals enter
the brain through the retinohypothalamic tract and reach the suprachiasmatic nucleus (SCN) where the master clock is located. The circadian
system in humans is able to entrain to non-photic factors as best exemplified by entrainment of totally blind persons. However, it is not known
whether the non-photic entrainment is achieved by the clock in the SCN
or other clocks located somewhere outside the SCN. Methamphetamine,
a CNS stimulant, is known to produce robust activity rhythms in bilaterally SCN lesioned rats and aperiodic clock mutant or Cry1/Cry2 double
knockout mice, indicating the existence of a behavior related oscillator(s) outside the SCN. Interestingly, the activity rhythm produced by
methamphetamine shows similar characteristics to those observed in the
human sleep rhythm, such as internal desynchronization from the circadian rhythms, a long endogenous period, circabidian (ca. 48 h) rhythm
and entrainment to non-photic factors. The underlying mechanism is regarded as a non-SCN clock. A feeding-associated oscillator is another
example of non-SCN clock. Activity rhythms in the circadian domain
were developed in rats and mice, when feeding is restricted to a fixed
time of day. The feeding-associated oscillation persists for several weeks
after the termination of restricted feeding. Using these model animals,
we are attempting to find out the site of oscillation of non-SCN clock.
The circadian clock in the suprachiasmatic nucleus (SCN) is composed
of multiple single-cell circadian oscillators, and a challenge now is to
learn how individual cells are assembled to create an integrated tissue
pacemaker that can orchestrate the temporal programs of whole organisms. By measuring SCN gene expression (in situ hybridization) as an
assay of clock activity, we have found that assembled cellular oscillators
can assume different configurations within the SCN, giving rise to unusual locomotor activity patterns. Thus, in hamsters maintained in constant light, splitting of the single circa-24 hr activity bout into two circa12 hr components appears to be the consequence of a paired SCN that is
reorganized into two oppositely-phased, left- and right-sided circadian
pacemakers. In rats exposed to an artificially short light-dark cycle, the
simultaneous expression of two stable circadian motor activity rhythms
with different period lengths corresponds to the desynchronization of
circadian pacemakers in the ventrolateral and dorsomedial subdivisions
of the SCN (as previously defined by regional differences in their cytoand chemo-architecture and topography of afferents and efferents).
These kinds of reconfigurations (left/right, dorsal/ventral) of regional
oscillators should provide a powerful approach for understanding intercellular coupling, tissue organization, and differential outputs of the
SCN in intact, behaving animals.
Massachusetts Medical School, Worcester, Massachusetts, Japan)
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Commemorative and illustrious individuals devoting to physiological sciences in Japan: Kojiro Matsuda and Yasuyoshi
Nisimaru [IUPS Symposium: History of Physiologist in Japan (held in Japanese)]
An Important Role of Circadian
Phosphorylation of CRY Proteins in the
Mouse Peripheral Clock
Fukada, Yoshitaka (Dept. Biophys. & Biochem., Graduate
School of Science, University of Tokyo, Tokyo, Japan)
Protein phosphorylation plays a crucial role for time-keeping mechanism of circadian clock systems. Several clock proteins undergo temporal change in phosphorylation in the mouse liver, a well-characterized
peripheral clock tissue, but it was unclear as to whether the central negative regulator CRYs are phosphorylated in vivo. We found that mCRY1
and mCRY2 are phosphorylated by MAPK in vitro and identified Ser265
and Ser557 of mCRY2 as in vitro phospho-acceptor residues. Similarly,
MAPK phosphorylated mCRY1 at Ser247, a site corresponding to
Ser265 of mCRY2. An effect of the Ser phosphorylation was investigated by mutating Ser247 of CRY1 and Ser265 of CRY2 to Asp, which resulted in attenuation of each CRY's ability to inhibit BMAL1:CLOCKmediated transcriptional activation. On the other hand, we found Ser557phosphorylated CRY2 accumulated in the liver during the subjective
night in parallel with CRY2 protein, and the phosphorylated form
reached its maximal level at late night preceding the peak-time of the
protein abundance by approximately 4 hrs in LD and DD conditions. The
Ser557-phosphorylated form of CRY2 was localized in the nucleus,
whereas CRY2 protein was located in both the cytoplasm and nucleus.
Functionally, Ser557-phosphorylation of CRY2 allowed subsequent
phosphorylation of the protein by GSK3β, resulting in efficient
degradation of CRY2 by a proteasome pathway. These results
demonstrate the important role of priming phosphorylation at Ser557 for
destabilization of CRY2 and illustrates a model that the circadian
phosphorylation of CRY2 contributes to its rhythmic degradation.
Commemorative and illustrious individuals
devoting to physiological sciences in
Japan: Kojiro Matsuda and Yasuyoshi
Arita, Makoto1,2; Kameyama, Masaki1,3; Nisimaru
(Yamada), Naoko1,4 (1SCCRE:The supporting Center for
Clinical Research and Education, Osaka, Japan; 2Yufuin
Kohseinenkin Hosp., Oita, Japan; 3Dept. Physiol., Grad. Sch. Med.
and Dent. Sci., Kagoshima Univ., Kagoshima, Japan; 4Dept.
Physiol., Oita Univ.Facul. Med., Oita, Japan)
1. Kojiro Matsuda (1908-1993): Dr. K. Matsuda devoted his academic
life to the development of cardiac electro-physiology/pathophysiology
and circulation physiology. He and his group have recorded, for the first
time, action potentials in specialized cardiac muscle tissues including
atrioventricular node and established physiological basis of impulse conducting system in the heart. (M. Kameyama)
2. Yasuyoshi Nisimaru (1897-1990): Dr. Y. Nisimaru established with
his numerous pupils (over 140 members) the concept of body fluid
circulation; blood (William Harvey), lymph (Claude Bernard) and tissue
fluid are linked and essentially one and the same extracellular fluid. He
was a great teacher and encouraged many, even outside his immediate
circle, for reasearch. He was one of the members who founded the
Physiological Society of Japan in 1922 (Naoko Nisimaru).
Commemorative and illustrious
individuals devoting to
physiological sciences in Japan:
Kojiro Matsuda and Yasuyoshi
Nisimaru [IUPS Symposium:
History of Physiologist in Japan
(held in Japanese)]
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Endocytosis of receptor tyrosine kinase: Novel mechanisms by new molecules
Endocytosis of vascular endothelial
growth factor receptor-1, Flt-1.
Maru, Yoshiro (Dept.Pharmacol. Tokyo Women 'S Medical Univ.
Tokyo, Japan)
Endocytosis of receptor tyrosine
kinase: Novel mechanisms by new
Vascular endothelial growth factor (VEGF) has two receptors with catalytic activity: Flt-1 and KDR. The uniqueness of Flt-1 is that it is also expressed in macrophages and plays an essential role in atherosclerosis.
Given that oxidized LDL and proteasome inhibitors appeared to downregulate the Flt-1 expression, regulation of Flt-1 on the cell surface as for
example a VEGF-trapping molecule for KDR may have a close linkage
with initiation of atherosclerotic plaque formation. We have shown that
Flt-1 is endocytosed upon binding to VEGF as in the case of other tyrosine kinase growth factor receptors. The minor autophosphorylation
site Y 1333 seems to be utilized for recruiting the c-Cbl/CD2AP complex to Flt-1. c-Cbl is an E3 ligase that ubiquitinates Flt-1 with subsequent degradation in proteasomes. Although CD2AP overexression
changes endosomal morphology and therefore it appears to be involved
in vesicle formation, the precise mechanisms and biological roles of endocytosis still remain to be elucidated. Inhibitors for heat shock protein
(Hsp) 90, which is assumed to stabilize the degrading molecule, induced
a seemingly VEGF-independent degradation of Flt-1. In this paper, we
hopefully discuss possible biological significance and molecular mechanisms of Flt-1 disappearance from the cell surface from the standpoint
of atherosclerosis.
Introduction: Regulation of endocytosis of
receptor tyrosine kinase
Receptor endocytosis triggers production
of PtdIns(3,4,5)P3 at endomembranes
Shimokawa, Noriaki; Londono, Marina; Qiu, ChunHong; Koibuchi, Noriyuki (Gunma Univ. Grad. Sch. Med.,
Sato, Moritoshi1,2; Umezawa, Yoshio1 (1Sch. Sci. Univ.
Gunma, Japan)
Cells are constantly exposed to various extracellular signals, which coordinate their growth, proliferation, differentiation, mortality and survival. Receptor tyrosine kinases (RTKs) are critical mediators between
ligands and cell interior. Such receptors include EGF, PDGF, FGF, HGF,
IGF, NGF, VEGF and M-CSF receptors. Immediately following activation of RTKs, these receptors are rapidly translocated from cell surface
into the endosomal compartment. Then, these are sorted into lysosomes
for degradation.Recently, evidence is accumulating that numerous adaptor proteins are involved in RTKs downregulation by internalization and
endocytosis. For example, we have been interested in the role of Cbl
(Casitas B-lineage lymphoma) that is a multi-adaptor protein with E3
ubiquitin ligase activity and mediate ubiquitylation of active RTK. We
have identified that polyubiquitylation of EGF receptor by Cbl ligase is
essential for its internalization and degradation. In this symposium, recent progress on the new mechanisms and adaptor molecules regarding
regulation of RTK endocytosis will be introduced by researchers from
five leading laboratories in this field.
Tokyo, Tokyo, Japan; 2PRESTO, JST, Saitama, Japan)
Phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) regulates diverse cellular functions including cell proliferation and apoptosis, and is
related to diabetes, cancer, etc.; however, little is known about exactly
when, where and how PtdIns(3,4,5)P3 is produced. We have developed
fluorescent indicators for PtdIns(3,4,5)P3 to reveal spatio-temporal regulations of PtdIns(3,4,5)P3 production in single living cells. After ligand
stimulations, PtdIns(3,4,5)P3 levels increased to a larger extent at the endomembranes, i.e. the ER and Golgi, than at the plasma membrane. This
PtdIns(3,4,5)P3 increase at the endomembranes was found to originate
from its in situ production at the endomembranes, a process stimulated
directly by receptor tyrosine kinases endocytosed from the plasma membrane to the endomembranes. The demonstration of PtdIns(3,4,5)P3 production through receptor endocytosis addresses a long-lasting question
about how downstream signaling pathways including Akt are activated
at intracellular compartments remote from the plasma membrane.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Regulation of plasma membrane localization of membrane transport proteins [YFI (Young Foreign Investigator) Symposium]
Regulation of growth factor receptor
downregulation by deubiquitination
Komada, Masayuki (Grad. Sch. Biosci. Biotech., Tokyo Tech,
Yokohama, Japan)
Ligand-activated receptor tyrosine kinases (RTKs) undergo endocytosis
and are transported via endosomes to lysosomes for degradation. This
process, known as receptor downregulation, is crucial to terminate the
cell proliferation signals produced by activated RTKs. During the process, ubiquitination of RTKs serves as a sorting signal for their trafficking from endosomes to lysosomes. The sorting of RTKs is executed by a
complex of two ubiquitin-binding proteins, Hrs and STAM, which localizes on the early endosomal membrane. STAM has been shown to interact with a deubiquitinating enzyme UBPY, also known as USP8. Here
we studied the role of UBPY in the downregulation of epidermal growth
factor receptor (EGFR).
Immunopurified UBPY deubiquitinated EGFR in vitro. Overexpression
of UBPY in EGF-stimulated cells reduced the ubiquitination level of
activated EGFR and delayed its degradation. Conversely, depletion of
UBPY by RNA interference resulted in elevated ubiquitination and
accelerated degradation of EGF-activated EGFR. Stimulation of cells
with EGF induced the association of UBPY with endocytosed EGFR on
Hrs-positive early endosomes, and this association required the
interaction of UBPY with the Hrs-STAM complex. On the other hand,
the endosomal localization of UBPY did not depend on the Hrs-STAM
complex. Together, we conclude that UBPY deubiquitinates activated
EGFR which is sorted by the Hrs-STAM complex on early endosomes,
thereby removing its sorting signal and regulating its downregulation
Regulation of plasma membrane
localization of membrane transport
proteins [YFI (Young Foreign
Investigator) Symposium]
Regulation of cell signalling by
Regulation of organic anion transporters
by PDZ proteins
Dikic, Ivan; Hoeller, Daniela; Bienko, Magda; Crosetto,
Nicola; Zapart, Gregorz; Haglund, Kaisa (Institute of
Anzai, Naohiko; Kanai, Yoshikatsu (Kyorin Univ. Sch. Med.,
Biochemistry II, Goethe University, Frankfurt, Federal Republic of
Organic Anion Transporters (OATs), belonging to SLC22 family, are
mainly localized in the renal proximal tubules and play important roles
in the detoxification and the secretion of xenobiotics such as drugs and
toxins and in the reabsorption of endogenous organic anions such as
urate and estrone sulfate. Recently, it has been reported that the transporters at the apical membrane of the proximal tubules that have PDZ
motif at their extreme C-terminus bind to the NHERF family proteins via
PDZ interaction. They exist and function on the network consisted of
such proteins beneath the plasma membrane (Anzai et al., Curr Opin
Nephrol Hypertens, 2005). Our yeast two-hybrid assays revealed that
urate transporter URAT1 and organic anion transporter OAT4, localized
at the apical membrane of the proximal tubules, interact with PDZ proteins. The interaction of PDZ proteins enhanced their transport activity
in the overexpressed mammalian cells through the increased surface protein expression (Anzai et al., J Biol Chem, 2004: Miyazaki et al., J Am
Soc Nephrol, 2005). To date, other transporters related to the renal organic anion handling have also been found to interact with PDZ proteins.
These results indicate that the organic anion transport molecular complex (organic anion transportsome) is formed through apical PDZ network and it may contribute to the renal organic anion handling as a
functional unit.
The attachment of a single ubiquitin, monoUb, to a substrate serves as an
important regulatory modification implicated in receptor endocytosis,
virus budding, gene transcription, DNA repair and replication, etc. The
discovery of Ub-binding domains (UBDs), such as UBA, UIM, CUE and
others, has indicated how monoUb can regulate such distinct cellular
functions. We have cloned two new Ub-binding domains named UBM
(Ub binding motif) and UBZ (Ub binding Zn finger) found in numerous
cellular proteins. Their functional and biophysical characterization will
be presented. In addition to binding Ub, several UBDs promote the
monoubiquitylation of host proteins. We have recently shown that
monoubiquitylation of the endocytic proteins Sts1, Sts2, Eps15 and Hrs
facilitates intramolecular interactions with the UBDs, thus preventing
them from binding to ubiquitylated cargoes. We mapped the in vivo
monoubiquitylation site in Sts2 and demonstrated its functional importance for EGF receptor endocytosis. We propose that monoubiquitylation of Ub-binding proteins represent a general regulatory mechanism
that inhibits their capacity to bind to and control functions of ubiquitylated targets in vivo.
Mitaka, Tokyo, Japan)
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Regulation of plasma membrane localization of membrane transport proteins [YFI (Young Foreign Investigator) Symposium]
Regulation of membrane localization of
renal sodium-dependent phosphate
Taketani, Yutaka1; Segawa, Hiroko2; Nashiki, Kunitaka1;
Takeda, Eiji1; Miyamoto, Ken-ichi2 (1Dept. Clin. Nutr. Inst.
Health Biosci. Univ. Tokushima Grad. Sch., Tokushima, Japan;
2Dept. Mol. Nutr. Inst. Health. Biosci. Univ. Tokushima Grad. Sch.,
Tokushima, Japan)
Phosphate (Pi) homeostasis is mainly regulated by control of Pi reabsorption in the kidney. Sodium-dependent Pi transport system localized
in brush border membrane of renal tubular cells is a responsible for the
Pi reabsorption. Until now, three isoform of the sodium-dependent phosphate transporter (NaPi-I, NaPi-IIa, NaPi-IIc) have been identified in the
brush border membrane of the renal proximal tubular cells. Among
them, NaPi-IIa is the most regulatable transporter by various hormone
and environmental changes. PTH is a potent inhibitor of NaPi-IIa and is
rapidly involved in the translocation of NaPi-IIa from plasma membrane
to intracellular compartments. Recent studies demonstrated that NaPiIIa can predominantly localize in the membrane microdomains (such as
lipid rafts or caveolae) of apical membrane of renal proximal tubular
cells, and NaPi-IIa can bind to actin cytoskeleton via NHERF-1/EBP50
and ezrin. Formation of the complex has thought to be important to determine the subcellular localization and hormonal regulation of the
NaPi-IIa. We identified ezrin is a target molecule for PTH signal. Repression of ezrin function inhibited both membrane targeting and PTH-dependent endocytosis of NaPi-IIa. These findings suggest that ezrin
would be a key molecule for both subcellular localization and hormonal
regulation of the NaPi-IIa.
Translocation of biliary transporters under
diseased or drug treatment condition
Ito, Kousei1; Sekine, Shuichi2; Horie, Toshiharu2;
Suzuki, Hiroshi1 (1Univ. Tokyo Hospital, Tokyo, Japan; 2Grad.
Sch. Pharm. Sci. Chiba Univ., Chiba, Japan)
ATP-binding cassette (ABC) transporters expressed on the bile canalicular membrane of the hepatocytes are rapidly internalized from and reinserted to the plasma membrane by a variety of stimulus including
oxidative stress, osmolarity change, drug treatment and so on. Such relocalization of the transporter molecules sometimes leads to cholestasis
or choleresis, although precise intracellular signaling pathway and final
molecular determinants involved in the specific transporter internalization is not elucidated. ABCC2/MRP2 and ABCB11/BSEP are both biliary transporters involved in bile flow, by excreting organic anions
(glutathione conjugates, glucuronide conjugates, reduced glutathine)
and bile salts, respectively, into bile. We are studying the intracellular
signaling pathway triggered by GSH depletion and finally leading to
Mrp2-specific internalization using isolated rat hepatocytes coulplets as
a experimental model. As a result, GSH depletion induced by ethacrynic
acid treatment produces nitric oxide (NO) followed by novel protein kinase C (nPKC) activation. Molecular mechanism regulating Mrp2-specific internalization are discussed in relation to canalicular scaffold
proteins and other canalicular ABC transporter molecules.
Molecular basis of the channels
responsible for K+ and water transport and
their specific localization in brain
Analysis of disease-causing mutants of
renal channels and their regulators.
Hibino, Hiroshi; Kurachi, Yoshihisa (Grad. Sch. Med. Osaka
Tokyo Medical and Dental University, Tokyo, Japan)
Univ. Suita, Japan)
In kidney, various types of channels and transporters present in the specific nephron segments are functioning to maintain body fluid balance.
Mutations of these membrane proteins have been found to cause some
human genetic diseases, such as Bartter syndrome and nephrogenic diabetes insipidus (NDI). These naturally occurring mutations sometimes
tell us important functional domains within proteins, especially in terms
of protein sorting. Recently, we found three frame-shift mutations in the
AQP2 gene in patients having autosomal dominant (AD)-type NDI. Previously, we have demonstrated that the AQP2 (763-772 del), a 10 nucleotide-deletion mutant, was mis-localized to the basolateral membrane in
MDCK cells. To analyze this sorting abnormality in vivo, we created
AQP2 (763-772 del) knock-in mice, which showed severely impaired
urine concentrating ability. Using this mouse model, we will discuss the
molecular pathogenesis of AD-NDI in vivo, and also the usefulness of
knock-in mouse models in the study of transportsome.
extracelluar K+ yielded by
The brain astrocytes transport excess
activation to regions of low K+ uni-directionally. This called "K+ buffering" is accompanied with water flux. Physiological coupling of these
fluxes is essential for proper brain function. Inwardly rectifying K+ (Kir)
channels are assumed to be crucial for K+ buffering. We found two types
of Kir channels, homomeric Kir4.1 and heteromeric Kir4.1/5.1, distributed on astrocytic membranes and were involved in K+ buffering.
Perivascular processes harbor the heteromer, which would secrete K+,
and perisynaptic processes differentially express either channel in a region specific manner, which may play a distinct role in K+ uptake. Because activity of Kir4.1/5.1 is dynamically regulated by intracellular pH
(pHi) change in a physiological range, K+ outflow and part of K+ influx
may be finely controlled by pHi. We further found that the two Kir channels occurred together with AQP4, only one water channel in astrocytes,
at the same membrane surface of the processes. Dystrophin associated
protein complex could specifically target the Kir and water channels to
the perivascular processes. Moreover, we have identified that "lipid raft"
microdomain selectively gathers not only apparatuses responsible for
water and K+ transport such as Kir4.1, Kir5.1 and AQP4 but also other
molecules including astroglical Cl– channel ClC-2 and glutamate transporter GLT-1. Accordingly, lipid rafts may serve as a functional microplatform synchronizing salt, water, and glutamate transports in
Uchida, Shinichi; Sohara, Eisei; Rai, Tatemitsu; Sasaki,
Sei (Department of Nephlorolgy, Graduate School of Medicine,
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: TRP channels: Regulatory aspects and physiology
Single particle analysis of TRPC3
Sato, Chikara; Mio, Kazuhiro; Ogura, Toshihiko (Neuro.
Sci. Inst., AIST)
TRP channels: Regulatory aspects
and physiology
TRPC3 plays important roles in neuronal differentiation and immune
cell maturation by mediating the cationic current in response to phospholipase C activation, Ca2+ depletion, and diacylglycerol stimulation. In
collaboration with Dr, Yasuo Mori (Kyoto Univ.), we purified the
TRPC3 channel as a glycosylated tetramer and observed the structure using electron microscopy for single particle analysis1). Negatively stained
specimens demonstrate homogeneous protein particles containing an internal cavity-like structure. These particle images were selected by automated pick-up programs2), aligned, and classified by the growing neural
gas network method3). Similarly oriented projections were averaged to
decrease the signal-to-noise ratio. The averaged images progress from
the top view to the side views, which are representative of their raw images. The top view confirmed the hypothesis of a four-domain structure,
and the side view demonstrates a large cytoplasmic domain with a
capped structure at the bottom, which is near a predicted locus of ion release. The total image of the protein is a blunt-edged trapezoid: both
width and height of the molecule are over 200 angstrom. This large dimension of TRPC3 is also supported by the Stokes radius (92 angstrom)
obtained from gel filtration chromatography.
1) Mio, K., Ogura, T., Hara, Y., Mori, Y. & Sato, C. The non-selective
cation-permeable channel TRPC3 is a tetrahedron with a cap on the large
cytoplasmic end. Biochem. Biophys. Res. Commun. 333, 768-777
2) Ogura, T. & Sato, C. J Struct Biol 146, 344-58 (2004).
3) Ogura, T., Iwasaki, K. & Sato, C. J Struct Biol 143, 185-200 (2003).
Regulation of the TRPV2 channel in
Nagasawa, masahiro; Kojima, Itaru (Inst.Mol.Cell.Reg.
Physiological significance of
diacylglycerol-acivated TRPC3 channels in
B cell receptor signalling
Gunma Univ. Maebashi, Japan)
Mori, Yasuo1; Numaga, Takuro2 (1Grad. Sch.Engineering,
The TRPV2 channel is expressed in various tissues including neurons,
neuroendocrine cells and blood cells including macrophages. We examined the regulation of the TRPV2 channel in macrophages. In serum-free
condition, immunoreactivity of TRPV2 was detected largely in cytoplasm. Addition of a chemotactic peptide fMLP induced translocation of
the TRPV2 to the plasma membrane. In accordance with this, fMLP increased the Cs+ current, which was inhibited by ruthenium red and the
transfection of the dominant-negative mutant of TRPV2. fMLP-induced
translocation of the TRPV2 was blocked by PI 3-kinase inhibitors and
pretreatment with pertussis toxin. When cytoplasmic calcium concentration ([Ca2+]c) was monitored by using fura-2, fMLP induced a rapid and
sustained elevation of [Ca2+]c, the latter of which was abolished by removal of extracellular calcium. Addition of ruthenium red or transfection of the dominant-negative mutant of TRPV2 did not affect the initial
rise but blocked the sustained phase of fMLP-induced [Ca2+]c response.
In stimulated macrophages, TRPV2 localized in the podosome, a microdomain involved in adhesion and migration, and colocalized with Rho
family small G proteins. Transfection of the dominant-negative Rac inhibited translocation of TRPV2. Finally, addition of ruthenium red or
transfection of dominant-negative mutant of TRPV2 inhibited chemotaxis of macrophage induced by fMLP. These results indicate that fMLP
induces translocation of TRPV2 by a PI 3-kinase dependent mechanism
and this translocation is important for sustained elevation of [Ca2+]c in
Kyoto Univ., Kyoto, Japan; 2Dept. Physiol. Sci., Sch. Life Sci., Grad.
Univ. Adv. Studies)
In B lymphocytes, B cell receptor (BCR)-activated Ca2+ signaling comprises initial transient responses followed by a Ca2+ entry-dependent sustained and/or oscillatory phase. BCR stimulation induces phospholipase
C γ2 activation and simulates Ca2+ influx across the plasma membrane
through multiple mechanisms, such as store-operated Ca2+ channels via
IP3-induced Ca2+ store depletion and cation/ Ca2+ channels directly
activated by diacylglycerol (DAG). Previously, we have revealed
requirement of store-operated Ca2+ channels for the generation of BCRinduced Ca2+ oscillations and subsequent gene expression. However, the
importance of DAG-activated channels is largely unknown in BCR
signalling. Canonical transient receptor potential (TRPC) 3 is known as
cation/ Ca2+ channels coupled with PLC γ2 and activated by DAG. In
this study, we have disrupted TRPC3 gene in DT40 B lymphocytes by
targeting method to study its impact on BCR signalling. Endogenous
TRPC3 formed DAG-activated Ca2+ channels in DT40 B lymphocytes.
BCR-induced Ca2+ oscillation and NF-AT activation were suppressed in
TRPC3-deficient cells. Furthermore, extracellular signal-regulated
kinase (ERK), one of the mitogen activated protein kinases (MAPK),
activation was reduced in TRPC3-deficient cells. This was attributable
to suppressed plasma membrane translocation of PKC βII, a signalling
components upstream of the ERK pathway. In conclusion, DAGdependent activation of TRPC3 plays an important role in BCRmediated Ca2+ and MAPK signalling.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: The effect of steroid hormone and related compounds on cellular proliferation: Studies using breast cancer cells
Molecular mechanisms of thermosensituve
TRP channel activity
Tominaga, Makoto (Sec. Cell Signaling, Okazaki Inst.
Integrative Biosci.)
Among the huge TRP super family of ion channels, some have been
proven to be involved in thermosensation. Insight into the molecular nature of temperature-gated ion channels came with the cloning of the capsaicin receptor, TRPV1 and the recognition that this ion channel protein
could be activated by elevated temperatures with a threshold near 43°C.
Three other TRPV channels, TRPV2, TRPV3 and TRPV4, have been
cloned and characterized as thermosensors. The threshold temperatures
for activation of these channels range from relatively warm (TRPV3 and
TRPV4) to extremely hot (TRPV2). In contrast to the four heat-sensitive
TRPV channels, TRPM8 and TRPA1, have been found to be activated
by cold stimuli. Most of the mammalian thermosensitive TRP channels
(thermoTRPs) identified to date can alternatively be activated by
chemical stimuli, such as capsaicin for TRPV1. We found that
temperature acts as a co-activator of thermoTRPs. In addition,
thermoTRPs have various mechanisms for their functional regulation
such as TRPV1 regulation through phosphorylation by PKC.
Furthermore, we found that TRPM2, phylogenetically close to TRPM8,
is a new thermoTRP activated by warm temperatures. We also found that
TRPV4 needs other cytosolic proteins for its functional regulation. I
summarize the recent progress in thermoTRPs research, especially about
molecular mechanisms of their functional regulation by focusing on our
own results.
The effect of steroid hormone and
related compounds on cellular
proliferation: Studies using breast
cancer cells
Novel regulatory mechanisms for the
vascular receptor-operated Ca2+ entry
channel TRPC6
Observation of hormone-responsive
growth using three-dimensional cultures
Inoue, Ryuji1; Jensen, Lars2; Geshi, Naomi1,2;
Takahashi, Shinichi1; Mori, Yasuo3; Ito, Yushi2 (1Dept.
The use of in vitro culture methods which faithfully reproduce the in vivo
behavior of cells is expected to add further information for prediction of
effectiveness of drugs used for treatment of mammary cancer. The conventional monolayer culture method, however, has not always been satisfactory in this respect. For example, only limited degrees of growth and
differentiation of mouse mammary epithelical cells have been observed
in vitro in response to mammogenic and lactogenic hormones. Analysis
of physicochemical microenvironment surrounding the mammary epithelial cells suggested that extracellular matrix as well as chemical mediators produced by the stromal cells of the mammary gland may be the
key factors for the hormone-responsiveness. Our studies led us to hypothesize that hepatocyte growth factor (HGF) may be the mammary
stroma-produced environmental factor which mediates the growth of adjacent epithelial cells. Use of three-dimensional collagen gel matrix culture method further enabled us to observe the hormone-responsive
growth as well as branching morphogenesis of mammary epithelial cells.
In the absence of homornes in the culture medium, the cells underwent
apoptosis. Similarly to the observations on mammary epithelial cells,
ventral prostate epithelial cells of the mouse grew in response to androgens under the three-dimensional collagen gel culture conditions in the
presence of HGF. These observations strongly support the hypothesis
that three-dimensional culture conditions allow the cells cultured in vitro
to behave as those in vivo.
Physiol., Sch. Med. Fukuoka Univ.; 2Dept. Pharmacol., Grad. Sch.
Med., Kyushu Univ.; 3Lab. Mol. Biol., Dept. Syn. Chem. & Biol.
Chem, Grad. Sch. Engineer., Kyoto Univ.)
TRPC6 (a canonical subfamily member of transient receptor potential
protein) is a predominant isoform expressed in vascular smooth muscle
and likely serves as an integrative non-voltage-gated Ca2+ entry channel
regulating the vascular tone and remodeling. Activation of this channel
occurs polymodally by stimulation of PLC-linked, G-protein-coupled
and tyrosine kinase receptors and mechanical forces. Although store depletion and diacylglycerol have been proposed to be important activating
signals, we have recently found that two novel mechanisms, i.e., Ca2+/
calmodulin (CaM)-dependent phosphorylation and generation of an
arachidonic acid (AA) metabolite, 20-hydroxyeicosatetraenoic acid (20HETE), strongly affect TRPC6 channel activities. Assuming a similar
membrane topology to TRPC1, the former may involve the phosphorylation of T487 on the II-III intracellular loop of TRPC6 channel presumably via CaM-dependent kinases bound to its C-terminus, which leads to
priming of the channel for opening in response to receptor stimulation.
In contrast, the latter requires the preceding activation of TRPC6 channel
by receptor stimulation, which appears to render the channel mechanosensitive through mechanical activation of phosphopliase A2 and subsequent metabolization of AA into 20-HETE via vascular smooth
muscle specific cytochrome P450 enzymes having ω-hydroxylase
activities. The both mechanisms seem to contribute to maintaining the
vascular tone.
Enami, Jumpei (Research Laboratory, Zenyaku Kogyo Co., Ltd.)
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: The effect of steroid hormone and related compounds on cellular proliferation: Studies using breast cancer cells
Age- and parity-dependent change in
biological characteristics of rat mammary
stem cells (clonogens)
Shimada, Yoshiya; Nishimura, Mayumi (Natl. Inst. Radiol.
Sci., Chiba, Japan)
It is hypothesized that stem cells are the targets for carcinogenesis. If
cancer arises from stem cells, cancer risk would depend on population
size and susceptibility to carcinogens of stem cells . Study on A-bomb
survivors shows clear age-related decline in the susceptibility to radiation-induced breast cancer. It is also known that women who undergo
full-term pregnancy have a reduced lifetime risk of breast cancer. These
results suggest that protection results from intrinsic effect of aging and
parity on breast tissues. We here examined change in the biological characteristics of rat mammary stem cells (clonogens) with aging and parity.
The results are as follows. (1) Total numbers of clonogens increased exponentially with a population doubling time of 4 days during pre-pubertal period. After puberty, it lengthened to 30 days. The total number of
clonogens in abdominal and inguinal mammary glands of 2 week-old
rats was 200, while that in 8 week-old and thereafter was more than
5,000. (2) The number of mammary clonogens in rats which underwent
pregnancy was less than 500, while that of nulliparous rats was 6,000.
(3) Prolactin treatment increased clonogen number by 8 folds in 8 weekold rats whereas it increased by just 2 folds in one year-old rats. (4) The
surviving fraction of clonogens before puberty after 5 Gy was 0.1, while
it was 0.3 after puberty. These results suggest that population size, response to prolactin and radiobiological characteristics of clonogens,
which change in age- and parity-dependent fashion, is associated with
susceptibility to radiation-induced mammary tumors.
Effectors of estrogen and tamoxifen
actions in breast cancer cells.
Iwasaki, Toshiharu; Koibuchi, Noriyuki (Dept. Integrative
Physiol., Gunma Univ. Grad. Sch. Med., Maebashi, Japan)
Estrogen receptor (ER) belongs to the nuclear receptor super family, and
is a key regulator of proliferation and differentiation in normal mammary gland and breast cancer cells. It has been reported that steroid and xenobiotic receptor (SXR), a new member of nuclear receptors, is
expressed in breast cancer cells. We investigated the role of SXR and
found a series of novel actions. (I) tamoxifen (TAM) activated SXR-mediated transcription of cytochrome P-450 3A4 (CYP3A4) and multidrug
resistance-1 (MDR-1) genes, which are involved in TAM metabolism.
Thus, SXR may be involved in TAM resistance by decreasing its local
concentration. (II) ER-mediated transcription was potentiated by SXR in
a receptor-concentration dependent manner in MCF-7 cells. We then further investigated the mechanism of SXR action. SXR did not bind with
ER or estrogen response element, and did not alter ER-coactivator binding. On the other hand, the binding between ER and silencing mediator
of retinoid and thyroid receptors (SMRT) was decreased by SXR in a
dose dependent manner. These results suggest that (III) SXR augmented
the ER-mediated transcription, by squelching limiting amount of SMRT.
These series of studies have shown that SXR expression in breast cancer
may alter the sensitivity to estrogen and its related compounds. SXR
may stimulate the development by potentiating estrogen action through
ER. It may decrease the effect of TAM by facilitating its metabolism.
Taken together, SXR may be an exacerbation factor of breast cancer.
Analysis of Estrogen-Responsive Genes
Involve in Growth and Progression of
Breast Cancer
Hayashi, Shin-ichi (Dept. Med. Technology, Sch. Med., Tohoku
Univ., Sendai, Japan)
Since estrogen plays an important role in the growth and progression of
human breast cancer, understanding the whole picture of estrogen signaling is a very important goal towards clarifying the biology of this disease. So far, we have studied the molecular mechanisms of estrogendependent breast carcinogenesis, specifically from the viewpoints of estrogen receptor (ER) gene expression and functional modulation of ER
in breast cancer. Recent several years, we are focusing the development
of new tools such as estrogen-responsive microarray and ERE-GFP reporter cells, which enable to characterize the estrogen-responsive genes
in breast cancer cells and estrogen signal-sensitivity in individual breast
cancer. We first identified estrogen-responsive genes by the comprehensive expression profiling in ER-positive breast cancer cells, and produced a custom-made estrogen-responsive microarray of narroweddown subset. Using this microarray, we studied several basic researches
for estrogen signaling such as the effect of estrogen-antagonists and endocrine-disruptors on estrogen-responsive gene expression profile. In
this study, we found that transcription factor EGR3 is the bona fide target
gene for ERa and involved in the estrogen-signaling pathway in breast
cancer cells. Furthermore, the expression of another new estrogen-responsive gene HDAC6 significantly correlated with survival of breast
cancer patients. In vitro study revealed that the HDAC6 caused the
deacetylation of a-tubulin in cytosol and induced cell motility in ER-positive breast cancer cells.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Organized spontaneous activities in the brain network—mechanism and significance
Neocortex-hippocampus interactions
through slow and fast network oscillations
Isomura, Yoshikazu1,2; Buzsaki, Gyorgy2 (1Neural Circuit
Theory, RIKEN BSI, Wako, Saitama, Japan; 2CMBN, Rutgers Univ.,
Newark, NJ, USA)
Organized spontaneous activities in
the brain network—mechanism and
The neocortex and the hippocampus are connected by way of the entorhinal cortex and the subiculum. To examine the ongoing network interactions among these cortical areas during neocortical slow (<1 Hz)
oscillations and hippocampal fast (80-250 Hz) oscillations, we recorded
intracellular potentials in single neocortical, entorhinal, subicular, and
hippocampal neurons, together with hippocampal field potentials and
multi-unit activity in adult rats, anesthetized with urethane and ketamine. We have found that 1) most entorhinal and subicular neurons displayed slow oscillations, including bimodal depolarizing (up) and
hyperpolarizing (down) states, in synchrony with neocortical slow oscillations, 2) no bimodal up-down distribution of the membrane potential
was present in hippocampal CA3 and CA1 neurons, 3) while hippocampal granule cells were directly driven by the up state (by way of the entorhinal input), CA3 and CA1 neurons discharged during both up and
down states, 4) gamma (30-80 Hz) and fast (ripple) oscillations were observed in the hippocampal CA1 area irrespective of the up-down transition, 5) hippocampal ripples and neocortical slow oscillations correlated
only weakly and at a long (sec) time scale. These observations suggest
that entorhinal and subicular regions are "neocortex-like" and distinct
from hippoocampal circuits that lack the necessary mechanisms for the
maintenance of slow oscillations; hippocampal networks can generate
self-organized gamma and ripple activities independent of the neocortical/entorhinal slow oscillations.
Structures and functions of spontaneous
activity in the cortex
Spontaneous activity, plasticity in cultured
cortical networks
Ikegaya, Yuji; Matsuki, Norio (Grad. Sch. Pharmaceut. Sci.,
Jimbo, Yasuhiko (Grad. Sch. Engng. Univ. Tokyo, Tokyo, Japan)
Univ. Tokyo, Tokyo, Japan)
Activity-dependent plasticity probably plays an important role in learning and memory as well as proper network formation during development. Though synaptic plasticity has been widely and extensively
investigated, little is known about its consequences in network activity.
We have applied micro-electrode arrays (MEAs) for neuronal ensemble
recording. The MEA is a dish for cell culture, on the surface of which
multiple micro-electrodes are embedded. Cortical neurons were taken
from E18 Wistar rat embryos and cultured on the MEAs. Spontaneous
activity started at about 3 days in vitro (DIV). Relatively long-lasting activity with loose network coupling was observed. Then the activity grew
up to periodic synchronized bursts with tight coupling. In about one
month, it reached a steady state. The steady-state activity was composed
of synchronized bursts at approximately 1 Hz, and some asynchronous
components. The spatial propagation patterns were not unique, but could
be classified into a few groups. The substrate-embedded electrodes
could also be used for stimulation. Several evoked activities were recorded and the effects of focal high frequency stimulation were evaluated. After the high frequency stimulation, some of the signal propagation
pathways were strengthened. The same high frequency stimulation
weakened some of the pathways. The correlation analysis revealed that
the pathways that were tightly correlated with the repeatedly activated
pathway were selectively potentiated. The next step will be to study how
spontaneous activity during development affects the network and single
cell properties.
The brain is continuously active. Spontaneous neuronal activity is prevalent in vivo and in vitro and could reflect intrinsic functional properties
of the microcircuit, so its dynamics may help reveal the basic logic of
network operations. However, it is largely unknown how such naturally
generated spikes are organized or how they can affect individual synaptic efficacy. We reconstructed spike patterns of many cortical neurons in
vitro and found that sequences of activity were reactivated in the same
spatiotemporal order. Spontaneous activity drifts with time, recruiting
different sets of cells, and thereby, sequences are replaced with novel
patterns. Patterns of spontaneous activity were predictable by training a
feedback neural network model with a past period of dataset. We also
sought to determine whether spontaneous activity alters synaptic
strength. When hippocampal slices were exposed to ACSF that mimicked the extracellular ionic compositions in vivo, cells started to exhibit
slow wave oscillations with rhythmic action potentials. After wash-out,
postsynaptic currents were altered at CA3 synapses. The direction of
synaptic plasticity was determined by the frequency of UP-DOWN state
alternations. When the modified ACSF was repetitively applied, identical cells generated different oscillation rhythms, and thus, changes in
synaptic efficacy varied from trial to trial. Therefore, spontaneous selfexcitation of cortical networks is non-randomly structured and can modify synaptic weights. Our talk provides a novel regimen of cortical operations, i.e., self-rewritable microcircuitry with ongoing plasticity.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: The circadian timing system: From clock gene expression to physiology
Involvement of endogenously released
ATP in the generation of network-driven
spontaneous oscillation in the neonatal
Kato, Fusao1; Kawamura, Masahito2; Yamaoka,
Masayoshi3 (1Lab. Neurophysiol., Dept. Neurosci., Jikei Univ.
Sch. Med., Tokyo, Japan; 2Dept. Phamacol., Jikei Univ. Sch. Med.,
Tokyo, Japan; 3Jikei Univ. Med. Sch., Tokyo., Japan)
The earliest organized intrinsic activity in the hippocampal network appears during the perinatal period, which is characterized by slow synchronized bursting of the pyramidal neurons and interneurons in forms
of giant depolarizing potentials (GDPs) in the slices and "sharp waves"
in vivo. These activities are proposed to be involved in the maturation of
the interneuronal networks (Ben-Ari et al., 2004). Since the generation
of GDP requires depolarisation of pyramidal neurons by GABA released
from interneurons and since exogenous ATP excites inhibitory interneurons in the CA3 through activation of P2Y1 receptors (Kawamura et al.,
J Neurosci, 2004), we examined whether activation of interneuronal purinoceptors by endogenous ATP and subsequent excitation of interneurons underlie the GDP generation. The spontaneous GDP activities were
recorded by patch-clamp recording from CA3 pyramidal neurons and by
imaging of [Ca2+]i with fluo-4 in the coronal hippocampal slice of the
rats (P4-8). The responses of GDPs to pharmacological manipulations of
ATP and adenosine receptors and their metabolism suggest a possible involvement of activation of interneuronal purinoceptors by endogenous
extracellular ATP in the maintenance of GDP, which might provide an
important mechanism linking on-going metabolic condition and largescale network activities in the early development.
Functional analysis of clock genes: from
clock gene expression to physiology
Ikeda, Masaaki1 (1Dept. of Physiol. Saitama Med. Sch.
Moroyama, Saitama, Japan; 2Mol. Clock Project, Saitama Med. Sch.
Res. Center for Genomic Medicine, Hidaka, Saitama, Japan)
Circadian clocks constitute a global regulatory system found in most eukaryotes. The center of the circadian rhythm is located in the suprachiasmatic nuclei (SCN) and informs the peripheral organs of the timing via
neuronal and hormonal pathways. It is driven by complexes of the transcription factors CLOCK and BMAL1, while CRY and PER oppose
CLOCK/BMAL1 activity, closing a negative feedback loop that results
in an approximately 24-hour rhythm. CLOCK/BMAL1 bind the E-box
in the promoter region of not only clock components, but also so-called
output genes of the circadian clock. Core clocks are located all over the
body and control the circadian expression of genes that regulate fat metabolism, the cell cycle, neural activity, and so on. In this symposium, we
will discuss the core clock mechanism and clock gene functions from the
cellular level to the level of the human body.
Neural functions and clock genes
Takumi, Toru (Osaka Bioscience Inst. (OBI), Suita, Japan)
The circadian timing system: From
clock gene expression to physiology
Several non-clock functions of clock genes have been discovered. In
mammals, the circadian system and stress systems, both centers of which
are located in the hypothalamus, are involved in an adaptation to predictable and unpredictable environmental stimuli, respectively. Although
the interaction and relationship between these 2 systems are intriguing
and have been studied in different ways since the "pre-clock-gene" era,
the molecular interaction between them largely remains unknown. I
show by systematic molecular biological analysis that acute physical
stress elevated only Period1 (Per1) mRNA expression in mouse peripheral organs. Although behavioral rhythms in vivo and peripheral molecular clocks are rather stable against acute restraint stress, the results of a
series of promoter analyses, including chromatin immunoprecipitation
(ChIP) assays, indicate that a glucocorticoid responsive element (GRE)
in the Per1 promoter is indispensable for induction of this mRNA both
in vitro and in vivo. These results suggest that Per1 can be a potential
stress marker and that there may exist a third pathway of Per1 transcriptional control in addition to the clock-regulated BMAL1/CLOCK-E-box
and light-responsive CREB-CRE pathways.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: The circadian timing system: From clock gene expression to physiology
Clock genes responsible for the circadian
rhythms in cytosolic calcium
concentrations in mice and Drosophila
Sleep-wake cycle and clock genes
Ikeda, Masayuki1; Kojiya, Tomoyoshi1; Ikeda, Masaaki2;
Goto, Junpei1; Matsumoto, Akira3; Matsumoto, Ken4;
Some people cannot adjust their sleep-wake cycle to socially-desired
time schedule, called circadian rhythm sleep disorders (CRSD). Recent
studies revealed that functional variations in human clock genes confer
susceptibility to CRDS, such as delayed sleep phase syndrome (DSPS),
advanced sleep phase syndrome (ASPS), and non-24-hour sleep-wake
syndrome (N-24). Missense variations in Period2 (Per2) gene and
Casein kinase1 delta (CK1δ) gene, each of which reduces
phosphorylation of PER protein, reportedly cause familial ASPS. We
have already reported that a missense variation in Per3 gene, which
presumably affect phosphorylation of PER3 protein, increases the risk
for DSPS and that a missense variation in CK1ε gene, which increases
the kinase activity, plays a protective role in the development of DSPS.
It is intriguing that all of the CRDS-susceptibility variations found so far,
as described above, seem to alter the phosphorylatrion of PER
proteins.Functional clock gene variations are also observed in
apparently normal subjects and likely to induce interindividual
differences in circadian period. Comprehensive genetic analysis for
variations of human circadian rhythmicity will make it possible to fully
understand the characteristics of each indivisual's internal clock, leading
to alleviation of health injury and economic loss induced by sleep
disorders or maladaptation to socially-desired time schedule, with which
a large number of people are afflicted in the modern society.
Yamamoto, Daisuke5 (1Department of Biology, Faculty of
Science, Toyama University, Toyama, Japan; 2Research Center for
Genomic Medicine, Saitama Medical School, Saitama, Japan;
3Department of Biology, Graduate School of Sciences, Kyushu
University, Ropponmatsu, Fukuoka, Japan; 4Research Group of
Chronogenomics, Mitsubishi Kagaku Institute of Life Sciences,
Tokyo, Japan; 5Graduate School of Life Sciences, Tohoku University,
Sendai, Japan)
We have shown that cytosolic Ca2+ concentration oscillates in circadian
fashion in cultured mouse suprachiasmatic nucleus (SCN) neurons and
in the in vivo Drosophila ventral lateral neurons (LNvs). Since these neurons play a critical role for the generation of behavioral rhythms, the circadian Ca2+ oscillations may mediate cellular output processes in
circadian pacemaker neurons in diverse systems. To further analyze the
function of clock genes for the circadian Ca2+ rhythm generations, the
present study examined over-expression of Bmal1 genes or of its dominant-negative genes in cultured SCN neurons. Also, Per01 mutant flies
carrying pdf-gal4/UAS-cameleon were generated to investigate the
function of clock genes in the Ca2+ dynamics of Drosophila LNvs. All of
these genetic manipulations caused reduction of circadian Ca2+ rhythms.
Therefore, we concluded that temporal patterns of cytosolic Ca2+ concentrations are regulated by the above clock gene families in circadian
pacemaker neurons.
Ebisawa, Takashi (Dept. Sleep Disord. Res., Grad. Sch. Med.,
Univ. Tokyo, Japan)
Clock gene and obesity
Oishi, Katsutaka; Ishida, Norio (Clock Cell Biol. Res. Group,
Consequences of a mutation in the murine
Per2 gene on physiological parameters
IBRF, AIST, Tsukuba, Japan)
Albrecht, Urs (Dept. of Med , Div. of Biochem, Univ. of Fribourg)
Recent studies of clock genes have revealed that an autoregulatory transcriptional feedback loop forms the core circadian rhythm generating
mechanism in mammals. Clock is the first clock gene identified in vertebrates by forward mutagenesis using N-ethyl-N-nitrosourea in a behavioral screening, and encodes a basic helix-loop-helix (bHLH)-PAS
transcription factor. Previously, we identified putative CLOCK target
genes in the mouse liver using microarray analyses and found that in addition to being a core component of the circadian oscillator, CLOCK is
involved in various physiological functions. We show here that serum
levels of triglyceride and free fatty acid were significantly lower in circadian Clock mutant ICR than in wild-type control mice, whereas total
cholesterol and glucose levels did not differ. Moreover, an increase in
body weight induced by a high-fat diet was attenuated in homozygous
Clock mutant mice. We also found that dietary fat absorption was extremely impaired in Clock mutant mice. Circadian expressions of cholecystokinin-A (CCK-A) receptor and lipase mRNAs were damped in the
pancreas of Clock mutant mice. We therefore showed that a Clock mutation attenuates obesity induced by a high-fat diet in mice with an ICR
background through impaired dietary fat absorption. I will also talk
about our recent findings that CLOCK is involved in the diabetes- and
obesity-induced cardiovascular diseases by increasing the expression of
plasminogen activator inhibitor-1 (PAI-1).
Living on earth has made us use the sun as reference and the 24-hour succession of light and darkness is probably the most pervasive epigenetic
influence in the evolution from a single cell organism to man. This periodic succession of light and darkness provided the base for relative timing of biological processes over the 24 hours of a day. Because energy
supply is the limiting parameter for survival, a system for optimal timing
of energy expenditure and uptake developed. The mechanism of this system took the shape of a cycle reflecting the recurrence of sunrise and
sunset, and is termed a " circadian clock" - a clock with a period of about
one day (latin: circa diem). The internalization of environmental time
within the organism not only allows organization of biological processes
along the 24-hour time scale but also prediction of recurring events, such
as availability of food and emergence of predators. Therefore it is not
surprising that alterations in the genetic machinery of the circadian clock
leads to alterations in biochemical and physiological processes.Data illustrating the influence of the Per2 gene on adaptation to changing lighting conditions, addiction, the aging process and food anticipation will be
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Molecular and cellular physiology of the metabolic control [Korea–Japan Joint Symposium]
Implication of Myosin Light Chain Kinase in
the Insulin-Stimulated GLUT4
translocation in Adipocytes
Jang, Yeon-Jin; Kim, Hye_Rim; Choi, Young_Ok; Song,
Young_Sook; Choe, Han; Leem, Chae_Hun (Department
of Physiology, University of Ulsan College of Medicine, Seoul,
Republic of Korea)
Molecular and cellular physiology
of the metabolic control [Korea–
Japan Joint Symposium]
In adipocytes, insulin stimulates glucose transport principally by promoting translocation of glucose transporter GLUT4 from an intracellular
compartment to the plasma membrane. Requirements for Ca2+/calmodulin during insulin-stimulated GLUT4 translocation have been demonstrated; however, the mechanism of action of Ca2+ in this process is
unknown. Recently, myosin II, whose function in non-muscle cells is
primarily regulated by phosphorylation of its regulatory light chain
(RLC) by the Ca2+/calmodulin-dependent myosin light chain kinase
(MLCK), was implicated in insulin-stimulated GLUT4 translocation.
We have investigated, using 3T3-L1 and 3T3-F442A adipocytes, the
possibility that MLCK may be involved in the insulin-stimulated translocation of GLUT4. Insulin significantly increases phosphorylation of
the myosin II RLC in a Ca2+-dependent manner. ML-7, a selective inhibitor of MLCK, as well as inhibitors of myosin II, such as blebbistatin and
2,3-butanedione monoxime, block insulin-stimulated GLUT4 translocation and subsequent glucose transport. In addition, suppression of
MLCK expression via stably expressing antisense-MLCK decreases insulin-stimulated glucose transport. Our studies strongly suggest that
MLCK may be a regulatory target of Ca2+/calmodulin and may play an
important role in insulin-stimulated GLUT4 translocation in adipocytes.
Role of the orphan nuclear receptor SHP on
the control of metabolic homeostasis
Choi, Hueng-Sik (Hormone Research Center, Chonnam National
University, Kwangju, Republic of Korea)
Small heterodimer partner (SHP; NR0B2) is a member of the large nuclear receptor family of transcriptional factors that lacks a conventional
DNA binding domain. Various studies have reported SHP to be a repressor of transcriptional activities of a number of nuclear receptors, including glucocorticoid receptor, estrogen receptor, androgen receptor,
thyroid hormone receptor, retinoic acid receptor, retinoid X receptor,
constitutive androstane receptor, pregnane X receptor, HNF4α, liver
receptor homologue 1, estrogen-related receptor-γ, Nur77 and liver X
receptor (LXR). The very broad range of receptors sensitive to inhibition
by SHP suggests a central role for SHP in modulation of nuclear receptor
signaling pathways. SHP is expressed in a wide variety of tissues,
including heart, brain, liver, spleen, adrenal gland, small intestine, and
pancreas. Moreover, human SHP gene is located on chromosome 1p36.1
and consists of two exons separated by an intron. SHP gene transcription
is regulated by several members of the nuclear receptor superfamily
including the bile acid receptor farnesoid X receptor, steroidogenic
factor-1, HNF4α, liver receptor homologue 1, estrogen receptor and
estrogen-related receptor-γ. Recent progresses on the elucidation of
molecular mechnism of SHP gene expression and function will give us
a chance to develop new drug therapies treating a variety of human
diseases including diabetes, obesity and disorder of lipid and cholesterol
Roles of Diet in Muscle Insulin Resistance
Kim, Jong-Yeon (Department of Physiology, Yeungnam
University College of Medicine, Daegu, Republic of Korea)
Dietary factors have been implicated in the development of hepatic and
peripheral insulin resistance. Differently composed diets can induce insulin resistance in different ways, but the mechanisms underlying these
phenomena are not yet clear. This study was conducted to evaluate
whether dietary composition change affects insulin resistance in the
skeletal muscles of rats fed high-carbohydrate diet or high-fat diet. We
assessed glucose transport in the skeletal muscles of rats in vitro. Diets
given were rat chow, high-starch (HT), high-sucrose (HS), high-fat highstarch (HFHT), high-fat high-sucrose (HFHS, HF), HF with fish oil
(HF+FO), and HF with linseed oil (HF+LO). Both of HS diet and HT
diet with or without high-fat depressed insulin-stimulated glucose transport compared with chow diet, but there were no significant difference
between groups. HF diet markedly decreased the insulin-stimulated glucose transport, and fish oil improved this partially, but linseed oil did not
significantly. Percent visceral fat pad mass, plasma insulin and triglyceride in high carbohydrate or high-fat diet groups that developed muscle
insulin resistance were much higher compared with chow diet group.
Fish oil and linseed oil decreased percent visceral fat pad mass, and fish
oil decreased plasma insulin and triglyceride. The composition of fat diet
was more important factor than that of carbohydrate diet to induce muscle insulin resistance assessed by glucose transport in vitro. Plasma triglyceride and insulin concentrations seemed to be important factors to
induce muscle insulin resistance in rats.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Molecular and cellular physiology of the metabolic control [Korea–Japan Joint Symposium]
Transcriptional Control of the
Differentiation of Pancreatic β Cells
Watada, Hirotaka (Department of Metabolism, Juntendo
Medical College, Tokyo, Japan)
The main role of pancreatic β cells is to secrete insulin in response to an
increase of the blood glucose level. To accomplish this, β cells express
numerous genes essential for glucose-responsive insulin secretion. To
allow the expression of such strictly selected multiple sets of genes,
various differentiation steps are required during pancreatic development.
As is the case for other types of cells, recent studies have identified
several transcription factors that control the activation and repression of
a large number of genes during pancreatic development and how these
factors function. Accumulation of such knowledge has revealed that
transcription factors orchestrate the intricate pathways of cellular
growth, death, and differentiation by direct regulation of gene
expression. Amongst the transcription factors in this well-organized
cascade, neurogenin 3 (Ngn3) plays a key role in determining the fate of
cells in the endocrine pancreas. We recently found how signals from
adjacent cells regulate the expression of Ngn3 in pancreatic precursor
cell. In addition, we found that Ngn3 regulates the expression of Pax4
and Nkx2.2 cooperated with HNF factors, thus induces β cell
Transcription factors that regulate insulin
sensitivity in the liver and metabolic
Shimano, Hitoshi (Dept. of Internal Medicine, Graduate School
of Comprehensive Human Sciences, University of Tsukuba, Tsukuba,
Liver plays a central role in energy metabolism depending upon nutritional states and hormones. Since long-term regulation of carbohydrate
and lipid metabolisms is controlled at the transcriptional level, hepatic
nutritional transcription factors play a pivotal role in energy metabolism.
SREBP-1c has been established as a transcription factor that controls
synthesis of fatty acids and triglycerides based upon our data from
SREBP-1c transgenic and knockout mice. SREBP-1c also regulates insulin sensitivity via direct regulation of IRS-2, a key insulin-signaling
molecule in the liver. Nutritional induction of hepatic SREBP-1c by dietary carbohydrates and saturated fatty acids well explains how liver
shifts metabolism from glycogen synthesis to lipogenesis in the feeding
cycle. To excess, SREBP-1c activation contributes to components of
metabolic syndrome such as dyslipidemia, diabetes, fatty liver, and insulin resistance, and finally leading to atherosclerosis as observed in our
murine models. Meanwhile, we have identified TFE3 as a strong activator of insulin signaling. TFE3 transcriptionally activates IRS2 and diversely participates insulin signaling and markedly ameliorates diabetes
in different models. TFE3 and FOXOs synergistically activate, and
SREBP-1c competitively suppresses IRS-2 promoter. Collectively, these
energy transcripton factors regulate carbohydrate-lipid metabolism, insulin signaling, and might be involved in metabolic syndrome and diabetes. Thus, these factors could be future therapeutic targets.
Ubc9 Regulates Insulin Sensitivity by
Promotion of GLUT4 Targeting to the
Insulin-Sensitive Storage Compartment
Shibata, Hiroshi (IMCR, Gunma University, Maebashi, Japan)
In muscle and adipose cells, insulin stimulates glucose uptake more than
several folds by recruiting the insulin-regulated glucose transporter,
GLUT4 from intracellular compartments to the plasma membrane.
While such large insulin stimulation of glucose transport coincides with
the expression of GLUT4 during differentiation of these cells, GLUT4
expression does not necessarily confer insulin sensitivity to glucose
transport in other types of cells. Previous studies have shown that in
muscle and adipose cells, GLUT4 is targeted to a unique GLUT4 storage
compartment (GSC) sequestered from the constitutive recycling pathway, whereas the significance and the mechanism of GLUT4 targeting
to GSC have remained obscure. We have recently found that Ubc9, the
SUMO conjugating enzyme, may be an important regulatory protein in
subcellular targeting and turnover of GLUT4. Adenovirus vector-mediated overexpression of Ubc9 in 3T3-L1 adipocytes substantially increased GLUT4, which was accompanied by promoted targeting of
GLUT4 to GSC, consequently leading to enhanced insulin responsiveness of glucose transport. On the other hand, siRNA-mediated depletion
of Ubc9 caused a marked down-regulation of GLUT4, with a selective
loss of GLUT4 in GSC, and significantly attenuated the insulin effect on
glucose transport. Interestingly, the turnover of GLUT4 was significantly retarded by targeting to GSC, but was accelerated by residing in the
recycling pathway. Thus, Ubc9 plays an indispensable role in acquisition
and maintenance of the insulin sensitivity of glucose transport in adipocytes.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Mechanisms for regulation of exocytosis and its physiological significance
Multiple types of Calcium channels and
their distribution in the presynaptic nerve
Kidokoro, Yoshiaki1 (1Institute for Molecular and Cellular
Regulation, Gunma University; 2Department of Physiology, David
Geffen School of Medicine at UCLA, USA)
Mechanisms for regulation of
exocytosis and its physiological
After exocytosis the synaptic vesicle (SV) membrane is recycled by endocytosis. Exocytosis requires external Ca and so does endocytosis,
which is supplied through voltage-gated Ca channels in the presynaptic
membrane. Multiple types of calcium channels in the presynaptic terminal are participating in these processes. Ca channels designated for exocytosis are highly localized at the release site. Other types of Ca channels
are probably diffusely distributed and may contribute to endocytosis.
Two types of endocytosis have been demonstrated in EM at the Drosophila neuromuscular junction, namely, active-zone endocytosis that occurs at the presynaptic active zone and non-active-zone endocytosis that
operates at the area away from the active zone. Recently we found that
two separate types of Ca channels support these two types of endocytosis. Non-active-zone endocytosis is blocked by low concentrations of La,
while active-zone endocytosis is inhibited by a spider toxin, PLTXII. Yet
another type of Ca channel encoded by the gene, cacophony, is specifically designated for exocytosis. This type of channel, cac-Ca channel, is
highly localized at the presynaptic active zone. The distribution of other
types of Ca channels is difficult to demonstrate, but physiological findings indicate that PLTXII-sensitive channels are located close to or within the active zone, while the La-sensitive channels reside away from it.
Ca is ubiquitously used a messenger. Its temporal and spatial characteristics mold its specific role.
Timing of synaphin/complexin action in
neurotransmitter release
Tokumaru, Hiroshi (Faculty of Pharmaceutical Sciences at
Kagawa Campus, Tokushima Bunri, Kagawa, Japan)
The release of neurotransmitters at synapses requires the temporally-ordered trafficking of synaptic vesicles. More than 20 proteins are involved in this process, including the SNARE proteins that participate in
membrane fusion during exocytosis. To discern the specific role that
each protein plays, it is necessary to sort out the temporal order in which
they interact. Here we consider the timing of action of synaphin (also
named as complexin), a SNARE-binding protein that plays an important
role in calcium-triggered neurotransmitter release. A light-activated
binding-site peptide was used to perturb the interaction of synaphin with
SNARE proteins at precise time intervals before neurotransmitter release. We find that this peptide inhibits neurotransmitter release within
180 milliseconds before synaptic vesicles fuse with the presynaptic plasma membrane. These results indicate that synaphin binds to SNARE
complex after synaptic vesicles dock but well before the fraction of a
millisecond required for calcium ions to trigger synaptic vesicle fusion.
These results are compatible with a role for synaphin in preparing transSNARE complexes for membrane fusion.
Bidirectional regulation of synaptic
efficacy through AMPA-receptor trafficking
in cerebellar Purkinje cell
Yamaguchi, Kazuhiko1; Tatsukawa, Tetsuya1,2;
Matsumoto, Azumi1 (1Lab. for Memory and Learning, BSI
RIKEN, Wako, Saitama, Japan; 2Lab.Cell.Neurobiol., Fac.of Life
Sci., Tokyo Univ. of Pharm. and Life Sci., Tokyo Japan)
Regulation of synaptic expression of AMPA type glutamate receptors
(AMPA-Rs) through a receptor-trafficking is one of the underlying
mechanism for synaptic plasticity in hippocampal and cerebellar neurons. In cerebellar Purkinje cell, induction of the long-term depression
(LTD) of parallel fiber (PF)-EPSC requires clathrin-mediated endocytosis, however, relation between the constitutive rapid trafficking and
LTD-induction was unclear. Suppressing exocytosis or endocytosis, we
addressed whether regulation of the rapid constitutive trafficking of
AMPA-Rs was underlying synaptic plasticity in cerebellar Purkinje cell.
Effects of intracellular infusion of tetanus toxin (TeTx), a blocker of
VAMP2, on PF-EPSC were analyzed in cerebellar slice using whole-cell
patch-methods. Infusion of TeTx reduced amplitude of PF-EPSCs to
60% within 20 min. Since infusion of botulinum neurotoxin C (a blocker
of syntaxin) reduced PF-EPSC amplitude to similar extent, contribution
of TeTx-insensitive receptor-trafficking was suggested to be negligible.
As a counterpart of constitutive elimination of synaptic AMPA-R, constitutive insertion of AMPA-Rs into PF-synapse was demonstrated by
blocking of dynamin. As for relation between constitutive trafficking of
AMPA-Rs and synaptic plasticity, LTD did not occlude with constitutive
elimination of AMPA-Rs at PF-synapse, suggesting that internalization
of synaptic AMPA-Rs during LTD did not belong to constitutively recycling pool of AMPA-Rs.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Integrative approaches to neural circuit function [YFI (Young Foreign Investigator) Workshop]
Analysis of presynaptic exocytosis and its
plasticity using mice genetically
expressing synaptopHluorin in the
Yawo, Hiromu; Araki, Rikita; Hikima, Takuya; Suyama,
Shigetomo; Ishizuka, Toru (Dept. Dev. Biol. Neurosci. Tohoku
Univ. Grad. Sch. Life Sci. Sendai, Japan)
The synaptic transmission is potentiated by the activation of adenylyl cyclase (AC) and protein kinase A (PKA) at hippocampal mossy fiberCA3 synapses. Although the AC/PKA activation was suggested to facilitate the transmitter release from MF presynaptic terminals, the most of
these classical evidences were indirect. In this study the presynaptic exocytosis was directly investigated in the hippocampal slice of a synaptopHluorin (SpH) transgenic mouse (TV-42 line) which expresses SpH
specifically at the mossy fiber terminals of hippocampus (Araki et al.
2005). The repetitive stimulation (10 Hz for 1 s) of mossy fiber bundle
transiently increased the SpH fluorescence in the presynaptic terminal.
The SpH fluorescence was sampled before and after application of forskolin (50 µM) and IBMX (100 µM), a combination which activates AC/
PKA. The AC/PKA activation increased the activity-dependent
increment of SpH (∆SpH) by 2.42 ± 0.49 (mean ± SEM, n = 29) on
average (p<0.001). However, both the rising and falling time course of
δSpH was not changed. The ∆SpH was also largely facilitated in the
presynaptic terminal of which it was null at baseline. It is suggested that
the AC/PKA activation facilitates the presynaptic exocytosis and that it
turns some presynaptically silent synapses into active.
Discharge properties of neurons in the rat
medial vestibular nucleus
Saito, Yasuhiko; Takazawa, Tomonori; Ozawa, Seiji
(Dept. Neurophysiol., Gunma Univ. Grad. Sch. Med., Maebashi,
Gunma, Japan)
The vestibular nucleus (VN) is a center for stabilizing gaze and posture
in response to the head rotation and tilt. VN neurons are physiologically
classified into regularly and irregularly discharging neurons on the basis
of the regularity of spacing of action potentials. The segregation of VN
neurons is involved in different response properties to head movements.
Although the discharge regularity has been considered to be attributed to
afterhyperpolarization (AHP), the relationships between the discharge
regularity and profiles of AHP are still unclear. In this study, we investigated discharge patterns of VN neurons using whole-cell patch clamp
technique not only in vitro slice preparations but in vivo preparations obtained from young rats. Previously, AHPs were classified into AHP
without a slow component [AHP(s-)], AHP with a slow component
[AHP(s+)], and AHP with a slow component and an afterdepolarization
[AHP(s+) with ADP]. Both in vitro and in vivo, neurons exhibiting
AHP(s+) fired more regularly than the other types of neurons. Application of 100 µM apamin to block Ca2+-dependent K+ channels abolished
the slow component of AHP(s+) and made regular discharges of neurons
exhibiting AHP(s+) to be irregular. These suggest that neurons
exhibiting AHP(s+) are regularly discharging neurons, whereas neurons
exhibiting AHP(s-) and AHP(s+) with ADP are irregularly discharging
neurons. The regular firings of neurons exhibiting AHP(s+) are
attributed to activation of apamin-sensitive Ca2+-dependent K+ channels.
Ref: Araki R. et al. (2005) genesis 42, 53-60.
Mechanisms for submillisecond
coincidence detection in the chick auditory
Kuba, Hiroshi (Grad. Sch. Med. Univ. Kyoto, Kyoto, Japan)
Integrative approaches to neural
circuit function [YFI (Young
Foreign Investigator) Workshop]
Localizing sound sources requires discriminating differences of sound
arrival time of a microsecond order between the two ears (interaural time
difference, ITD). In nucleus laminaris (NL) of birds, neurons calculate
ITDs by detecting the coincidence of binaural synaptic inputs. We utilized slice-patch recordings, immunohistochemistry and computer simulations to explore the acuity and cellular mechanisms of coincidence
detection in NL neurons of the chick. At 40 °C, the avian body
temperature, the acuity of coincidence detection was high enough to
account for the animal behavior. This acuity was achieved by the
acceleration of EPSP time course due to the activation of Kv1.2mediated low-threshold K+ conductance. In NL, neurons are tuned to a
specific frequency of sound (characteristic frequency, CF), and are
arranged so that the CF decreases from rostro-medial (high-CF) to
caudo-lateral (low-CF) direction. Along this tonotopic axis, NL neurons
were specialized morphologically and functionally depending on their
CF. In the high- and middle-CF neurons, dendrites were short and
expression of Kv1.2 channels was strong, which made the EPSP time
course rapid and improved the coincidence detection. In the mid-high CF
neurons, the process of generating spikes was also specialized; the axon
initial segment was myelinated and Nav channels were clustered at some
distance from the soma (20-50 µm) in the axon. Theoretical model
predicted that this unique distribution of Nav channels in the axon is
essential for making the high-frequency generation of action potentials
and enhancing the ITD detection.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Physiological studies on environmental health—Vulnerability of biological functions to xenobiotic chemicals
Surround inhibition of nociceptive
transmission in the superficial spinal
dorsal horn through activation of tactile C
afferent fiber. -In vivo patch-clamp analysis
of modality dependent synaptic
responsesFurue, Hidemasa; Kato, Go; Yasaka, Toshiharu;
Yoshimura, Megumu (Dept of Integrative Physiol, Grad. Sch.
Med. Sci., Kyushu Univ., Fukuoka, Japan)
Disinhibition such as a loss of inhibitory interneurons or a shift in the
transmembrane anion gradient especially in the substantia gelatinosa
(SG) of the spinal dorsal horn is thought to be a crucial etiology for
chronic pain syndromes. However, there is little direct evidence to elucidate the natural inhibitory mechanism for nociceptive transmission because of difficulties in recording inhibitory synaptic responses from
small size SG neurons in vivo. In this study, whole-cell recordings were
obtained from SG neurons in vivo and in slice preparations to analyze
how inhibitory synaptic inputs modulate noxious transmission and the
underlying neuronal circuits. SG neurons in vivo responded to cutaneous
pinch accompanied with a barrage of EPSCs. On the other hand, touch
evoked a barrage of IPSCs during the stimulation and the receptive fields
were larger than those of pinch-evoked EPSCs. After cessation of a brief
touch, a burst of IPSCs lasted for about 10 s in some cells. The number
of action potentials generated by pinch was decreased by the simultaneous stimulation of touch applied to the surrounding area. In slice experiments, activation of C fiber was required to elicit the burst of
inhibitory response and large islet cells known as an inhibitory interneuron received C fiber inputs. The results suggest that innocuous stimulation even brief touch sufficiently suppresses noxious sensation in the SG
through activation of C fibers.
Physiological studies on
environmental health—
Vulnerability of biological functions
to xenobiotic chemicals
Transgenic approach to cerebellar cortical
Indoor air quality and sick house syndrome
Watanabe, Dai (Osaka Bioscience Institute, Suita, Japan)
Sick house syndrome is a disorder of nerve function, mainly affecting the
central nervous system/autonomic nervous system, caused by a sensitivity reaction induced by exposure to trace amount of deleterious chemical
substances present in the living environment. Diagnosis is not easy because pathophysiological understanding of the syndrome is not sufficiently complete.
In the cerebellar circuit, Golgi cells receive inputs from granule cells and
in turn terminate their axons on granule cell dendrites. Since Golgi cells
are the only element that controls the activity of granule cells, Golgi cells
are thought to play an important role in information processing via feedback mechanisms.First we investigated the role of Golgi cells by selective ablation using the immunotoxin-mediated cell targeting technique.
The elimination of Golgi cells caused severe acute motor disorders.
These mice gradually recovered but retained a continuing difficulty in
performing fine movements. Electrophysiological analyses indicated
that disruption of Golgi cells not only eliminates GABA-mediated inhibition but also attenuates functional NMDA receptors in granule cells.
These results demonstrate that synaptic integration involving GABA inhibition and NMDA receptor activation is essential for motor coordination. Next we investigated the synaptic mechanisms of postsynaptic
metabotropic glutamate receptor subtype 2 (mGluR2) on Golgi cell dendrites, using whole-cell patch-clamp recording of green fluorescent protein-positive Golgi cells of wild-type and mGluR2-deficient mice.
Postsynaptic mGluR2 was activated by glutamate released from granule
cells and hyperpolarized Golgi cells via G protein-coupled inwardly rectifying K channels. This hyperpolarization induced long-lasting silencing of Golgi cells, the duration and extents of which were dependent on
stimulus strengths. Postsynaptic mGluR2 thus senses inputs from granule cells and plays a pivotal role in spatiotemporal modulation of mossy
fiber-granule cell transmission.
Sakabe, Kou (Sch. Pharm, Kitasato Univ. Tokyo, Japan)
In this syndrome, functional assessment of nerve function is especially
relevant. For example, the electronic iriscorder is useful as one of the
tests of autonomic nerve functioning in this syndrome. There are many
cases of this syndrome in which some abnormality and/or instability of
pupillary light reaction, that is primarily caused by functional
abnormality of autonomic nerve function, has been observed.
Furthermore, evaluation of eye movement by Electro-Oculograph is also
very useful, as many patients have some disorder of smooth pursuit
movement. Modulation Transfer Function, which evaluates the higher
optical center (visual cortex) is also useful, and a decrease in Visual
Contrast Sensitivity is often observed.
Genetic polymorphism testing of drug metabolizing enzymes, such as
CYP, GST, NST, and PON1 are useful in evaluating an inherited
sensitivity to chemical substances in the patient. From our latest
investigation, differences such as absence, decrease in concentration,
and delay in induction of these enzymes are observed in some of the
patient groups which are obviously different from healthy people. This
is important knowledge leading to possible specification of the genes
active in expression of this syndrome.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Physiological studies on environmental health—Vulnerability of biological functions to xenobiotic chemicals
Modulation of functional development of
brain by polychlorinated biphenyls
Effects of environmental chemicals on
emotional behaivor and the brain
Koibuchi, Noriyuki (Gunma Univ. Grad. Sch. Med., Maebashi,
Aou, Shuji1; Fujimoto, Tesuya1; Fueta, Yukiko2; Ishidao,
Toru2; Hori, Hajime2; Kubo, Kazuhiko3 (1Dept. Brain Sci.
Gunma, Japan)
Polychlorinated biphenyl (PCB) is an environmental chemical that may
cause adverse health effects. Previous studies have shown that developing central nervous system is one of the most vulnerable organs against
its exposure. However, the molecular mechanisms of PCB action have
not yet been fully understood. Since PCB exposure induces abnormal
brain development similar to those seen in perinatal hypothyroid animal,
we have been studied the effect of PCB/dioxin on thyroid hormone (TH)
receptor (TR)-mediated transcription. We have previously identified that
PCB may not competitively bind to TR ligand binding domain. Instead,
it partially dissociated TR from TH-response element located on the promoter region of target gene. The mechanisms of such dissociation in not
well known. However, our recent findings have indicated that PCB may
bind to DNA binding domain of TR, which may alter the structural conformation of TR protein.In addition to PCB action on TR, we have also
studies the effects of PCB on several other nuclear receptors. It may affect to estrogen receptor (ER)-, and steroid and xenobiotic receptor
(SXR)-mediated transcription, but not to glucocorticoid or progesterone
receptor action. Further, PCB may also act to neuronal membrane to induce an increase in intracellular calcium concentration, which then stimulate the expression of calcium-induced transcription factors such as cJun.These results indicate that PCB may act at multiple systems to alter
the gene expression profile of neuronal cells that may affect the normal
brain development.
Eng., Kyushu Inst. Technol., Kitakyushu, Japan; 2Depts.
Med.Technol. & Environm. Manage,, Sch. Health Sci., Univ. Occup.
Environm. Health, Kitakyushu, Japan; 3Dept. of Otorhinolaryngol.,
Chidoribashi Hospital, Fukuoka, Japan)
The effects of environmental chemicals on sexual differentiation of exploratory behavior and emotional behaviors and brain were investigated.
We exposed bisphenol A (BPA, 0.05-5 ppm) to mother rats pre-, peri- or
postnatal period or 1-bromopropane (1-BP, 700 ppm) for 6 h/day during
prenatal period. In the open field test, control females explored more frequently than males. This sex difference was not affected by neonatal
BPA treatment but was abolished by pre- and perinatal treatment or 1BP. The time spent in open arms in the elevated plus maze test decreased
by neonatal or prenatal BPA treatment although sex difference was not
clearly affected. In the forced swimming test, both prenatal and neonatal
BPA exposures increased immobility time, an index of depressive behavior, in male rats and reduced immobility latency in both sexes. The
duration of immobility decreased in the forced swimming test and the
sex difference were disappeared by 1-BP. These findings suggest that
prenatal BPA or 1-BP exposure is more effective to impair sexual differentiation of exploratory behavior than neonatal BPA exposure but neonatal period is also important for development of emotional behavior
Central neurotoxicity induced by 1bromopropane, a substitute for specific
Fueta, Yukiko1; Ueno, Susumu2; Ishidao, Toru1;
Yoshida, Yasuhiro3; Hori, Hajime1 (1Sch. Hlth. Sci. Univ.
Occupational/Environ. Hlth. Kitakyushu. Japan; 2Dept. Pharmacol.
Sch. Med. Univ. Occupational/Environ. Hlth. Kitakyushu. Japan;
3Dept. Immunol. Sch. Med. Univ. Occupational/Environ. Hlth.
Kitakyushu. Japan)
1-Bromopropane (CH3CH2CH2Br 1-BP) is a newly introduced substitute for specific chlorofluorocarbons whose production was prohibited
because of depletion of ozone layers, and is mainly used for degreasing
agents and spray adhesives. Although case studies in the USA and China
have demonstrated that 1-BP could adversely affect the human nervous
system, the underlying mechanism for the effects of 1-BP inhalation exposure on the CNS has not been understood. We investigated the effects
of 1-BP exposure on the CNS using different models of exposure. 1-BP
potentiated GABA but inhibited ACh responses in Xenopus oocytes expressing GABAA and nicotinic ACh receptors, respectively, and enhanced recurrent inhibition in the rat hippocampus. On the other hand,
hippocampal disinhibition was observed in a concentration-dependent
manner (200 - 1500 ppm) after chronic inhalation of 1-BP in the rats and,
at the highest concentration of 1-BP inhalation, epileptic potentials were
evoked in the dentate gyrus. Moreover, prenatal exposure to 1-BP vapor
resulted in enhanced stimulation/response (S/R) curve of population
spikes in the CA1 area of PND 11-15 rats and reduced S/R curve of field
excitatory postsynaptic potentials in the CA1 area in adults (6-8w).
These results suggest that 1-BP inhalation exposure disrupts neuronal
excitability of the hippocampal formation.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Trafficking and localization of the NMDA receptor
Down-regulation of drebrin A expression
suppresses homeostatic synaptic
targeting of NMDA receptors
Shirao, Tomoaki (Dept. of Neurobiol. and Behav., Gunma Univ.
Grad. Sch. of Med., Maebashi, Japan)
Trafficking and localization of the
NMDA receptor
Drebrin is a major F-actin binding protein in the brain. We have recently
demonstrated that the expression of drebrin A (neuron specific isoform)
is rapidly upregulated in parallel with synapse formation, and that it governs the targeting of postsynaptic density (PSD) protein PSD-95 to synapses. Immunoelectron microscopy demonstrated that drebrin A is first
appeared at the submembranous regions of developing excitatory
postsynaptic sites at the initial stage ofsynapse formation. To determine
the role of drebrin A on excitatory synapse formation, we analyzed
whether the suppression of drebrin A expression affects filopodia-spine
morphology and synaptic targeting of NMDA receptors in cultured hippocampal neurons. Suppression of developmentally programmed upregulation of drebrin A by antisense treatment significantly decreased the
density and width of filopodia-spines. Immunocytochemistry showed
that the antisense treatment did not attenuate synaptic clustering of
NMDA receptors under condition that permitted spontaneous activities,
but inhibited the accelerated targeting of NMDA receptors into synapses
by its antagonist AP5. These results indicate that drebrin A upregulation
play a pivotal role in spine morphogenesis and activity-dependent synaptic targeting of NMDA receptors.
Regulation of molecular organization and
morphology of postsynapses by Cupidin/
Synaptic localization and left-right
asymmetrical allocation of NMDA
Furuichi, Teiichi1; Mizutani, Akihiro2; Shoji, Satoshi1;
Mikoshiba, Katsuhiko2; Shiraishi-Yamaguchi, Yoko1
Shigemoto, Ryuichi1,2,3 (1Nat. Inst. Physiol. Sci. Okazaki,
Japan; 2Sokendai; 3SORST, JST)
(1RIKEN BSI, Wako, Japan; 2Inst. Med. Sci., Univ. Tokyo, Tokyo,
Homer is a postsynaptic scaffold protein with the N-terminal target binding and C-terminal self-assembly domains. Homer multimers likely link
their targets, including proteins related to the Glu receptor and Ca2+ signaling (mGluR1a/5, Shank, IP3R) and to the actin cytoskeleton (Drebrin
and Cdc42), at postsynaptic density (PSD). The Homer family consists
of three long-form Homers H1b/c, Cupidin/H2, and H3. A natural dominant-negative, short-form H1a with only the N-terminal domain is also
activity-dependently expressed. In hippocampus, H1b/c and Cupidin/H2
predominate in CA1 region, whereas H3 is largely localized in CA2CA3 region. In cultured hippocampal cells, dendritic clustering and synaptic targeting of long Homers coincide with those of NMDAR and
PSD-95 throughout development. Overexpression of long Homers increases mature-shape spines, whereas that of H1a alters PSD target contents and spine morphology. In cerebellum, H1b/c and Cupidin/H2 are
concentrated in PSDs of granule cells. In cultured granule cells, their
clustered distribution is changed by NMDAR-mediated Ca2+ influx, and
H1a has a neuroprotective action against excess Glu exposure probably
by interfering a target linkage via long Homers, which inhibits NMDAR
activity. On the other hand, H3 is exclusively localized in Purkinje cells.
mGluR1a binding and dendritic localization of H3 is controlled by its
Ca2+-dependent phosphorylation states. Thus, Homer is involved in synapse formation and function by regulating molecular organization of
PSD and spine morphology.
NMDA receptors play a key role in synaptic plasticity in the hippocampal CA1 pyramidal cells. Two subunits of NMDA receptors NR2A and
NR2B have distinct expression patterns in development and may contribute differently to induction of long-term potentiation and depression.
We discovered input-dependent left-right asymmetry of NR2B subunit
allocation in Schaffer collateral (Sch)- and commissural fiber- pyramidal
cell synapses. However, it has not been known if NR2B has such asymmetrical distribution in Sch-interneuron synapses, and if NR2A has also
asymmetry to neutralize the NR2B asymmetry in pyramidal cell synapses. Here, we investigated distribution of NR2A and NR2B in single
synapses by postembedding immunogold and SDS-digested freeze-fracture replica labeling methods. To facilitate the detection of NR2B density difference, we utilized NR2A knockout mice, which have a simplified
NMDA receptor subunit composition.The labeling density for NR2B but
not NR1 in Sch-CA1 pyramidal cell synapses was significantly different
between the left and right hippocampus with opposite directions in strata
oriens and radiatum. No significant difference in NR2B density, however, was detected in CA1 stratum radiatum between the left and right Schinterneuron synapses. Immunoblot analysis of PSD fractions from CA1
radiatum confirmed significant difference in protein amount for NR2B
but not for NR1 and NR2A between left and right hippocampus. These
results indicate that the asymmetry of NR2B distribution is target-cell
specific and unique to this subunit.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Environmental factors during development affect behavioral patterns: Critical period and molecular mechanisms
Roles of NMDA receptor phosphorylation
in the amygdala
Watabe, Ayako M.1; Nakazawa, Takanobu3; Yamamoto,
Tadashi3; Manabe, Toshiya1,2 (1Inst. Medical Science, Univ.
Tokyo, Tokyo, Japan; 2CREST, JST, kawaguchi, Japan; 3Inst.
Medical Science, Univ Tokyo, Tokyo, Japan)
While NMDA-type ionotropic glutamate receptor (NMDAR) is widely
accepted as a key regulator for certain forms of memory and learning and
LTP induction, not much is known about how NMDAR function is regulated at physiological circumstances. The NR2B subunit of the NMDAR is tyrosine phosphorylated in brain, with Tyr-1472 as a major
phosphorylation site. Phosphorylation of neural proteins is one of the
main mechanisms underlying dynamic changes in neural functions, so
we investigated the physiological significance of NR2B phosphorylation
in neuronal plasticity and learning behavior. Mice with a knockin mutation of the Tyr-1472 site to phenylalanine (Y1472F) showed impaired induction of amygdaloid long-term potentiation and fear-related learning.
Basic properties of synaptic transmission were normal in YF/YF mice,
suggesting that impaired LTP in YF/YF mice is not caused by direct
modification of NMDAR current properties but is associated with some
intracellular signaling downstream from NMDAR activation. In fact,
CaM kinase II, a key regulator for synaptic plasticity was undetectable
in NMDAR complex of YF/YF mice. Electron microscopic analyses revealed that NMDAR localization at synapses was impaired in YF/YF
mice, presumably resulting in altered NMDAR complex of YF/YF mice.
These results strongly argue that phosphorylation of Tyr-1472 regulates
NMDAR localization at synapses leading to modulating synaptic plasticity and fear-related learning.
Critical role of cross modal integration of
sensory cues for the development of peer
Nakamura, Shun1; Koshiba, Mamiko1,2 (1Natl Inst Neurosci,
Tokyo, Japan; 2CREST, JST, Kawaguchi, Japan)
Human is a social animal and unique to communicate with language. We
want to understand the neurobiological basis of this feature in the light
of comparative neuroethology. Social animals, like primates and birds,
could communicate with conspecific mates by vocalization. We have established the developmental model of social communication with domestic chick which is a precocial bird and can grow itself without parent
care. This allows us to investigate the development of peer relationship
without considering the effect of parent-infant relationship. We reared
chicks under grouped or individual condition, and compared socialization between two conditions. We measured the association and calling
behavior as indicating socialization. The chicks reared as a group for 814 days after hatching showed socialization. In contrast, the chicks
reared under socially deprived condition showed strong fear response in
the novel environment and could not develop socialization. We, next,
tested uni-modal social deprivation, that is, either visual or vocal cue was
presented during individually rearing. Under these conditions, the fear
response was suppressed when the behavior was tested on 8-14th day.
Socialization, however, was different between the sensory social cues
presented. Only vocally communicated chick could develop socialization. These results suggest that social interaction during infant is critical
to develop peer relationship and mutual vocalization is important. Now,
we are localizing the neuronal substrate for the development of peer relationship
Olfactory imprinting: Molecular
mechanism of olfactory learning in pups
Okutani, Fumino1; Kaba, Hideto1,2 (1Dept. Integr. Physiol.
Kochi Med. Sch, Nankoku, Japan; 2Dept. Adapt. Develop. NIPS,
Okazaki, Japan)
Environmental factors during
development affect behavioral
patterns: Critical period and
molecular mechanisms
Young rats prior to eye opening depend on somatosensory and olfactory
function for survival, as they can learn their dam's odor and approach her
without visual information. In order to establish olfactory learning, the
pairing of odor and tactile stimulation is crucial. Noradrenergic activation through the locus coeruleus by a somatosensory stimulus is implicated in olfactory learning. Within the olfactory bulb (OB), the
noradrenergic innervation modulates the efficacy of dendrodendritic
synapses between the mitral and granule cells. At the dendrodendritic reciprocal synapses, mitral cell activity is inhibited by GABA released
from the granule cells. It is noteworthy that disinhibition of the mitral
cells is a crucial step in the formation of an olfactory memory. We previously showed that intrabulbar infusion of the GABA receptor antagonist,
bicuculline facilitated olfactory learning. These results implicate the OB
as a critical site for olfactory learning. Since the transcription factor,
CREB is well known to be involved in plasticity, we examined whether
CREB is involved in olfactory learning. Behavioral pharmacology
shows that only long-term olfactory memory was prevented by CREB
antisense infusion, but short-term memory was intact. Western blot analyses reveal that P-MAPK/ERK was increased for 1 hour after odor exposure paired with shock, followed by increase of P-CREB lasting for 6
hours. These may be evidence suggesting that synaptic plasticity in the
OB underlies aversive olfactory learning.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Research frontiers in cardiovascular physiology—New original concepts
Factors affecting rhythm entrainment and
stress responsiveness by periodic
maternal deprivation
Honma, Sato; Honma, Ken-ichi (Grad. Sch. Med. Hokkaido
Univ., Sapporo, Japan)
The life of newborn rats totally depends on their maternal care. Although
the first several days of their life are characterized by stress-hyporesponsiveness, absence of mother acts as a strong stressor which overrides it
and results in long-lasting stress-vulnerability. Periodic maternal deprivation (MD) for the first few days act as a strong non-photic time cue and
entrains pups' circadian clock. However, little is known as to the mechanisms how the MD affects on the pups' circadian clock and stress-responsiveness in the adulthood. We imposed newborn rats to MD of
various durations at different time in the light phase, at different period
in the postnatal life, with or without keeping pups warm. In addition, by
restricting food access of mother rats to 2h (RF), behavioral rhythms of
mothers were modified without depriving them from pups. We measured
clock gene expression rhythms in the suprachiasmatic nucleus of the
fetes and neonates, and behavioral rhythms after weaning. At 8 weeks of
age, stress responsiveness was examined by measuring plasma corticosterone levels after exposing to mild stress of cage exchange. 12 h MD
during the light phase in day1-6 completely reversed the circadian
rhythms of clock gene expression. The behavioral rhythms after weaning
were also shifted depending on the phase of MD. Rats exposed to MD
exhibited hyper responsiveness to the novelty stimuli, which was abolished by MD with warming. These results suggest that the maternal care
is important to entrain pups' clock, while heat loss due to isolation is critical for the stress hyper-responsiveness in adulthood.
Research frontiers in
cardiovascular physiology—New
original concepts
Molecular mechanism of stress
vulnerability and resilience induced by
early environment
Morinobu, Shigeru1; Takahashi, Terumichi1; Iwamoto,
Yasuyuki1; Yamawaki, Shigeto1; Okuno, Hiroyuki2; Bito,
Haruhiko2 (1Dept. Neuropsychiat. Grad. Sch. Biomed.
Sci.Hiroshima Univ., Hiroshima, Japan;
2Dept.Neurochem.Grad.Sch.Med. Univ. Tokyo, Tokyo, Japan)
Although an early adversity is a major risk factor for the vulnerability to
stress later in life, the mechanism of the stress vulnerability remains to
be unknown. It is known that while neonatal isolation (NI) induces stress
vulnerability in adult rats, environmental enrichment (EE) following NI
leads to resilience. We examined whether NI induced the susceptibility
to learned helplessness (LH) (animal model of depression) and EE ameliorated this susceptibility. Pups were individually isolated from postnatal day 2 to 9. After weaning, EE was administrated until the beginning
of LH session. In adulthood, we measured the number of escape failures
and escape latency 24 hours after exposure to inescapable shock session.
Behavioral analyses revealed that whereas the population of LH in NI
rats was significantly higher than that in sham rat, EE markedly decreased the population of LH in NI rats. We tried to identify genes involved in the molecular mechanism underlying the susceptibility to LH
using a cDNA array, and real-time PCR. The comparison of hippocampal gene expression between NI-LH and sham-nonLH rats revealed the
significant decrease in LIMK1 mRNA in NI-LH. EE prevented the decrease in the expression of LIMK mRNA in the hippocampus of NI rats.
These findings suggest that LIMK may play an important role in stress
vulnerability developed by an early environment.
Blood Pressure Control -Two significances
and Two mechanismsNishida, Yasuhiro; Hirakawa, Haruhisa; Hiruma,
Megumi; Kemuriyama, Takehito (National Defense Medical
College, Physiology II, Tokorozawa, Japan)
It is well established that blood pressure (BP) is controlled by feedback
mechanisms, e.g. via baroreceptor reflex. The feedback mechanism supports blood pressure homeostasis to save life at sudden orthostasis or
massive hemorrhage. We have shown that sinoaortic denervation abolished BP resetting to a higher level at daily physical activity. This indicates that the baroafferent signal supports active resetting of BP during
exercise, suggesting that BP should be controlled not only homeostatically by feedback but also homeodynamically by feedforward mechanisms. The above mechanisms are involved in the short-term control of
BP. Resting level of BP is thought to be relatively constant for life-long
in health subjects. Lesion of the area postrema, which is a site for neurohumoral interaction with vasopressin and angiotensin II, did not show
any abnormality in day-to-day control of BP. In animal-models with saltsensitive hypertension, vascular endothelial functions or renal functions
are impaired but central sympathetic control is preserved, although the
central nNOS systems are upregulated to inhibit sympathetic activation.
These data lead to the possibility that main causes for abnormality in
long-term control of BP might be present in the effector organs but not
in the central control system, rather that the central sympathetic system
functions to compensate hypertension. These data suggests that BP
should be regulated by feedback and local mechanisms homeostatically
for saving life and by feedforward mechanisms homeodynamically for
helping organ-functions.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Research frontiers in cardiovascular physiology—New original concepts
Resetting of Cardiovascular Regulation by
Emotional Stimuli
Kuwaki, Tomoyuki1,2; Zhang, Wei1 (1Dept. Molec. Integ.
Physiol., Chiba Univ. Grad. Sch. Med., Chiba, Japan; 2Dept.
Autonom. Physiol., Chiba Univ. Grad. Sch. Med., Chiba, Japan)
The word "homeostasis" implies a regulatory mechanism that stabilizes
the biological parameters around an operating point during the resting
condition. During exercise or stressful condition, however, a new operating point will be set. This operating point is different from that under
the resting condition and should be suitable for the bodily demand during
the new condition. To explore neural mechanisms of such resetting of the
operating point, we have recently focused on the stress-induced defense
response because stressor induces not only cognitive, emotional and behavioral changes but also autonomic changes. These changes include increases in blood pressure, heart rate, muscular blood flow, respiratory
frequency, and tidal volume and suppression of the baroreceptor reflex
and pain sensitivity. Although research on the neural circuits underlying
such autonomic changes has implicated the hypothalamus in the defense
response against stressors, neurotransmitters in this multifacet and coordinated response have not been revealed. In my talk, I will summarize
our recent discovery of possible contribution of orexin as a master switch
to elicit multiple efferent pathways in the defense response and discuss
future directions.
Koganezawa, Tadachika1,2; Paton, Julian FR2; Terui,
Naohito1 (1Dept. Physiol., Inst. Basic Med. Sci., Grad. Sch. Comp.
Human Sci., Univ. Tsukuba, Tsukuba, Japan; 2Dept. Physiol., Sch.
Med. Sci., Univ. Bristol, Bristol, UK)
A major source of excitatory drive to the pre-ganglionic sympathetic
neurons originates from the rostral ventrolateral medulla (RVLM).
RVLM sympathetic premotor neurons (RVLM neurons) have spontaneous activity in vivo. For the generation of RVLM neuron activity, "Network theory" and "Pacemaker theory" have been suggested by in vivo
and in vitro studies. However, it is still unclear which one is right. We
studied how RVLM sympathetic premotor neurons generate their activity using an in situ arterially perfused preparation (the working heartbrainstem preparation). We recorded activities of the thoracic sympathetic chain and RVLM neurons. The firing response of RVLM neurons
during hypoxic-induced gasping is heterogeneous with some inhibited
and others persisting. The finding that some RVLM neurons continue to
fire after blockade of fast synaptic transmission is consistent with the hypothesis that they may have intrinsic pacemaker activity. Since some
RVLM neurons remained responsive to hypoxia after blockade of fast
excitatory and inhibitory synaptic transmission, they may be directly
sensitive to hypoxia. Some RVLM neurons may be central oxygen sensors and exhibit pacemaker activity during hypoxia to ensure high levels
of sympathetic activity and hence maintenance of arterial pressure.
Role of the intracranial vasodilative system
that regulates cerebral parenchymal
Hotta, Harumi (Tokyo Metropol. Inst. Gerontol. Tokyo, Japan)
Cerebral blood flow is vital for the survival and function of the brain. In
particular, the hippocampus and cerebral cortex are very sensitive to
transient ischemia. The hippocampus and cerebral cortex receive cholinergic vasodilative fibers that originate in the medial septum and the nucleus basalis of Meynert (NBM), respectively, of the basal forebrain (see
a review by Sato et al., 1995, Alzheimer Dis. Assoc. Disord. 9: 28). Recently, we showed that increases in blood flow in the hippocampus and
cerebral cortex in rats during activation of the vasodilative system, either
by pharmacological (i.v. nicotine) or physiological (electrical stimulation of the NBM) methods, can prevent delayed death of hippocampal
and cortical neurons following transient ischemia. Stimulation of the
NBM increased the diameter of cortical parenchymal microvessels during stimulation. In addition, after the end of stimulation, an increase in
the concentration of brain-derived neurotrophic factor (BDNF) in the
cortical extracellular fluid was observed. From these findings, we suggest that activation of the intracranial vasodilative system provides protection against ischemia-induced delayed neuronal death by inducing
increases in both the diameter of parenchymal microvessels and the release of an endogenous neuroprotective factor, BDNF. We also showed
that activation of the vasodilative system occurred during passive somatosensory stimuli and active movements such as walking. Thus, this intracranial vasodilative system may contribute to the beneficial effect of
physical activity on cognitive brain functions.
Mechanisms of generation for sympathetic
spontaneous discharge on the
cardiovascular center in the medulla
oblongata; the study by an in situ arterially
perfused preparation
Regulation of cardiac function by cAMP/Ca
Ishikawa, Yoshihiro; Minamisawa, Susumu (Grad. Sch.
Med. Yokohama City Univ. Yokohama, Japan)
Catecholamine signal is a major mechanism of regulating cardiac function. Norepinephrine released from the synaptic terminal binds to beta
adrenergic receptors, leading to the activation of the stimulatory G protein and thus adenylyl cyclase. Cyclic AMP generated by adenylyl cyclase activates protein kinase A, which initiates multiple
phosphorylation reactions within cardiac myocytes. A major impact of
catecholamine stimulation is the enhancement of Ca cycling within myocytes. In the past decade, multiple molecules have been identified that
are involved in Ca cycling. Transgenic studies using mouse models have
elucidated the function of such molecules. Indeed, a growing body of evidence has shown that Ca cycling and Ca-dependent signaling pathways
play a pivotal role in cardiac hypertrophy and heart failure. In addition,
recent studies identified that mutations of the genes encoding sarcoplasmic reticulum proteins cause human cardiomyopathies and lethal ventricular arrhythmias. The regulation of Ca homeostasis via the SR
proteins may have potential therapeutic value for heart diseases such as
cardiomyopathy, heart failure and arrhythmias. Similarly, molecular
mechanisms of catecholamine signal have been elucidated and the diversity of cAMP signal within the heart has been demonstrated. For example, it is now well known that the heart expresses multiple isoforms of
adenylyl cyclase. The role of each adenylyl cyclase isoform is different
in regulating cardiac function and the viability of cardiac myocytes under normal and pathological conditions. We will summarize our recent
progresses in the study of this pathway in the heart.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: New stream of system biology by novel bioactive substances and hormones [Science Council of Japan Symposium]
Physiological roles of adiponectin and
adiponectin receptors
Yamauchi, Toshimasa; Kadowaki, Takashi (Grad. Sch.
Med. Univ. Tokyo, Tokyo, Japan)
Adiponectin/Acrp30 is a hormone secreted by adipocytes that acts as an
antidiabetic and anti-atherogenic adipokine. We reported that AdipoR1/
R2 serve as receptors for adiponectin and mediate increased fatty-acid
oxidation and glucose uptake by adiponectin. Moreover, obesity was associated with decreased plasma adiponectin levels as well as decreased
expression levels of AdipoR1/R2, the latter reduced adiponectin sensitivity, both of which finally lead to insulin resistance.
New stream of system biology by
novel bioactive substances and
hormones [Science Council of
Japan Symposium]
In this study, to clarify the physiological and pathophysiological roles of
AdipoRs in vivo, we studied the effects of adenovirus-mediated
upregualtion of AdipoRs in the mice liver. Here we show that
adenovirus-mediated expression of AdipoR1 in the liver of db/db mice
increased adiponectin effect such as increased activation of AMP kinase
by adiponectin, decreased molecules involved in gluconeogenesis and
increased fatty-acid oxidation, thereby ameliorating diabetes. Moreover,
adenovirus-mediated expression of AdipoR2 in the liver of db/db mice
increased adiponectin effect such as increased PPARalpha target genes
including molecules involved in fatty acid oxidation and energy
dissipation, thereby ameliorating diabetes. These data raised the
possibility that AdipoR1 may be more tightly linked to activation of
AMP kinase pathway, while AdipoR2 may be more tightly linked to
activation of PPARalpha pathway.
Adiponectin receptor agonists and adiponectin sensitizers should serve
as versatile treatment strategies for obesity-linked diseases such as
diabetes and metabolic syndrome.
Regulatory role of leptin-AMP kinase
system in body energy metabolism
Novel functions of appetite-regulating
Minokoshi, Yasuhiko; Suzuki, Atsushi; Okamoto, Shiki;
Shiuchi, Tetsuya; Lee, Suni; Saito, Kumiko (Natl. Inst.
Kojima, Masayasu (Institute of Life Science, Kurume University,
Kurume, Fukuoka, Japan)
Physiol. Sci., Okazaki, Japan)
Feeding is a basic behavior that is necessary for life. Long-term lack of
food results in death. It is well accepted that appetite is controlled by the
brain and that feeding behavior is regulated by complex mechanisms in
the central nervous system, in particular the hypothalamus. However, recent identifications of novel neuropeptides and peptide hormones develop a paradigm in appetite regulatory mechanisms in the central nervous
system. In this presentation, I will discuss the two appetite-regulating
peptides, ghrelin and neuromedin U. Ghrelin is a growth-hormone releasing and appetite-stimulating hormone secreted mainly from stomach. On the other hand, neuromedin U (NMU) is a potent appetitesuppressing peptide. Moreover, we recently revealed that ghrelin directly acts on osteoblast cells to regulate bone formation, and NMU is involved in the regulation of peripheral inflammation. Thus, both ghrelin
and NMU are more than appetite regulators, but have multifaceted roles
in, for example bone formation and inflammation.
Leptin is an adipocyte-secreted hormone that regulates body energy metabolism. We have recently shown that leptin stimulates fatty acid oxidation in skeletal muscle by activating α2 AMP-activated protein kinase
(AMPK). Leptin exerts this effect directly at the level of muscle and
through the hypothalamic-sympathetic nervous system. In contrast,
hypothalamic α2 AMPK activity is inhibited by anorexigenic hormones
(leptin and insulin), a melanocortin (MC) receptor agonist (anorexigen),
high glucose and refeeding. AGRP (orexigenic neuropeptide), fasting
and MC4 receptor-KO obese mouse increase hypothalamic AMPK
activity. Expression of dominant-negative (DN) and constitutively
active (CA) AMPK in the hypothalamus is sufficient to change food
intake, body weight and expression of orexigenic neuropeptides such as
NPY, AGRP and MCH. CA-AMPK blocks leptin-induced suppression
of food intake.
We recently examined the signaling pathway of leptin's effects on
AMPK, using muscle and neuronal cell lines that express leptin receptor
Ob-Rb. Leptin activates α2 but not α1 AMPK in muscle cells through
activation of ataxia telangiectasia mutated and calcium/calmodulindependent protein kinase kinase β. Furthermore, cellular localization of
α2 AMPK is changed in response to leptin. In contrast, leptin suppresses
α2 AMPK activity and NPY expression in neuronal cells.
Thus, leptin reciprocally regulates AMPK activity in neuronal and
muscle cells. Our data indicate that leptin-AMPK system plays a critical
role in peripheral and central regulation of body energy metabolism.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Diverse functions of the gap junctions and their molecular mechanisms
Regulation and dysregulation of
metabolism by new bioactive factors
derived from muscle and fat
Matsuda, Morihiro1; Nishizawa, Hitoshi2; Fukuhara,
Atsunori1; Shimomura, Iichiro1,2 (1Grad. Sch. of Frontier
Bioscience, Osaka Univ. Osaka, Japan; 2Grad. Sch. of Med. Osaka
Univ. Osaka, Japan)
Skeletal muscle and fat tissue are involved in the homeostasis of glucose
metabolism. Here, we introduce new bioactive factors derived from skeletal muscle and fat tissue.
Musclin was identified via signal sequence trap of mouse skeletal
muscle cDNAs. Musclin protein contained a region homologous to
nariuretic peptide family. Its mRNA was expressed almost exclusively
in skeletal muscle of mice, and regulated by nutritional changes.
Recombinant musclin protein significantly attenuated insulin-stimulated
glucose uptake and glycogen synthesis in myocytes.
A newly identified adipocytokine, visfatin, is highly enriched in the
visceral fat of both human and mice and whose expression level in
plasma increases during the development of obesity. Visfatin exerted
insulin- mimetic effects in cultured cells and lowered plasma glucose
levels in mice. Heterozygous knockout mice of visfatin had modestly
higher levels of plasma glucose relative to wild type littermates.
Surprisingly, visfatin binds to and activates the insulin receptor.
Diverse functions of the gap
junctions and their molecular
Production of ROS increased selectively in fat tissue of obese mice. In
cultured fat cells, oxidative stress caused dysregulated production of
adipocytokines, including adiponectin, PAI-1, IL-6, and MCP-1. In
obese mice, treatment with NADPH oxidase inhibitor reduced ROS
production in fat tissue, attenuated the dysregulation of adipocytokines,
and improved diabetes.
Further study on the physiological role of these factors may lead to new
Disecting behaviors from orphan GPCRs
Interaction between gap and tight junctions
Yanagisawa, Masashi (Univ. of Texas Southwestern Med. Ctr.,
Howard Hughes Med. Inst., Dallas, TX, USA)
Sapporo Med. Univ., Sapporo, Japan)
To be filled in...
Kojima, Takashi; Sawada, Norimasa (Dep. Path. Sch. Med.
It is thought that gap junctions may be closely associated with tight junctions. However, the mechanisms are still undefined. We found that Cx32
but not Cx26 was closely related to tight junctional proteins in primary
cultured rat hepatocytes (Exp. Cell Res. 263, 193-201, 2001) and that
Cx32 formation and/or Cx32-mediated intercellular communication
could induce expression and function of tight junctions in a mouse hepatic cell line (Exp. Cell Res. 276, 40-51, 2002). When we performed
cDNA microarray analysis of Cx32-transfectants, compared to parental
cells derived from Cx32-deficient hepatocytes, an increase in expression
of membrane-associated guanylate kinase with inverted orientation -1
(MAGI-1), which is known to be localized at adherens and tight junction
regions, was observed (Cell Tissue Res. 319, 341-347, 2005). More recently, we performed to express short interfering RNA (siRNA) for
Cx32 in primary cultured rat hepatocytes which highly expressed Cx32
and tight junction proteins and examined changes in expression of tight
junction proteins and activated MAP-kinase. Down-regulation of Cx32
was associated with a decrease of claudin-1 and an increase of claudin2. Furthermore, up-regulation of phosphorylated MAP-kinase was observed by the siRNA. Cx32 expression may in part regulate expression
of tight junctions through the signal transduction pathway such as MAPkinase.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Diverse functions of the gap junctions and their molecular mechanisms
Chemical gating of gap junction channels:
role of carboxyl terminal of connexin43 as
a regulatory domain.
Gap junctions and cell death: Changes in
connexin localization during apoptosis in
cells expressing Cx43-GFP
Seki, Akiko1; Hagiwara, Nobuhisa1; Steven, Taffet3;
Mario, Delmar2; Kasanuki, Hiroshi1 (1Dpt. Cardiology, Tokyo
Oyamada, Masahito1; Zhou, Wuxiong1; Oyamada,
Yumiko1,2; Takamatsu, Tetsuro1 (1Grad. Sch. Med. Kyoto
Women's Medical University; 2Dpt. Pharmacology, SUNY Upstate
Medical University, NY, U.S.A.; 3Dpt. Imuunology and microbiology,
SUNY Upstate Medical University, NY, U.S.A.)
Pref. Univ. Med., Kyoto, Japan; 2Meiji Univ. Orient. Med., Nantan,
It has been known that intracellular acidification leads to gap junction
channel closure. This phenomenon is called "chemical gating", which
may be one of the causes of lethal arrhythmia during cardiac ischemia.
Chemical regulation of Cx43 follows a ball-and-chain model, in which
the carboxyl terminal (CT) domain acts as a gating particle that binds to
a receptor affiliated with the pore. However, the location of the "receptor" for the CT has been unknown.Electrophysiological analysis shows
that Cx43 channels reside in three states; closed (C), open (O) or residual
(R). Since the R state is eliminated by truncation of the CT, it is hypothesized that the R state results from the interaction of the CT with the receptor. Recently, we showed in vitro that there is an intramolecular
interaction of the CT with a region in the cytoplasmic loop of Cx43 (amino acids 119-144; dubbed "L2"). To determine the function of the L2,
Cx43 channels were recorded in the presence of a peptide corresponding
to the L2 region, delivered via the patch pipette. This manipulation eliminated the R state in a manner similar to that observed after truncation of
the CT, indicating that L2 peptide competitively inhibits the interaction
between the CT and the native L2 region. Thus, we propose that the L2
acts as a "receptor" that interacts with the CT during channel gating.
Gap junctions are considered to play an important role in moderating cell
death including apoptosis. However, the basic phenomena underlying
when and where the alterations of gap junctions occur during apoptosis
have not been well documented. In this study, To answer these questions,
we analyzed the spatiotemporal changes of Cx during UV light-induced
apoptosis using Cx43-EGFP-expressing HeLa cells, and compared them
with those of mitochondrial membrane potential (MMP) using tetramethylrhodamine ethyl ester (TMRE) and nuclear morphological observation using Hoechst 33342. At 2 hr post-UV-irradiation, a third of the cells
became TMRE-negative, i.e., they showed the loss of MMP, but with
slight nuclear fragmentation, and high percentages of linear Cx43-EGFP
plaques were found among both TMRE-positive and TMRE-negative
cells. At 4 hr post-UV-irradiation, the percentage of these linear plaques
was decreased, and both punctate and diffuse localization of Cx43EGFP were noted in the cytoplasm of TMRE-negative cells without nuclear fragmentation. At 8 hr post-irradiation, punctate cytoplasmic localization of Cx43-EGFP was noted in TMRE-negative cells with nuclear
fragmentation. Treatment with the caspase inhibitor Z-VAD-FMK
blocked nuclear fragmentation and partially preserved both gap junctional plaques and MMP. These results indicate that, during apoptosis,
Cx mobilization into the cytoplasm occurs after MMP depolarization but
before nuclear fragmentation and that this alteration partly depends on
Degradation of connexin 43 induced by
phosphorylation and dephosphorylation
EDHF responses and Gap Junction
Imanaga, Issei1; Hai, Lin2; Ogawa, Koichi3 (1General
Med. Univ., Sapporo, Japan)
Research Center of Medical Sciences.Fukuoka
Univ.Sch.Med.Fukuoka, Japan; 2Dept.Physiol.Fukuoka Univ.Sch.
Med.Fukuoka, Japan; 3Dept.Anat.Fukuoka Univ.Sch.Med.Fukuoka,
Connexins are expressed in vascular endothelial and smooth muscle
cells. However, the roles of connexins in the regulation of arterial tone
are unclear. In this symposium, we would like to introduce recent evidence that connexins mediate endothelium dependent arterial relaxation
caused by endothelium-derived hyperpolarizing factor (EDHF). The
molecular identity of EDHF is not convincing. We assessed that whether
NO, PGI2, K+, anandamide, H2O2 or EET act as EDHF. However, none
of that act as EDHF in rat mesenteric artery. Recent studies suggest that
gap junctional communication between endothelium and smooth muscle
may account for EDHF responses. In rat mesenteric artery, endotheliumdependent relaxation and hyperpolarization by EDHF were inhibited by
gap junction inhibitors. RT-PCR experiment showed that connexin 37,
40, 43 & 45 were expressed in the artery. In immunohistochemistory,
connexin37, 40 & 43 were expressed in endothelium and connexin 43
was expressed in smooth muscle cells. EDHF-mediate hyperpolarization
and relaxation were correlated with serum estrogen level. The expressional levels of connexin40 & 43 were also dependent on estrogen level.
These results suggest that EDHF is not a molecule and its responses are
mediated by gap junctional communications. Connexin may play a pivotal role in the regulation arterial tone in physiological and pathophysiological states in especially small arteries.
In cardiac muscle, the gap junction greatly contributes to intercellular
impulse propagation. The remodeling of the gap junction is induced in
pathological myocardium and influences the cardiac function. We investigated remodeling of connexin 43 (Cx43) which is dominant in the ventricular muscle cells, in reference to phosphorylation and
dephosphorylation of the protein, using methods of electrophysiology,
immuno blot and immunohistochemistry in adult guinea-pig and rat
hearts.An activation of PKA promoted the PKA-mediated phosphorylation of Cx43 in association with an increase in the electrical intercelluar
coupling and in expression of Cx43. In hypoxia, intracellular Ca-overload or acidosis, the PKA-mediated phosphorylation of Cx43 was inhibited in association with a suppression of the intercellular coupling and of
expression of Cx43. These deteriorated changes of Cx43 were alleviated
by PKA-activators. In the diabetic or PMA-treated heart, the PKC-mediated phosphorylation of Cx43 was augmented in association with an
inhibition of the intercellular coupling and of expression of Cx43. These
effects of an activation of PKC were ameliorated by a treatment of PKCinhibitors, proteasome inhibitors or lysosome inhibitors. These results
indicate that Cx43 hyperphosphorylated by PKC is highly susceptible to
proteolytic degradation. It is concluded that Cx43 is up-regulated by
PKA and down-regulated by PKC, and the remodeling of Cx43 is essentially induced by an excess activation of PKC.
Fukao, Mitsuhiro; Tohse, Noritsugu (Dept. Phys., Sapporo
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Mechanisms of synapse development, maintenance and plasticity [YFI (Young Foreign Investigator) Symposium]
Connexins in human endometrium and
correlation to carcinogenesis
Saito, Tsuyosi1; Sazuki, Takahiro1; Horie, Miyabi1;
Fujimoto, Takashi1; Yamasaki, Hiroshi2 (1Dept. Obstet.
Gynecol. Sapporo Med. Univ. Sapporo Japan; 2Life Sci. Sch. Sci .
Tech. Kwansei Gakuin)
There are several lines of evidence suggesting that connexin expression
is suppressed and/or aberrantly localized in pre-cancerous lesions in several organs and many, if not all, tumor-promoting agents have been
shown to inhibit gap junctional intercellular communication (GJIC) of
cultured cells as well as those in vivo, suggesting that the loss of GJIC
enhances clonal dispersion, causing loss of the growth-suppression signals from the surrounding cells. For endometrial carcinogenesis, it may
be concluded that the loss of GJIC caused by the suppressed expression
and the aberrant localization of connexin support the clonal evolution of
endometrial cancer cells originating in the hyperplasia cells. In the
present study, GJIC of IK-ER1, which overexpresses ER-alpha was
markedly reduced in the estradiol-containing medium and the reduction
was found to be inhibited by ICI182.780, a pure anti-estrogen substrate,
as demonstrated by Lucifer-Yellow dye-transfer assay. Western blot
analysis indicated that the expression of both Cx26 and Cx32 also decreased in E(+) and the reduction was inhibited by adding ICI182.780.
These results supported the result of the dye-transfer assay. Thus, estrogen, which suppresses connexin expression of endometrial epithelium
and causes cell proliferation, may act as a tumor-promoting agent for endometrium.
Simultaneous observation of pre- and
postsynaptic morphological changes in
hippocampal slice culture
Umeda, Tatsuya1; Ebihara, Tatsuhiko2; Okabe,
Shigeo1,2,3 (1Department of Cell Biology, Tokyo Medical and
Dental University, Tokyo, Japan; 2AIST, Neuroscience Research
Institute, Tsukuba, Japan; 3CREST, JST, Kawaguchi, Japan)
Dendritic spines are highly motile structures, but how spines move while
keeping their contacts with presynaptic varicosities is not clear. To analyze movements of these synaptic structures simultaneously, we labeled
CA1 pyramidal cells with green fluorescent protein and CA3 pyramidal
cells with rhodamine-dextran in hippocampal slice cultures. We obtained
stable expression of GFP in a limited population of CA1 pyramidal neurons by using transgenic mice with a Cre-loxP recombination system.
CA3 pyramidal neurons of the slice cultures were loaded with
rhodamine-dextran by electroporation. Labeled varicosities of CA3 pyramidal cells and spines of CA1 pyramidal cells were visualized using
two-photon microscopy to detect close association of the two components. Time-lapse imaging revealed that they performed rapid morphological changes without losing their contacts. The extent of overall
structural changes between varicosities and spines was correlated, while
the direction of short-term volume changes was regulated independently.
Furthermore, dendritic morphological changes induced by electrical
stimulation had little effect on their association. These results suggest
that synaptic junctions provide stable attachment sites functioning to
correlate presynaptic and postsynaptic motility.
Neuronal phenotype and chronic network
activity both influence the susceptibility of
hippocampal neurons to NMDA-induced Factin reorganization at synapses
Shiraishi-Yamaguchi, Yoko1,3; Mosbacher, Johannes2;
Halpain, Shelley3 (1RIKEN BSI, Wako, Saitama, Japan;
Inst., Basel, Switzerland; 3TSRI, La Jolla, California,
Mechanisms of synapse
development, maintenance and
plasticity [YFI (Young Foreign
Investigator) Symposium]
Actin cytoskeletal organization in dendrites and dendritic spines are believed to form a molecular basis for the morphological plasticity at brain
synapses. We here demonstrate that actin cytoskeleton of hippocampal
neurons is rapidly rearranged by N-methyl-D-aspartate (NMDA) receptor activation in both neuronal phenotype and developmental activitydependent manner. In rat hippocampus primary cultures, a certain population (20-30%) of neurons showed drastic redistribution of its dendritic
filamentous (F-) actin after a stressful NMDA stimulation (50µM for 30
s). The NMDA-induced actin rearrangement correlated with changes of
spine morphology and disruption of several postsynaptic components
like Homer1b/c, GluR1 and NMDAR2A, even though the synaptic
contacts seemed to stay preserved. Immunohistochemical
characterization showed that NMDA-susceptible cells did not express
calbindin-D28k. Reduction of network activity by chronic tetrodotoxin
application resulted in an increased number of calbindin-D28k -negative
and NMDA susceptible cells. Exogeneous calbindin expression in these
neurons could recover their resistance to NMDA induced F-actin
redistribution. These data indicate that F-actin organization is diverse
among different populations of neurons, which are selectively sensitive
to hyper excitatory input. Such neuronal type-specific heterogeneity also
points toward specific molecular mechanisms that contribute to
cytoskeletal regulation in dendrites.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Mechanisms of synapse development, maintenance and plasticity [YFI (Young Foreign Investigator) Symposium]
Role of drebrin in dendritic spine
morphogenesis and dual regulation of
drebrin dynamics by AMPA and NMDA
Takahashi, Hideto (Dept. of Neurobiol. and Behav., Gunma
Univ. Grad. Sch. of Med., Maebashi, Japan)
Dendritic spines represent the developmentally-regulated and activitydependent pleomorphism based on actin cytoskeleton. However, molecular mechanisms governing the pleomorphism are unclear. First, we find
that during development, synaptic drebrin clustering in dendritic filopodia is required for spine morphogenesis. Drebrin clustering with actin filaments occurs at postsynaptic sites of axon-filopodia contact. The
drebrin clustering precedes and governs synaptic PSD95 clustering and
spine morphogenesis. Second, using fluorescence recovery after photobleaching (FRAP) technique and immunocytochemistry, we find that
activities of AMPA receptors (AMPA-Rs) and NMDA-Rs orchestrate
drebrin dynamics for synaptic clustering of drebrin and PSD95. AMPAR blockade reduces binding capacity of drebrin within spines, observed
as a reduction of unrecoverable fraction. Consequently, Chronic AMPAR blockade inhibits synaptic clustering of drebrin and PSD95. NMDAR blockade facilitates transport of drebrin into spines, observed as a reduction of time constant. Further, chronic NMDAR blockade promotes
synaptic targeting of NMDA-Rs but inhibits that of PSD95. Finally, we
find that drebrin is involved in activity-dependent synaptic NMDA-R
targeting. Drebrin-A knockdown inhibits the accelerated targeting of
NMDA-Rs into synapses by NMDA-R blockade despite no effect on
NMDA-R localization under conditions of spontaneous activities. In
conclusion, activity-regulated actin-cytoskeletal system based on drebrin is critical for the diversity of spine structure.
Synaptotrophin, a novel neurotrophin
required for synaptic integrity and
information processing
Iijima, Takatoshi; Matsuda, Keiko; Kondo, Tetsuro;
Yuzaki, Michisuke (Sch. Med. Keio, Tokyo, Japan)
Learning and memory formation requires continuous synaptic plasticity
at both the functional and structural level. The stability of synapse is
maintained by bidirectional signals between pre- and post-synaptic molecules in response to synaptic activity. However, very little is known
about molecules that are involved in such a transsynaptic action. Synaptotrophins (Sptn1-Sptn4) structurally belong to C1q/tumor necrosis factor (TNF) family. Recently, Sptn1 has been revealed to be glycoprotein
secreted from cerebellar granule cell, and to regulate synaptic plasticity
and synaptic integrity between parallel fiber-Purkinje cell (PF-PC) synapse. sptn1-null mice are ataxic, and exhibit molphological abnormalities of PF-PC synapse [e.g., naked spine, mismatched Postsynaptic
density (PSD)]. Whereas sptn1 mRNA is predominantly expressed in the
cerebellum, other members of synaptotrophin family, of which structure
are highly similar to that of Sptn1, are expressed in not only cerebellum
but also other brain regions. We hypothesized that the transsynaptic action of the secreted synaptotrophins plays a critical role in structural remodeling of synapses in various central nervous systems. Here we
present the expression and the biochemical characteristic of Synaptotrophin family.
Role of phospholipase Cβ as a coincidence
detector for retrograde endocannabinoid
Hashimotodani, Yuki1; Ohno-Shosaku, Takako2; Kano,
Masanobu3 (1Dept. Cellular Neurophysiol., Grad. Sch. Med. Sci.,
Kanazawa Univ., Kanazawa, Japan; 2Dept. Impair. Stud., Grad. Sch.
Med. Sci., Kanazawa Univ., Kanazawa, Japan; 3Dept. Cellular
Neurosci., Grad. Sch. Med., Osaka Univ., Osaka, Japan)
Endocannabinoids (eCB) mediate retrograde signal at various brain regions. Postsynaptic release of eCB can suppress neurotransmitter release
through activating presynaptic CB1 receptor and cause short-term or
long-term synaptic plasticity. The eCB release is induced by strong increase in postsynaptic [Ca2+]i or activation of Gq/11-coupled receptors.
Furthermore, coincidence of [Ca2+]i elevation and receptor activation
markedly enhances eCB release. Phospholipase C (PLC) is involved in
biosynthesis of the major eCB 2-arachidonoylglycerol. To determine the
role of PLC in eCB release, we used cultured hippocampal neurons and
monitored the eCB release by measuring CB-sensitive synaptic currents.
We found that the receptor-driven eCB release was absent in PLCβ1knockout mice. This PLCβ1-mediated eCB release was dependent on
physiological levels of [Ca2+]i. We measured PLCβ1 activity in intact
neurons by using exogenous TRPC6 channel as a biosensor for the PLC
product diacylglycerol. The receptor-driven TRPC6 currents were
absent in PLCβ1-knockout mice and showed a similar [Ca2+]i
dependence to that of receptor-driven eCB release. These results indicate
that PLCβ1 serves as a coincidence detector for triggering eCB release
in the hippocampus. PLCβ contributes to various neuronal signaling.
Therefore, Ca2+ dependency of PLCβ may play an important role in
various synaptic modulations and plasticity.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Application of physiological study to various branches: Physiological approach to music and dance [Associates of Young
"We are alive." at the Rave Party.
Seino, Eiichi (SEINO Eiichi Office, Balearic Sunrise Org., Tokyo,
Rave is the outside dance music party, started in 1988 in Britain, and has
spread all over the world.
Now it's one of the biggest youth culture.
And before all, it's a strong and personal experience of sound, dance and
Application of physiological study
to various branches: Physiological
approach to music and dance
[Associates of Young Researchers
What Rave gave the youth is the shout & feeling of "We are alive."
But what's that?
And for me, it's a "big somewhere" related to the writing.
Where is there?
Is it the place religion calls "a holy", or Timothy Leary's LSD revolution,
or alcoholic junkie's blue devil?
Nobody explain yet, but Rave clearly gives us.
"We are alive."
I do dance, I do science.
Oshio, Ritzka1,2,3 (1Nat. Inst. Phys. Sci. Div. Cereb. Integr., Aichi,
Japan; 2Dept. cell biophys. Sch. Med. Nagoya Univ., Aichi, Japan;
3SORANOMADO project)
Observation, invention, experiment, control, speculation, discussion and
presentation...these are processes that a dance work is born. Movements
come from nature both inside and outside me. To pick up and compose
such movements, what I need to do everyday is sharpen my senses and
hone my skill. It is same for doing science, isn't it?
Sharpen senses, make imagination from what we sense and express the
internal image as a work...these are the processes of artistic expression.
These processes are composed of every physiological functions
beginning from the sensory inputs. When I do dance, I need to act on
natural physiological limits such as joint angle, muscle mobility and so
on. In other words, these limitations are the origins of the characteristic
dance movement. Such limitation is the very source of beauty and
confidence for movements. Audible sound, visible color, actable
movement...these physiological limitations have important meanings for
both artistic expression and impression. With these physiological
limitations, we can converge and decide the artwork and can share
impressions over the cultures and age universally.
Many people believe that art is too subjective to be a scientific object.
Indeed,individual experience of art is hard to be described in objective
way. However, I believe it still should have a great importance to study
about art in physiological paradigm, because art is the work of the human
beings limited physiologically.
I was in the scene from 80's, and wrote some books about the rave
culture, dance music and trance, dancing high and ecstasy, the party as a
temporary autonomous zone etc.
How can physiology approach music?: A
case study on the hypersonic effect
Honda, Manabu (Nat. Inst. Neurosci., Nat.Cent. Neurol. Psychi,
Tokyo, Japan)
In the Western modern framework, which had Cartesian dualism as one
of its vital origins, music, which induces beauty and pleasure in human
mind, and physiology, which illuminates physical mechanism of human
body, were considered to belong to exclusively independent domains.
Physiological approaches to music, therefore, are basically challenges
against the paradigm dating back to Descartes and inevitably involve essential difficulties in practice.
As a successful instance of physiological approach to music, I will
examine the discovery of the "hypersonic effect" in this presentation.
The hypersonic effect is the phenomenon that imperceptible highfrequency component of air vibration above human audible range
activates neural circuit of beauty and pleasure, and makes the sound
more comfortable to hear. Regarding the phenomenon, there had long
been a serious disagreement between artists and researchers. It may be
easy to recognize that the critical factor for the discovery of the
hypersonic effect, beyond this historical conflict, was the fact that the
discoverer, Tsutomu OOHASHI, was a distinguished artist, Shoji
YAMASHIRO, at the same time. Sharing scientific ability and artistic
sensibility in one single personality, however, was just a necessary
condition but not a sufficient condition for this discovery. This paper
will introduce "Eiffel-Tower, Pyramid, and volcanic-islands models of
human activity" that realized the physiological approach to music.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: The function of neocortical inhibitory interneurons
Regulation of inhibitory synaptic
transmission by presynaptic glutamate
receptors in visual cortex
Komatsu, Yukio (Dept. Visual Neurosci., Res. Inst. Environ.
Med., Nagoya Univ., Nagoya, Japan)
The function of neocortical
inhibitory interneurons
We studied the roles of presynaptic glutamate receptors in synaptic transmission and plasticity in cortical inhibitory connections. Whole-cell recording was conducted from layer 2/3 pyramidal cells in visual cortical
slices of young mice. Inhibitory postsynaptic currents (IPSCs) were recorded at the reversal potential of excitatory synaptic transmission. The
frequency of miniature IPSCs (mIPSCs) recorded in the presence of tetrodotoxin was increased by bath application of glutamate, AMPA and
NMDA, while it was decreased by the AMPA receptor antagonist
NBQX and the NMDA receptor antagonist APV. These compounds did
not change mIPSC amplitude, suggesting that AMPA and NMDA receptors are present at the presynaptic terminal of inhibitory synapses and
their activation facilitates inhibitory synaptic transmission. Indeed, application of either APV or NBQX decreased the amplitude of evoked IPSCs considerably with accompanying increases in both paired-pulse
ratio and coefficient of variation of IPSC, consistent with presynaptic action of these antagonists. High-frequency stimulation produced longterm potentiation (LTP) of IPSCs in normal solution. The magnitude of
LTP decreased in the presence of NBQX and LTP did not occur in the
presence of NBQX and APV, suggesting that AMPA and NMDA receptors both contribute to the facilitation of LTP production. These results
indicate that both AMPA and NMDA receptors are present at the presynaptic terminal of inhibitory synapses in layer 2/3 pyramidal cells and that
their activation facilitates inhibitory synaptic transmission and LTP.
Nonpyramidal cells and their wiring in
cortical microcircuit
Kubota, Yoshiyuki; Kawaguchi, Yasuo (Div. Cerebral
Thalamocortical innervation to inhibitory
neurons in the cortex: relevance to
cholinergic control of cortical network
Circuitry, NIPS)
Kimura, Fumitaka (Div Neurophysiol. Osaka Univ. Grad. Sch.
Activities of cortical pyramidal cells are regulated by GABAergic nonpyramidal cells with temporally and spatially differentiated inhibitotry
wiring. Cortical inhibitory synapses were believed to make synaptic contacts mainly on soma and/or proximal dendrites of pyramidal cells, however the latest our finding shows approximately 1/3 of axon terminals of
cortical nonpyramidal cells, such as double bouquet cell, Martinotti cell
and neurogliaform cell, make synaptic contact on spine head, which also
receive an asymmetrical input, called double innervated (DI) spine.
From morphological point of view, the inhibitory synapse on DI spine
probably has a vetoed function to the asymmetrical excitatory input. We
studied morphological properties of the double innervated spines using
vesicular glutamate transporters (VGLUTs) positive terminals, which
show complementary distribution in cortex, VGLUT1 positive terminals
were mostly originated from cortical pyramidal cells and VGLUT2 positive terminals mostly from thalamic projection neurons. This complementary localizations permitted to study an origin of the excitatory
terminals on the DI spines. We observed 291 VGLUT1 innervated and
442 VGLUT2 innervated spine heads and found the target of these inhibitory synapses were almost exclusively DI spines received VGLUT2
positive excitatory synapse. Forty four (9.6%) out of 442 spine heads innervated by VGLUT2 positive synapses received symmetrical synaptic
input and only 2 (0.7%) out of 291 VGLUT1 innervated DI spines were
found. These results indicated that part of thalamo-cortical efferent fibers were vetoed by inhibitory synapse selectively at spine head.
Med., Suita, Japan)
Mammalian cortex receives dense cholinergic innervation from basal
forebrain cholinergic neurons, but how acetylcholine (ACh) regulates
cortical circuits is still unclear. Recent experiments favor the hypothesis
that instead of producing simple facilitation or inhibition, ACh serves to
shift the cortical circuits into a condition where cortical neurons are influenced predominantly by afferent inputs from thalamus rather than
other cortical inputs. This is achieved by muscarinic suppression of intracortical connections and nicotinic facilitation of thalamocortical inputs, both presynaptically. Indeed, excitatory postsynaptic potentials
(EPSPs) in layer 4 neurons elicited by thalamic stimulation were enhanced or in some cases "unsilenced" in the presence of a nicotinic agonist. Optical recordings further supported facilitatory effect of ACh, but
it also revealed that this facilitation was followed by suppression only in
layer 4. By comparing the sensitivity to a nicotinic agonist between excitatory and inhibitory cells, we found that inhibitory neurons were more
susceptible to nicotine. Incidentally, we also found that thalamic activation of GABAergic neurons preceded that of excitatory neurons in a given barrel, which effectively works to produce a feedforward inhibition
on excitatory relay cells. Thus, exploiting such intrinsic network property, ACh not only facilitate thalamic input to cortex, but also restrict the
excitation of postsynaptic cells to a narrow window of time by selectively enhancing thalamic innervation to inhibitory neurons in the cortex.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Dysfunction of intracellular trafficking and neuropathophysiology
GABA uptake determines critical period
onset in mouse visual cortex
Iwai, Youichi1; Lester, Henry2; Hensch, Takao1 (1Neuronal
Circuit Dev, RIKEN BSI, Wako, Japan; 2California Inst. Tech.,
Pasadena, CA, USA)
Strengthening GABA-A receptor α1 subunit-mediated inhibition with
diazepam triggers ocular dominance (OD) plasticity prematurely
(Fagiolini et al., 2004). Yet, the endogenous determinant of critical
period (CP) induction remains unknown. Several biochemical analyses
intriguingly reveal an elevated GABA uptake with a peak before the CP
onset. The main GABA transporter (GAT1) localized to inhibitory axon
terminals may be responsible for this transient activity based on its
developmental expression profile. Here, we tested directly whether loss
of GAT1 function regulates CP onset. In GAT1 knockout (KO) mice,
neuronal response strength in visual cortex was normal, but prolonged
discharge reflecting weak intracortical inhibition was lost earlier,
suggesting an accelerated maturation of GABA function in vivo. Brief
monocular deprivation (MD) revealed that OD plasticity of GAT1 KO
mice was prematurely activated and rapidly eliminated. When CP onset
is delayed by genetic disruption of GABA synthesis, infusion of a GAT1
inhibitor into visual cortex concomitant with MD restored robust OD
shifts. Similarly, natural CP delay by dark-rearing is counteracted by
diazepam treatment (Iwai et al., 2003) and was also prevented by loss of
GAT1. Interestingly, western blot analysis showed that 2d diazepam
treatment in the dark triggers a reduction of GAT1 expression, similar to
the normal developmental decline in the light. Indeed, GAT1
heterozygous mice mimic such a downregulation and also exhibited an
accelerated CP. These findings indicate that GAT1 expression acts as a
"brake" to delay CP onset.
Dysfunction of intracellular
trafficking and
Inhibitory neuronal circuitry underlying
visual object recognition in area TE
Vesicular transport of Alzheimer's disease
related proteins and neurodegeneration
Tamura, Hiroshi (Grad. Sch. Front. Bio. Osaka Univ., Osaka,
Suzuki, Toshiharu (Grad. Sch. Pharmaceutical Sci. Hokkaido
Univ. Sapporo, Japan)
Neurons in area TE of the monkey inferior temporal cortex respond selectively to images of particular objects and are considered to be a neural
basis for visual object recognition. The mechanism of generation of the
stimulus selectivity, however, is largely unknown. We addressed the role
of inhibitory TE neurons in this process by examining their visual response properties and interactions with adjacent target neurons. We applied cross-correlation analysis to spike trains simultaneously recorded
from pairs of adjacent neurons in anesthetized macaques (Macaca fuscata). Neurons whose activity preceded a decrease in activity from their
partner were presumed to be inhibitory neurons. Most inhibitory neurons
responded to a variety of visual stimuli in our stimulus set, which consisted of several dozen geometrical figures and photographs of objects,
with a clear stimulus preference. On average, 10% of the stimuli increased firing rates of the inhibitory neurons. Degree of stimulus selectivity of inhibitory neurons was similar to that of excitatory neurons.
Although inhibitory neurons occasionally shared the most preferred
stimuli with their target neurons, overall stimulus preferences were less
similar between adjacent neurons with inhibitory linkages than adjacent
neurons with common inputs and/or excitatory linkages. These results
suggest that inhibitory neurons in area TE are activated selectively and
exert stimulus-specific inhibition on adjacent neurons, contributing to
shaping of stimulus selectivity of TE neurons.
Dysfunctions of vesicular transport are linked to neurodegenerative disease, including Alzheimer disease (AD). Amyloid β-prrotein precursor
(APP) has been implicated in the development and progression of AD.
Recent reports suggest that APP functions as cargo receptor for kinesin
I. APP interacts with kinesin light chain (KLC) indirectly via JNKinteracting protein 1b (JIP1b). We have reported that APP associates
with Alcadein, a novel type I membrane protein, in neuron through their
cytoplasmic interaction with X11-like (X11L) protein. We also found
that Alcadein associates with KLC directly, thus Alcadein and APP/
JIP1b competed for KLC. Alteration in APP- and Alcadein-transport
system in neuron impairs the vesicle trafficking in axon, suggesting that
inappropriate assignment of these cargos leads to neuronal malfunction
and the degeneration in future.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Dysfunction of intracellular trafficking and neuropathophysiology
Functions of Presenilins in Mediating
Protein Trafficking
Xu, Huaxi (Burnham Inst. La Jolla, CA, USA)
Cdk5 is a membrane-associated protein
kinase whose mislocalization induces
neuronal cell death
Alzheimer's disease (AD), the most common form of senile dementia, is
characterized by excessive production and accumulation of neurotoxic
β-amyloid (Aβ) peptides which are proteolytically derived from βamyloid precursor protein (APP) via β- and γ-secretase cleavages.
Experimental evidence from several groups including our own has
demonstrated that the production of Aβ occurs largely in the trans-Golgi
network (TGN) where APP molecules predominantly reside. Mutations
in presenilins genes are associated with the majority of familial AD
likely through a mechanism of increase Aβ42 production. Presenilins
(PS, PS1 and PS2) along with their associated proteins including
nicastrin (Nct), PEN2 and APH1 are essential for the γ-secretase activity.
The precise functions of Nct, APH-1 and PEN-2 have not been fully
elucidated. Recent studies including ours suggest that PEN-2 mediates
endoproteolysis of PS1, while APH-1 and Nct play regulatory roles in
maintaining the stability of PS1 and the complex. PS1 knockout mice
exhibit pre-neonatal lethality and PS1 has also been shown to affect
numerous physiological functions including calcium homeostasis,
skeletal development, neurite outgrowth, apoptosis, synaptic plasticity,
tumorigenesis. These data strongly indicate critical physiological roles
of PS1 addition to its essential role in γ-secretase activity. We and others
have reported that PS1 plays an important role in intracellular trafficking
(especially from the TGN to the plasma membrane) of select membrane
proteins including APP, PEN2 and nicastrin. The detailed cell biological
mechanism for PS-mediated protein trafficking will be discussed.
Hisanaga, Shin-ichi; Asada, Akiko; Saito, Taro (Lab. of
Aβ generation and APP trafficking in
Alzheimer's disease
Kinoshita, Ayae (Dept.Health Sci. Fac.Med.Kyoto Univ, Kyoto,
Alzheimer's disease is a slowly progressive neurodegenerative disorder
which causes severe dementia. Amyloid-beta peptide (Aβ) deposition in
senile plaques is one of the pathological hallmarks in Alzheimer's
disease. Aβ peptide is derived from the amyloid precursor protein (APP)
by proteolytic processing by beta-secretase which cleaves APP at the Nterminus of Ab, and by gamma-secretase which cleaves at the Cterminus of Aβ. In spite of extensive research, the precise subcellular
localization of Ab generation has not been identified yet. Using the
recently developed fluorescence resonance energy transfer (FRET)
approach and pulse-chase ELISA, we examined the subcellular
localization of interactions between APP and beta-secretase. Our data
showed a close APP-BACE interaction in early endosomes, and
highlight the cell surface as an additional potential site of APP-BACE
interaction. Furthermore, we identified a novel interaction between LRP,
an endocytic receptor for APP, and beta-secretase, in the early
endosomes and on the cell surface. The interaction between LRP and
beta-secretase was not detected when cholesterol was depeleted,
suggesting that LRP encounters beta-secretase in the lipid raft of the
membranes. Taken together, we propose that APP interacts with betasecretase in the lipid rafts of the cell membrane and in early endosomes,
and that LRP may be a scaffold protein which links APP and BACE
upon endocytosis. We believe that investigation of the interaction
between APP and its secretases helps us understand the mechanisms of
Aβ generation and pathogenesis of Alzheimer's disease.
Mol. Neurosci., Tokyo Metro. Univ., Hachioji, Tokyo, Japan)
Cyclin-dependent kinase 5 (Cdk5) is a multifunctional Ser/Thr protein
kinase activated by binding to its activator p35 or p39, which is expressed predominantly in neurons. Cdk5 is shown to be involved in neuronal migration during brain development, synaptic activity in matured
neurons, and neuronal cell death in aged brains. However, exact roles of
Cdk5 in those neuronal activities have not been answered yet. Cellular
localization would be critical to understand the detailed functions of
Cdk5/p35 or Cdk5/p39. p35 or p39 activator controls the cellular localization as well as the kinase activity. The Cdk5/p35 and Cdk5/p39 complexes bind to plasma membranes and Golgi apparatus via
myristoylation at the N-terminal Gly of p35 or p39 that may compartmentalize the active Cdk5 complexes in the cytoplasm. When Gly is mutated to Ala, Cdk5/p35 and Cdk5/p39 become soluble in the cytoplasm
and then is translocated into nucleus. This mislocalization is observed at
the time of neuronal cell death. For example, endoplasmic reticulum
(ER) stress deregulates the Cdk5 activity by cleavage of p35 to p25 with
calpain. The cleavage of p35 changes the cellular distribution of active
Cdk5, stabilizes the p25/Cdk5 complex, and stimulates the kinase activity of Cdk5, thereby allowing potentially aberrant phosphorylation of
neuronal proteins, which would adversely affect the survival of neurons.
We would also like to discuss on its localization in relation to membrane
trafficking in living neurons.
Critical role of calpain-dependent
cleavages of amphiphysin I in regulation of
synaptic vesicle trafficking during
Tomizawa, Kazuhito; Wu, Yu-Mei; Matsui, Hideki (Dept.
Physiol., Okayama Univ. Grad. Sch. Med., Okayama, Japan)
Clathrin-mediated endocytosis plays a key role in the recycling of synaptic vesicles in nerve terminals and amphiphysin I is one of the components of the molecular machinery involved in this process. Amphiphysin
I mediates invagination and fission of synaptic vesicles in cooperation
with dynamin. We found that amphiphysin I was cleaved to three fragments by treatment with high KCl (80 mM) and by high-frequency electrical stimulation in the mouse hippocampal slices. The cleavage sites
were localized in the CLAP domain. The cleaved amphiphysin I was unable to interact with dynamin and disrupted the co-polymerization into a
ring formation with dynamin and liposome in a cell-free system. The
calpain-dependent cleavages inhibited clathrin-mediated endocytosis.
Finally the amphiphysin I cleavages were found in the hippocampus of
kainate-treated FVB/N mice and the cleavages inhibited the neural hyperexcitation of the mice. I will review these findings and discuss the
role of calpain-dependent cleavages of amphiphysin I in protecting neurons against excitotoxicity and hyperexcitation.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Kickoff symposium of the Journal of Physiological Sciences in succession of the Japanese Journal of Physiology
Novel roles of anion channels in
physiology and pathophysiology
Okada, Yasunobu; Shimizu, Takahiro; Inoue, Hana;
Sabirov, Ravshan; Takahashi, Nobuyuki; Dutta, Amal K;
Liu, Hongtao
Kickoff symposium of the Journal
of Physiological Sciences in
succession of the Japanese Journal
of Physiology
DiPolo, Reinaldo1; Beauge, Luis2 (1Laboratorio de
Fisiologia Celular, IVIC, Caracas Venezuela; 2Instituto de
investigaciones Medicas M.y.M Ferreyra, Cordoba Argentina)
The Na-Ca exchangers family of membrane transporters is widely distributed in cells and tissues of the animal kingdom and constitutes one of
the most important mechanisms for extruding calcium from the cell. Two
basic properties characterize them: 1-Their activity is not predicted by
thermodynamic parameters of classical electrogenic counter-transporters (dependence on ionic gradients and membrane potential), but is
markedly regulated by transported (Na and Ca) and non-transported ionic species (protons and other monovalent cations). These modulations
take place at specific sites in the exchanger protein located at extra, intra
and trans-membrane protein domains. 2- Exchange activity is also regulated by the metabolic state of the cell. The mammalian and invertebrate
preparations share MgATP in that role; the squid has an additional compound, phosphoarginine. This presentation emphasizes the interrelations
between ionic and metabolic modulations of Na-Ca exchange, focusing
mainly in two preparations where most of the studies have been carried
out: the mammalian heart and the squid giant axon. A surprising fact that
emerges when comparing the MgATP related pathways in these two systems is that although they are different (PIP2 in the cardiac and a soluble
cytosolic regulatory protein in the squid), their final target effects are essentially similar: sodium-calcium-proton interactions with the exchanger. A model integrating both ionic and metabolic interactions in the
regulation of the exchanger will be discussed.
Anion channels play a stabilizing role in excitability in muscle and neuronal cells and a Cl– transporting role in epithelial cells. Recent investigations have revealed their more general functions including cell volume
regulation and cell proliferation. Here, we present additional roles that
have been found in our laboratory. First, the volume-sensitive outwardly
rectifying (VSOR) Cl– channel, which is ordinary activated by cell
swelling, plays an inductive role of apoptotic cell death. An apoptotic inducer rapidly activated the VSOR current without cell swelling and
thereby induced apoptotic volume decrease (AVD) in epithelial and cardiac cells. Second, the same channel is involved in excitotoxic neuronal
cell death. Stimulation with NMDA induced activation of the VSOR Cl–
channel, varicosity formation, somatic swelling and eventually necrotic
death in cortical neurons. Third, the maxi-anion channel with a singlechannel conductance of around 400 pS serves as the release pathway of
ATP, which is an extracellular signal for cell-to-cell communication, in
mammary cells, kidney macula densa cells, cardiomyocytes and astrocytes activated by a variety of stimuli. Forth, the maxi-anion channel
also mediates glutamate release from cortical astrocytes under ischemic
conditions. Molecular understanding of physiological or pathoplysiogical functions of these anion channels will progress after identification of
their molecules.
Cardiovascular modules in the cerebellum
Nisimaru, naoko (Dept. Physiol. Facult. Med. Univ. Oita, Oita,
The cerebellum is involved in the control of not only motor but also autonomic functions. I will summarize roles of the cerebellum in cardiovascular control. I propose that the cerebellum contains five distinct
modules (cerebellar corticonuclear microcomplexes) dedicated to cardiovascular control. First, a discrete rostral portion of the fastigial nucleus and the overlying medial portion of the anterior vermis (Lobules I, II
and III) conjointly form a module that controls the baroreflex. Second,
anterior vermis also forms a microcomplex with the parabrachial nucleus. Third, a discrete caudal portion of the fastigial nucleus and the overlying medail portion of the posterior vermis (lobules VII and VIII) form
another module controlling the vestibulosympathetic reflex. Forth, the
medial portion of the uvula may form a module with the nucleus tractus
solitarius and parabrachial nucleus. Fifth, the lateral edge of the nodulus
and the uvula, together with the parabrachial nucleus and vestibular nuclei, forms a cardiovascular microcomplex that control the magnitude
and /or timing of sympathetic nerve responses and stability of the mean
arterial blood pressure during changes of head position and body posture. Another region of the flocculus, which has recently been found to
be related to cardiovascular control, will be also discussed (Nisimaru and
Ito, 2005).
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Transgenic approach to mammalian neurophysiology—Functional dissection with genetic and electrophysiological tools [YFI
(Young Foreign Investigator) Workshop]
Inherited cardiomyopathies as a troponin
Morimoto, Sachio (Kyushu Univ. Grad. Sch. Med., Fukuoka,
Synaptic connections and rhythmic
activity of Renshaw cells in GAD67-EGFP
knock-in mouse
Nishimaru, Hiroshi1; Yanagawa, Yuchio2; Ole, Kiehn3
Troponin, one of the sarcomeric proteins, plays a central role in the
regulation of contraction in vertebrate skeletal and cardiac muscles.
More than two hundred of mutations in the cardiac sarcomeric proteins,
including myosin heavy/light chains, actin, troponin, tropomyosin, myosin-binding protein-C, and titin/connectin, have been found to cause
various types of cardiomyopathy in human since 1990, and over sixty
mutations in cardiac troponin subunits have been identified in the hypertrophic (HCM), dilated (DCM) and restrictive (RCM) cardiomyopathies. To explore molecular mechanisms for the pathogenesis of these
cardiomyopathies, recombinant mutants of human cardiac troponin subunits were exchanged into permeabilized rabbit cardiac muscle fibers
and their effects on the Ca2+-dependent force generation in cardiac muscle were examined. Most mutations in cardiac troponin subunits associated with HCM had Ca2+-sensitizing effects. In contrast, DCM-linked
mutations in cardiac troponin T decreased the Ca2+ concentrations required for force generation, strongly suggesting that changes in the Ca2+
sensitivity of force generation in cardiac muscle in opposite directions,
i.e. Ca2+-sensitization and desensitization, play important roles in the
pathogenesis of these two distinct forms of cardiomyopathy. RCMlinked mutations in cardiac troponin I had much greater Ca2+-sensitizing
effects on force generation than HCM-linked mutations, suggesting that
HCM and RCM-linked mutations in troponin subunits share a common
feature of increased Ca2+ sensitivity of cardiac myofilament, but more
severe change in Ca2+ sensitivity is associated with the clinical phenotype of RCM.
(1Neurosci. Res. Inst., AIST, Tsukuba, Japan; 2Gunma Univ.,
Maebashi, Japan; 3Karolinska Inst., Stockholm, Sweden)
One of the first functionally identified groups of inhibitory neurons in
the mammalian central nervous system are Renshaw cells (RCs, Renshaw 1946). RCs are excited by axon collaterals from motor neurons
(MNs), and provide recurrent inhibition of MNs (Eccles et al. 1954). It
has been shown by studies using cat spinal cord since then that, 1) excitatory synaptic inputs from MNs are mediated by acetylcholine (Curtis
and Ryall 1966) and 2) these inputs are the main driving force for RC activity during locomotion (Noga et al 1987). We examined the physiological nature of RCs in detail using visually guided whole cell recording in
isolated spinal cord preparations from glutamic acid decarboxylase
(GAD) 67-EGFP knock-in mouse neonates. Among the GFP-positive
cells in the lumbar ventral horn, RCs were uniquely identified by electrically stimulating the adjacent ventral root to evoke a short latency EPSC
and by filling the cell with alexa-dyes to confirm its expression of calbindin-28k after recording. The short latency EPSCs that were reduced to
20-40% of control in amplitude by nicotinic receptor blockers and further reduced by blocking ionotropic glutamate receptors indicating that
glutamate is also mediating synaptic inputs to RCs from MNs. During
the locomotor-like rhythmic activity evoked by bath-application of 5-HT
and NMDA, RCs fired rhythmically and modulated not only by excitatory synaptic inputs but also inhibitory ones. These results show that
such technique is a powerful tool to reveal the neuronal mechanisms of
motor control.
In vitro and in vivo analyses of the neuronal
network involved in the regulation of sleep/
wakefulness using transgenic mice
Yamanaka, Akihiro1,2 (1Basic Med. Sci. Univ. Tsukuba, Tsukuba,
Japan; 2ERATO Yanagisawa Orphan receptor project JST)
Transgenic approach to
mammalian neurophysiology—
Functional dissection with genetic
and electrophysiological tools [YFI
(Young Foreign Investigator)
Orexin A and B are a pair of neuropeptides implicated in the regulation
of sleep/wakefulness and energy homeostasis. The regulatory mechanism of orexin neurons is poorly understood since the small number of
orexin neurons is sparsely distributed in the lateral hypothalamus. We
made the following transgenic (Tg) mice to study the physiological role
in the regulation of sleep/wakefulness.Orexin/cameleon Tg mice, in
which orexin neurons specifically express calcium sensing protein, were
used for calcium imaging to screen what kind of neurotransmitter affects
the activity of orexin neurons.Orexin/EGFP Tg mice, in which orexin
neurons express EGFP, were used for electrophysiological studies to reveal intracellular mechanisms involved in the activation or inhibition response. Orexin/GFP::TTC Tg mice, in which orexin neurons express a
retrograde tracer, were used for immunohistochemical studies to reveal
which neurons directly innervate orexin neurons. These studies using Tg
mice revealed how orexin neurons are regulated by afferent neurons.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Physilogical approaches to lombic and hypothalamic circuits for emotion, learning and behavior
Sodium-sensing mechanism in the brain:
from molecular to behavior
Hiyama, Takeshi Y.1,2 (1Nat'l Inst. for Basic Biol., Okazaki,
Japan; 2The Graduate Univ. for Advanced Studies (SOKENDAI),
Okazaki, Japan)
Dehydration causes an increase in the sodium (Na) concentration and osmolarity of body fluid. For Na homeostasis of the body, controls of Na/
water-intake and -excretion are of prime importance. However, the system for sensing the Na level within the brain that is responsible for the
control of Na/water-intake behavior remains to be elucidated. Using
physiological and behavioral techniques in combination with genetic
manipulations, we have demonstrated that a sodium channel Nax is indispensable for the Na-level-sensing in the brain. We previously showed
that Nax channel is preferentially expressed in glial cells in the circumventricular organs (CVOs) and that Nax-knockout mice ingest saline in
excess under dehydrated conditions. Subsequently, we demonstrated
that Nax is a Na-level-sensitive Na channel. Recently, we confirmed the
physiological role of the Nax in vivo by infusion of hypertonic Na solution to the cerebral ventricle. The infusion induced prompt intake of water and aversion to salt in wild-type mice. In contrast, such aversive
behavior was not observed in the knockout mice. When Nax cDNA was
introduced into the brain of the knockout mice with an adenoviral expression vector, only animals with a transduction of the Nax gene into the
subfornical organ (SFO) among the CVOs recovered salt-avoiding behavior under dehydrated conditions. These results clearly indicate that
the SFO is the center of the control of salt-intake behavior in the brain,
where the Na-level-sensitive Nax channel is involved in sensing the
physiological increase in the Na level of body fluids.
Physilogical approaches to lombic
and hypothalamic circuits for
emotion, learning and behavior
Synaptic transmission in the basal ganglia
in the process of neuronal repair with
grafted neuroepithelial stem cells
Implications of abnormal temporolimbic
and prefrontal morphology in development
of schizophrenia
Momiyama, Toshihiko (Lab. Cereb. Struc, NIPS, Okazaki,
Suzuki, Michio1,2 (1Dep. Neuropsychiatry, Univ. Toyama,
Toyama, Japan; 2CREST, JST, Tokyo, Japan)
The fate of grafted neuroepithelial stem cells in the mature brain environment was assessed to confirm their feasibility in the functional repair
of damaged neural circuitry. The neuroepithelial stem cells were harvested from the mesencephalic neural plate of enhanced-GFP-carrying rat
embryos, and implanted into the striatum of normal adult rat or Parkinson's disease model rat. The differentiation pattern of donor-derived
cells was monitored immunohistochemically. The functional abilities of
the donor-derived cells and communication between them and the host
were investigated using host-rat brain slices incorporating the graft with
whole-cell patch-clamp recording. Vigorous differentiation of the neuroepithelial stem cells into mostly neurons was noted in the short-term
with positive staining for tyrosine hydroxylase, suggesting that the donor-derived cells were following their genetically programmed fate. In
the long-term, the large number of donor-derived neurons was sustained,
but the staining pattern showed appearance of medium spiny or cholinergic neurons, suggesting that some neurons were following environmental cues. Some donor-derived astrocytes were also seen in the graft.
Firing pattern and membrane properties suggest the presence of both
dopaminergic and non-dopaminergic neurons in the donor-derived neurons. Glutamatergic and GABAergic post-synaptic currents could be
evoked by electrical stimulation applied in the host region. Neuroepithelial stem cells are therefore an attractive candidate as a source of donor
material for intracerebral grafting in functional repair.
In order to clarify the implications of morphological brain changes in development of schizophrenia, we have made extensive comparisons of
brain morphology using MRI between established schizophrenia and
schizotypal disorder, a schizophrenia-spectrum disorder without overt
and sustained psychotic episode. Compared with controls, bilateral volumes of the amygdala, hippocampus and posterior superior temporal gyrus were reduced comparably in both schizotypal and schizophrenia
patients. Total prefrontal grey matter was smaller bilaterally in schizophrenia patients than in controls, whereas schizotypal patients had larger
right prefrontal grey matter than controls. In schizophrenia patients, the
bilateral superior frontal, inferior frontal and straight gyri, and the left
middle frontal gyrus were smaller than those in controls, while schizotypal patients had larger bilateral middle frontal gyri and smaller right
straight gyrus. In white matter, decreased volume of the anterior limb of
the internal capsule, a fiber bundle connecting the frontal cortex and
thalamus, was found bilaterally in schizophrenia but only on the right in
schizotypal disorder. These findings suggest that volume reductions in
the medial and postero-lateral temporal regions are the common morphological substrates for the schizophrenia-spectrum which presumably
represent the vulnerability. Additional widespread involvement of the
prefrontal cortex might lead to the loss of inhibitory control in other
brain regions and play a critical role in the manifestation of overt psychosis.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Physilogical approaches to lombic and hypothalamic circuits for emotion, learning and behavior
Social cognition related neural responses
in the monkey amygdala
The role of the primate amygdala in
processing emotional facial expressions.
Hori, Etsuro1,2; Tazumi, Toru1,3; Kobayashi,
Tsuneyuki1,2; Umeno, Katsumi1,2; Ono, Taketoshi1,2;
Nishijo, Hisao1,2 (1System Emotional Science, Grad. Sch. Med.
Gothard, Katalin M. (Department of Physiology , University of
Univ. Toyama, Toyama, Japan; 2CREST, JST, Kawaguchi, Japan;
3Dept. Human Pychol. Seisen Univ. Hikone, Japan)
The previous neuropsychological studies demonstrated that the human
amygdala (AM) increased its response to emotional facial expressions
and gaze direction toward to the subject, and suggested that the AM is
essential in social cognition. In the present study, the monkey AM neuronal activity was recorded during performance of a delayed non-matching to sample task using human photos with various facial expressions
and gaze direction. Some neurons were further tested with various human actions such as approaching toward the monkey. Autonomic activity (pupil radius) of the monkey, which reflected emotional expression,
was simultaneously recorded. The results indicated that the AM neurons
differentially responded to various emotional expressions and/or gaze
directions. These facial expression-differential neurons were most sensitive to those of the familiar persons to the monkeys. These results suggest that social cognition might develop based on learning through social
interaction, and the AM is involved in such learning. Activity of other
AM neurons increased when the experimenter approached toward the
monkey, or when the experimenter moved its arm or leg. Pupil radius
also increased during this approaching. These results suggest that the
AM is essential in primate social cognition as well as emotional expression.
Arizona, College of Medicine, Tucson, Arizana, USA)
The primate amygdala plays an important role in differentiating between
facial expressions, yet the neural properties underlying this process are
largely unknown. We recorded from the monkey amygdala neural responses to images of monkey faces, human faces, and objects. Most neurons differentiated between these image categories, yet monkey faces,
human faces, and objects were equally likely to elicit stimulus-selective
responses. In certain animals threatening faces appeared to elicit increased firing rates compared to neutral or appeasing faces suggesting a
processing bias in favor of stimuli that signals potential danger. Neural
responses to monkey faces were further examined to determine whether
the observed changes in firing rate can be best accounted for by face
identity or facial expression. The majority of neurons responded to
unique combinations of identity and expression suggesting that in the
amygdala identity and facial expressions are merged into a single representation. This representation might carry information about the emotional and social significance of facial expressions encountered during
social interactions. The amygdala is also involved in orchestrating overt
behavioral and autonomic responses to images with emotional value. We
recorded skin conductance response, heart rate, and facial muscular activity in conjunction with neural responses in the amygdala and found
correlations between stimulus-selective neural activity and peripheral
autonomic responses.
Regulation of the hippocampal function by
the supramammillary nucleus of the
Sekino, Yuko1,2 (1Div. Neuronal Network, Inst. Med. Sci. Univ.
Tokyo, Tokyo, Japan; 2CREST,JST, Kawaguchi, Japan)
It is a continuing question how emotions enhance memory formation. To
answer the question, we study the connection between the hippocampus
and the hypothalamus, which is involved in Papez circuit. We have hypothesized that the hippocampal activity is enhanced by direct inputs
from the supramammillary nucleus (SuM) of the hypothalamus to the
dentate gyrus (DG) and the CA2 region. Immunocytochemistry of Fos
positive neurons (FN) demonstrated that the SuM-hippocampal pathway
was activated when animals were exploring a novel environment. Number of FN in SuM and the hippocampus increased when rats were placed
in an open field. SuM lesions significantly suppressed the increase of FN
in the entire hippocampus. Small lesions in the lateral SuM significantly
suppressed the increase of FN in the ipsilateral CA2 compared with the
control side, although there was no difference in the number of FN between both sides of DG. These data suggest that CA2 neurons is specifically activated by ipsilateral inputs from SuM, while DG is activated by
bilateral inputs from SuM. Since SuM is related to anxiety, anxiety enhances the neuronal activity of the dentate granule cell and CA2 neurons
and results in the enhancement of memory formation.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Current status of Japanese Guideline and Regulation on Animal Experimentation [Symposium Organized by Physiological
Society of Japan: Symposium on animal ethics and animal supply (held in Japanese)]
The Notification System for the Importation
of Animals
Kobayashi, Kazuto (Inst. Biomed. Sci., Fukushima Med. Univ.
Sch. Med., Fukushima, Japan)
Current status of Japanese
Guideline and Regulation on
Animal Experimentation
[Symposium Organized by
Physiological Society of Japan:
Symposium on animal ethics and
animal supply (held in Japanese)]
The Ministry of Health, Labour, and Welfare introduced the Notification
System for the Importation of Animals, which is effective from 1st September 2005, to prevent the outbreaks of human infectious diseases derived from imported animals. Any importers of animals, including
experimental animals or transgenic animals, are required to submit a
written declaration giving the specified information on the animals, such
as their species name and quantity, to the quarantine station of the Ministry of Health, Labour, and Welfare. This declaration has to be accompanied by a health certificate issued by the government authorities of the
exporting country certifying that the animals are free from the infectious
diseases specified for each species. Procedure to issue the health certificate by the government authorities varies among the exporting countries.
For example, in the United States the animal facility that stores the exporting animals publishes the heath certificate corresponding to the regulation proposed by the Ministry of Health, Labour, and Welfare, and
sends the certificate to the USDA office located in each state, where the
official veterinarians endorse the certificate. In addition, the Ministry of
Health, Labour, and Welfare is asking to register the health certificate
form for the government authorities in other countries that have not yet
responded to the notifying system for importation of animals. Researchers who are planning to import the experimental animals need to understand the current situation of the importation and correspond to this new
system to ensure the smooth importation of animals into Japan.
Current status of Japanese Guideline and
Regulation on Animal Experimentation
Tamaoki, Norikazu (Central Institute for Experimental Animals
Kawasaki, kanagawa, Japan)
Scientific institution in Japan established an institutional animal care and
use committee for reviewing animal experimentation protocols and advise the institutional director to improve the welfare of laboratory animals according to the administrative guidance "Notification Concerning
Animal Experimentation Conducted by Universities etc."(1987). In
2004, Science Council of Japan proposed a new regulation rule of animal
experimentation to promote the public understanding of ethical and scientific animal experimentation. Major points of revision are as follows:1. Establishment of a guideline for animal experimentation
commonly applicable to the all scientific institutions in Japan.2. Establishment of an objective evaluation system on the institutional self regulation for animal experimentation. Establishment of new regulation
system is now on going. On the other hand, Amended Law for the Humane Treatment and Management of Animals (2005) stipulated the 3Rs
principle in animal experimentation.For the development of health research for humans as well as animals, balance between science and animal welfare is indispensable.(Former chair of Committee on Laboratory
Animal Science, Science Council of Japan)
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Gene manipulation for research of cardiovascular system
The involvement of Fyn tyrosine kinase
and membrane rafts in the signal
transduction of abnormal vascular smooth
muscle contraction
Kishi, Hiroko1,2; Guo, Fengling1,2; Kawamichi, Hozumi1;
Miao, Jinying1; Morikage, Noriyasu1; Xu, Dan1,2; Wang,
Chen1; Kajiya, Katsuko1; Kobayashi, Sei1,2 (1Dept. Mol.
Physiol., Yamaguchi Univ. Sch. Med., Ube, Japan; 2Mol. Cell. Dig
Bioreg. Grad. Sch. Med.. Yamaguchi Univ, Ube, Japan)
Gene manipulation for research of
cardiovascular system
Rho-kinase (ROK)-mediated Ca2+ sensitization plays a pivotal role in
abnormal vascular smooth muscle (VSM) contraction such as vasospasm. Previously we identified sphingosylphosphorylcholine (SPC) and
Src family tyrosine kinase (Src-TK) as an upstream signaling molecule
of ROK-mediated Ca2+ sensitization. Since VSM contains both Fyn and
c-Src among Src-TK, we analyzed which Src-TK was truly important for
the Ca2+ sensitization mediated by SPC/ROK pathway. Immunofluorescent study showed that SPC induced the translocation of Fyn, but not cSrc, to plasma membrane in cultured VSM cells and eicosapentaenoic
acid, a specific inhibitor of SPC-induced VSM contraction, blocked the
translocation of Fyn. The siRNA which specifically knockdown Fyn diminished SPC-induced contraction remarkably in cultured VSM cells. In
β-escin permeabilized VSM strips, constitutively-active Fyn, which was
expressed by baculovirus system, induced Ca2+ sensitization and
dominant-negative Fyn blocked Ca2+ sensitization. In confocal study,
SPC induced the translocation of Fyn to plasma membrane where it
colocalized with caveolin-1, a membrane-raft-associated protein. A
functional proteomics approach identified p160 and its phosphorylation
site as a possible target of Fyn. Those findings suggested that membrane
rafts and its associated Fyn played an essential role in ROK-mediated
Ca2+-sensitization of VSM contraction.
Lentiviral vector-mediated SERCA2 gene
transfer improves the heart failure and left
ventricular remodeling induced by
myocardial infarction in rat
Arai, Masashi1; Niwano, Kazuo1; Ikeda, Yasuhiro2;
Miyoshi, Hiroyuki3; Kurabayashi, Masahiko1 (1Gunma
Univ Grad Schl Med, Maebashi, Japan; 2Yamaguchi Univ Schl Med,
Ube, Japan; 3BRC, RIKEN Tsukuba Inst, Tsukuba, Japan)
Introduction Reduced gene expression of SERCA2 impairs calcium
handling and represents a hallmark of heart failure. Unlike adenovirusor adenoassociated vectors, lentivirus can stably integrate into host genome of terminally differentiated cardiac myoctes and induces permanent expression. We developed lentivirus-based SERCA2 gene transfer
system and examined its feasibility as a therapy for heart failure. Results
The therapeutic effect of Lenti-SERCA2 vector (1x1011 IU/300g BW)
was compared with the Lenti-β-Gal control vector in the failing heart
induced by myocardial infarction (MI) in rats. Echocardiography
revealed that Lenti-SERCA2 introduction prevented an increase in left
ventricular diameter and a decrease of fractional shortening by 10-15%
compared with Lenti-β-Gal group rats from days 30 to 180. Pressurevolume analysis demonstrated that Lenti-SERCA2 introduction
improved systolic (dP/dt max, 7677 vs 3028 mmHg/sec; Emax, 0.68 vs
0.37) and diastolic function (tau, 18.4 vs 22.6). Northern and Western
blot analyses revealed that SERCA2 mRNA and protein were elevated
and the BNP mRNA was significantly decreased in Lenti-SERCA2
group. Finally, SERCA2 gene transfer prevented the expansion of MI
region and decreased the mortality rate. Conclusion Our study showed
that the SERCA2 gene was successfully integrated into hearts and
supports the premise that a lentivirus-based SERCA2 gene therapy
improves heart failure.
Identification of phosphoinositide 3-kinase
C2α as an essential signaling molecule in
Ca2+-dependent Rho activation and myosin
phosphatase inhibition by using RNA
interference-mediated gene silencing
Takuwa, Yoh; Yoshioka, kazuaki; Wang, Yu;
Mohammed, Ali Azam; Takuwa, Noriko; Sugimoto,
Naotoshi (Grad. Sch. Med. Kanazawa Univ, Kanazawa, Japan)
Excitatory receptor agonists such as noradrenaline stimulate the activity
of the small G protein Rho and inhibit myosin phosphatase (MP) through
mechanisms involving Rho kinase-dependent phosphorylation of the
MP regulatory subunit MYPT1 in VSM. We have recently demonstrated
that a novel, Ca2+-dependent mechanism for Rho activation and myosin
phosphatase (MP) inhibition is operating in receptor agonist- and membrane depolarization-induced vascular smooth muscle (VSM) contraction. We found that phosphoinositide 3-kinase (PI3K) was required for
Ca2+-dependent Rho activation; the PI3K inhibitors wortmannin and
LY294002 inhibited all of the Ca2+-dependent Rho activation, MYPT1
phosphorylation, MP inhibition, MLC phosphorylation and contraction.
We tried to identify a PI3K isoform by adopting RNA interference and
cultured VSM cells. The selective down-regulation of the expression of
class II alfa isoform (PI3K-C2α), but not the class I p110α, by a specific
siRNA markedly inhibited Rho kinase-dependent MYPT1
phosphorylation, MLC phosphorylation and contraction in differentiated
VSM cultures. Noradrenaline as well as membrane depolarization
stimulated the activity of PI3K-C2α, but not p110α, in a Ca2+-dependent
manner. Thus, these observations unveiled a novel role of the PI3K-C2α
as an upstream regulator of Rho and consequently MP and contraction.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Gene manipulation for research of cardiovascular system
A innovative anti-inflammatory therapeutic
strategy for regeneration of
atherosclerosis with biodegradable gene
eluting stent
Primary role of inositol 1,4,5-trisphosphate
receptor type 1 in agonist-induced aortic
contraction in mice
egashira, kensuke (Cardiovasc Med. Kyushu University)
Japan; 2Calcium Oscillation Project, ICORP, JST, Tokyo, Japan)
Recent evidence suggests that stent-associated inflammation is a prominent feature in animals and humans, and thus can be a promising nextgeneration target for prevention of restenosis. We have shown great benefit of anti-monocyte chemoattractant protein-1 (MCP-1) therapy by
systemic transfer of an N-terminus deletion mutants of human MCP-1
(called 7ND) gene for prevention of restenotic changes in animals.
Therefore, to translate our achievement on MCP-1 pathobiology to clinic, we tested the hypothesis that stent-based local delivery of 7ND gene
reduces in-stent neointimal formation. Bare, polymer-coated, and 7ND
plasmid-coated stents were implanted in iliac arteries of hypercholesterolemic rabbits (n=8-10 each) and cynomolgus monkeys (n=7-10 each).
7ND gene-eluting stents attenuated stent-associated monocyte infiltration/activation and neointimal formation (about 30% reduction) in rabbits. In monkeys, significant reduction of neointimal formation was
noted 1, 3, and 6 month after stenting, indicating long-term benefits of
7ND gene ES in monkeys. No evidence of incomplete healing process
was noted in 7ND-eluting stent sites. In conclusion, anti-MCP-1 strategy
with 7ND gene-eluting stents was strikingly effective in reducing experimental restenosis in rabbits and monkeys. Our finding in nonhuman primates has significant clinical significance, implying that this antiinflammation strategy targeting MCP-1 might be a promising therapy
against human restenosis.
Contraction of vascular smooth muscles is under the regulation of sympathetic activity and vasoactive hormones. It is known that release of
Ca2+ from inositol 1,4,5-trisphosphate (IP3)-sensitive stores causes the
initial phase of agonist-induced vasocontraction. In the present study,
phenylephrine (PE)-induced contraction was measured in thoracic aortas
isolated from the wild-type (WT) and IP3 receptor type 1 knockout
(IP3R1-KO) mice, in order to specify the IP3 receptor subtype responsible for the agonist-induced contraction. PE (10–8 - 10–6 M)-induced aortic contraction in the IP3R1-KO mice was greatly diminished, compared
to that in WT mice, and lacked the steep contraction which was invariably seen in WT aortas immediately after PE application at 10–6 M. But,
high K+-induced contraction was indistinguishable between WT and
IP3R1-KO aortas. Immunoblotting analysis demonstrated the presence
of three IP3 receptor subtypes (IP3R1, IP3R2 and IP3R3) in WT mouse
thoracic aorta; however, abundance of each subtype was in the order of
IP3R1 > IP3R3 >> IP3R2. These results indicate that IP3R1 constitutes
the Ca2+ release channels critical to vasocontraction regulated by sympathetic activity and vasoactive hormones.
Nakamura, Takeshi1,2 (1Juntendo Univ. Sch. Med., Tokyo,
Novel Signaling Mechanism of Angiotensin
II Type 2 Receptor in the Cardiovascular
Senbonmatsu, Takaaki (Saitama Medical School, Saitma,
The role of Angiotensin II (Ang II) in the regulation of the cardiovascular system under normal and pathologic conditions have been well documented. A variety of angiotensin converting enzyme inhibitors
(ACEIs) and angiotensin II receptor blockers (ARBs) are selected as first
choice medicine for hypertension or heart failure. Although two major
subtypes Ang II receptors, exist type1 (AT1) and type2 (AT2), most studies and treatments have focused on AT1 coupled events. Previous reports
indicated that AT2 plays a role in essentially growth suppression such as
through the tyrosine phoaphatase SHP-1 and MKP-1 activation. However, a detailed signaling mechanisms of these responses still remain unclear. Interestingly, an increasing number of recent reports indicate that
AT2 plays a role in growth promoting similar to the AT1 function. We reported that AT2 gene-deleted mice lose the ability to develop cardiac hypertrophy in response to pressure overload or to chronic Ang II
stimulation, and also found a novel signaling mechanism of AT2 mediated by the transcription factor promyelocytic leukemia zinc finger
(PLZF) leading to cardiac hypertrophy. PLZF is selectively expressed in
the heart, but not in the kidney or aorta. Upon Ang II stimulation, AT2
and PLZF are internalized, and PLZF translocates into the nucleus,
whereby nuclear PLZF activates phosphoinositide 3-kinase (PI3K) regulatory subunit 85α leading to cardiac hypertrophy. However, in the
absence of PLZF, Ang II evoked SHP-1 activation leading to growth
suppression via AT 2. These results suggest that AT2 may have dual
switching functions mediated by PLZF.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Motor control mechanism by the cerebellum
Role of the cerebellum in the acquisition
and consolidation of motor memory
revealed by long-term adaptation of ocular
reflex paradigm
Nagao, Soichi1; Shutoh, Fumihiro1,2; Ohki, Masafumi1,3
(1RIKEN BSI, Saitama, Japan; 2Grad. Univ. Tsukuba, Tsukuba,
Ibaragi, Japan; 3Grad. Univ. Tokyo, Tokyo, Japan)
Motor control mechanism by the
Adaptation of ocular reflexes is a prototype of cerebellum-dependent
motor learning. Two different views are proposed for its neural mechanisms: one that the memory of adaptation is formed within the cerebellar
flocculus through the cerebellar long-term depression (LTD) of parallel
fiber-Purkinje cell synapses, and the other that the memory is formed
within the vestibular nuclear neurons using signals mediating through
the flocculus. We developed a long-term adaptation paradigm adaptation
of mouse eye movements. We revealed that the memory trace of motor
learning induced by short-term (day-long) training is located within the
cerebellar cortex, while that induced by long-term (week-long) training
in the cerebellar or vestibular nuclei, by evaluating the effects of reversible pharmacological shutdown of the cerebellar cortex. These results
suggest that the memory trace of motor learning is initially formed in the
cerebellar cortex, and later shifts transsynaptically to cerebellar/vestibular nuclei for consolidation. We further revealed that LTD plays a critical
role in both the acquisition and consolidation of memory by pharmacological and gene-knockout mouse experiments.
Identification of loci involved in the
memory of chronic motor learning of the
vertical vestibuloocular reflex in squirrel
Hirata, Yutaka1; Blazquez, Pablo2; Highstein, Stephen2
(1Dept. Computer Science, Chubu Univ. Kasugai, Aichi, Japan;
2Washington Univ. School of Med., St. Louis, MO, USA)
The vestibuloocular reflex (VOR) stabilizes vision during head turn by
counter-rotating the eyes in the orbit. Its gain (eye velocity/head velocity) can be modified by visual-vestibular mismatch, but following cerebellar inactivation, the gain cannot be further modified. Thus, the VOR
has been a model system to study potential cerebellar roles in motor
learning. The cerebellum may have different roles in acute versus chronic VOR motor learning, because cerebellar inactivation entirely eliminates any acutely learned component, but it only partially eliminates the
memory of long-term gain change, suggesting multiple loci for the
chronic memory. To pinpoint these, a series of experiments in which activities of cerebellar Purkinje cells (PCs) and their target neurons in dorsal Y group (YNs) were recorded before and after chronic VOR motor
learning. The sensitivities of PCs to both vestibular (V) and efference
copy (E) signals changed with learning, YNs changed their sensitivities
to V modalities, and these changes are asymmetric for gain increase and
decrease. Computational modeling revealed significant changes in 1) V
pathway to cerebellar flocculus (FL), 2) direct V pathway to YNs after
gain increase, and in 3) E pathway to FL, 4) direct V pathway to YNs, 5)
pathway from PCs to YNs, and 6) V pathway excluding those through
FL and YNs after gain decrease. The results suggest involvement of several loci in chronic learning and different neuronal mechanisms for gain
increase and decrease.
Feed-forward associative motor learning
by the cerebellum
Fujita, Masahiko (Fac. Eng. Hosei Univ., Japan)
There are some unresolved problems in motor learning theory. One is determining the source of a learning signal, sometimes called a motor error
signal. Another one is the credit assignment problem of the motor error,
since the erroneous performance perceived by a subject is due to the actions of many elementary motor units. The feed-forward associative
learning theory attributes the source to the movement system itself.
When a subject performs a corrective movement after his primary movement, the proposed neural learning device learns to associate the primary
motor command with the corrective motor command by using a placecoding system. In the subsequent trials, the primary movement will involve a correction due to the participation of this mechanism, thus resulting in better performance. The device consists of many adaptive units
each of which is specialized for a particular elementary motor unit, and
naturally resolves the assignment problem. The theory assumes three
conditions, namely, that a motor center and the learning device share the
same place-encoded motor information; the motor center issues a command and a learning signal simultaneously from the same unit; and a
learning signal issued with a corrective command has a heterosynaptic
interaction with the previous primary command. The cerebellum is a reasonable candidate for the device satisfying these conditions. The reaction time of a corrective movement, usually 100-300 ms, almost satisfies
the coincidence condition for long-term depression of the granule-toPurkinje synapses. As an application, this theory is demonstrated to account for behavioral results regarding saccadic adaptation.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Functional coupling between neuron and glia
Mechanism of cerebellar function studied
using mutant mice
Hirano, Tomoo1,2 (1Dept. Biophys., Grad. Sch. Sci., Kyoto Univ.,
Introduction: Importance of glia-neuron
functional coupling in higher order brain
Kyoto, Japan; 2CREST, JST, Kawaguchi, Japan)
Kudo, Yoshihisa (Tokyo Univ. of Pharm. and Life Sci. Hachioji,
The cerebellum plays critical roles in motor control and learning. Relative simplicity of the cerebellar cortical circuit has prompted the study
on how it works, which has made the cerebellum one of the best characterized structure of central nervous system. However, respective role of
each component or its function such as a particular type of synapse, neuron or synaptic regulation has been elusive. We have been addressing
these issues using several types of mutant mice, and here I present our
recent data on the GluRδ2 (glutamate receptor δ2 subunit) knockout
mice (δ2-/-). GluRδ2 is a molecule related to ionotropic glutamate
receptor, which is specifically expressed at parallel fiber (PF) - Purkinje
neuron (PN) synapses. The δ2-/- show impairment in the long-term
depression, synaptic stabilization of PF-PN synapses and elimination of
surplus climbing fiber (CF) inputs resulting in the multiple innervation
to a PN, and also show motor discoordination and motor learning failure.
We studied the eye movements and found that δ2-/- show involuntary
spontaneous eye movements and large phase delay in the optokinetic
response (OKR). We have been analyzing the mechanism of these
abnormal motor regulations by simultaneous recording of eye
movements and PN activity. Our results suggest that the enhanced CF
activity in δ2-/- largely disturbed the normal pattern of PN activity
regulating eye movement, that highlights the importance of synaptic
inputs balance on a PN in motor regulation.
Tokyo, Japan)
The concept for glial cells as non-excitable- supporting elements has
been accepted until almost the end of 20th century without doubt. However, total number of glial cells in human brain was found to be far larger
than that of neuronal cells, and the ratio of glial cells to neurons in the
brain was found to be higher in the highly developed animals than that
in primitive animals. Those evidences suggested that the cells may be required for establishing higher order brain function. The dynamic feature
of the cells has been revealed by the Ca2+ imaging techniques. Since then
astrocytes have been recognized as dynamic cells and are regarded as intimate collaborators with neuronal cells. The concept of"tri-partite synapse" has been put forward to explain the possible participation of
astrocytes into the synaptic transmission and information processing in
the brain. However, the interaction among neuronal cells and astrocytes
may not be such small scale. The transmission of the Ca2+ waves from
an astrocyte to the other has been found to be performed through gapjunctions and also specific transmitters and receptors system. Thus astrocytes themselves form a wide network among them, which may be woven into the neuronal networks and construct large and highly organized
information processing system. Neuronal networks as main system in
the brain information processing may be controlled slowly and widely
by astrocytes networks. This "glia-neuron functional coupling" will establish a higher order brain function and its deficit will cause brain dysfunctions.
Identification of peri-interneuronal glial
cells and its modulatory effects on neurons
in the hippocampus of rat
Yamazaki, Yoshihiko1; Hozumi, Yasukazu2; Kaneko,
Kenya1; Fujii, Satoshi1; Miyazaki, Keita1; Sugihara,
Toshimichi1; Kato, Hiroshi1 (1Dept. Neurophysiol., Yamagata
Univ. Sch. Med., Yamagata, Japan; 2Dept. Anat. and Cell Biol.,
Yamagata Univ. Sch. Med., Yamagata, Japan)
Functional coupling between
neuron and glia
Recent studies have demonstrated the existence of direct interactions between neurons and glial cells. To evaluate these interactions, we focused
on interneuron/peri-interneuronal glial cell pairs in the hippocampal
CA1 region, because of the close proximity of these two cells. Based on
the electrophysiological, morphological and immunohistochemical
studies, the peri-interneuronal glial cells were classified into astrocytes
and oligodendrocytes, and we worked with the peri-interneuronal astrocytes (PNAC) in this study. Excitatory postsynaptic currents (EPSCs) recorded in an adjacent interneuron were suppressed by the depolarizing
current injection into the PNAC. These suppression of EPSCs accompanied the increase of paired-pulse ratio and were blocked by the application of adenosine A1 receptor antagonist, indicating the involvement of
presynaptic adenosine A1 receptors. Moreover, PNAC depolarization
modified the directly induced firing of the interneuron. These results
demonstrate directly modulatory effects of the PNAC on neuronal activities.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Functional coupling between neuron and glia
Regulation of excitatory synaptic
transmission by glial glutamate
Alexander disease model mice
Takatsuru, Yusuke; Takayasu, Yukihiro; Iino, Masae;
Ozawa, Seiji (Dept. Neurophysiol., Gunma Univ. Grad. Sch.
Alexander disease is caused by heterozygous mutation in glial fibrillary
acidic protein (GFAP). The pathological hallmark is the presence of astrocytic GFAP aggregation called Rosenthal fibers (RF). To understand
the pathophysiology of Alexander disease and utilize those results to the
understanding of normal function of astrocytes in vivo, we have established transgenic mice that express hunan GFAP R239H mutant under
the control of mouse GFAP promoter. Immunohistochemistry using human GFAP specific antibody, SMI21, showed that this immunoreactivity
was present only in S100 beta positive astrocytes and ependymal cells.
Some astrocytes possessed the aggregations with SMI immunoreactivity
that was co-labeled by small heat shock protein, alpha B crystalline and
HSP25. Contrary to the case of human, transgenic mice showed no abnormality of myelin formation and structure. To examine whether the
presence of human GFAP aggregation altered brain functions, we challenged kainic acid administration to transgenic and wild type mice and
studied the susceptibility of convulsion and the vulnerability of hippocampal cell damage. Transgenic mice were more susceptible to systemic
administration of kainic acid (20 mg/kg) than wild type mice as revealed
by the number of mice showing tonic-clonic convultion: 8 out of 11 for
transgenic mice and 2 out of 14 for wild type mice. Transgenic mice
were more vulnerable to excitotoxicity of kainic acid as revealed by the
appearance of Fluoro Jade positive dying hippocampal neurons after 24
hours administration. We concluded that Alexander disease model mice
compromised the protective function against kainic acid excitotoxicity.
Med., Maebashi, Japan)
Glial glutamate transporters, GLAST and GLT-1, are co-localized in processes of Bergmann glia (BG) wrapping excitatory synapses on Purkinje
cells (PCs). Although GLAST is expressed 6-fold more abundantly than
GLT-1, no change is detected in the kinetics of climbing fiber (CF)-mediated excitatory postsynaptic currents (CF-EPSCs) in PCs in GLAST(/-) mice compared to the wild-type mice (WT). The prolongation of the
decay kinetics of CF-EPSCs in GLAST(-/-) mice is found only in the
presence of cyclothiazide (CTZ), which attenuates the desensitization of
AMPA receptors. We attempted to clarify the mechanism(s) underlying
this unexpected finding using a selective GLT-1 blocker, dihydrokainate
(DHK), and a novel antagonist of glial glutamate transporters, (2S,3S)3-[3-(4-methoxybenzoylamino)benzyloxy]aspartate (PMB-TBOA). In
the presence of CTZ, DHK prolonged the decay time constant (τw) of
CF-EPSCs in WT, indicating that GLT-1 plays a partial role in the
removal of glutamate. The application of 100 nM PMB-TBOA, which
inhibited CF-mediated transporter currents in BG by ∼80%, caused no
change in τw in WT in the absence of CTZ, whereas it prolonged τw in
the presence of CTZ. This prolonged value of τw was similar to that in
GLAST(-/-) mice in the presence of CTZ. These results indicate that
glial glutamate transporters can apparently retain the fast decay kinetics
of CF-EPSCs if a small proportion (∼20%) of functional transporters is
preserved, and that GLT-1 alone in GLAST(-/-) mice is sufficient to
keep the fast kinetics of EPSCs in the absence of CTZ.
Ikenaka, kazuhiro1,2; Tanaka, Kenji1,2 (1Nat'l Inst. Physiol.
Sci.,Okazaki, Japan; 2SOKENDAI, Hayama, Japan)
The role of neural-glial communication in
dynamic remodeling of the extracellular
Matsui, Ko; Jahr, Craig (Vollum Institute, Oregon Health and
Science University, Portland, Oregon, USA)
Neural-glial communication has been assumed to be mediated by spillover of transmitter from the synaptic cleft. In the cerebellum, Bergmann
glia cell (BG) processes encase synapses between presynaptic climbing
fiber (CF) and parallel fiber elements and postsynaptic Purkinje cell
(PC) spines and glutamate released from these fibers can activate Ca2+permeable AMPA receptors on BGs. Quantal responses recorded from
BGs were not coincident with quantal responses recorded in adjacent
PCs sharing the same CF input. By combining electrophysiological recordings and quantitative immunogold electron microscopic analysis,
high-concentration (1.5 mM) rapid-transients (0.5 ms) of glutamate were
estimated to underlie BG quantal events. We propose that exocytosis can
occur from ectopic release sites located directly across from BG membranes. Ectopic release may be necessary to activate low affinity AMPA
receptors on BGs, which may provide a geographical cue to guide BG
membrane to surround active synapses and ensure efficient glutamate
uptake. We have recently started to employ two-photon microscopy to
study the result of such neural-glial communication. Morphological refinement of BG processes occurs within a few days in early development
and rapid motility and spontaneous remodeling of extracellular space by
BG protrusions were observed in minutes. Synaptic activation leads to
Ca2+ influx at the tip of the protrusions via Ca2+-permeable AMPA receptors. We are currently probing the mechanisms that manipulate the
motility and refinement of BG processes and their effect on synaptic
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Current topics of stem cell biology—focusing on neural stem cells
Generation of Cerebellar Neuron
Precursors from Embryonic Stem Cells
Muguruma, Keiko; Su, Hong-Lin; Matsuo-Takasaki,
Mami; Watanabe, Kiichi; Sasai, Yoshiki (Neurogenesis and
Organogenesis, CDB, RIKEN)
Current topics of stem cell
biology—focusing on neural stem
We report in vitro generation of Math1+ cerebellar granule cell precursors and Purkinje cells from ES cells by using soluble patterning signals.
When neural progenitors induced from ES cells in a serum-free suspension culture are subsequently treated with BMP4 and Wnt3a, a significant proportion of these neural cells become Math1+. The induced
Math1+ cells are mitotically active and express markers characteristic of
granule cell precursors (Pax6, Zic1 and Zipro1). After purification by
FACS and co-culture with postnatal cerebellar neurons, ES cell-derived
Math1+ cells exhibit typical features of neurons of the external granule
cell layer, including extensive motility and a T-shaped morphology. Interestingly, differentiation of L7+/Calbindin-D28K+ neurons (characteristic of Purkinje cells) is induced under similar culture conditions but
exhibits a higher degree of enhancement by Fgf8 rather than by Wnt3a.
This is the first report of in vitro recapitulation of early differentiation of
cerebellar neurons by using the ES cell system.
Control of proliferation and differentiation
of neural progenitor cells in the cerebellum
Regulation of plasticity of neural cells by
methy-CpG binding proteins
Ishizaki, Yasuki (Dept. Mol. Cell. Neurobiol., Gunma Univ. Grad.
Sch. Med., Maebashi, Japan)
Kohyama, Jun; Namihira, Masakazu; Nakashima,
Kinichi (NAIST,Nara, Japan)
During CNS development, neural stem cells (NSCs) give rise first to various kinds of specified precursor cells, which proliferate extensively before terminally differentiating into either neurons or glial cells. It is still
not clear, however, whether the precursor cells are irreversibly determined to differentiate into their particular cell types. Neither is it clear
how proliferation of the precursor cells are terminated, although it is
widely accepted that control of the proliferation plays a crucial role in
determining the number of neurons or glial cells. We have been addressing these issues using the developing mouse cerebellum as a model system. We found that cerebellar granule cell precursors (GCPs) can
differentiate into astroglial cells when exposed to Shh and BMP. This indicates that GCPs are not irreversibly committed to neuronal development, but can be induced to differentiate into astroglial cells by
appropriate extracellular signals. We also examined the role of cyclindependent kinase inhibitors in the control of proliferation of GCPs.
Among the inhibitors we examined, only p27 was expressed at significant levels in cells of the granule cell lineage in the developing cerebellum. We found that there was an inverse correlation between BrdU
uptake and p27 expression by GCPs. Even in the presence of saturating
amounts of Shh, a potent mitogen, the cells eventually stopped dividing
and differentiated, expressing p27 strongly. These results suggest that
there is an intracellular mechanism that stops GCP division and causes
GCPs to differentiate and that p27 is part of this mechanism.
It has become apparent that epigenetic modification plays a critical role
in the regulation of lineage-specific gene expression. We have previously reported that the change in DNA methylation at the promoter of astrocytic genes, such as glial fibrillary acidic protein (GFAP), controls the
switch from neurogenesis to astrocytogenesis in the developing telencephalon. The methylated promoter at midgestation undergoes demethylation as gestation proceed, corresponding to the onset of
astrocytogenesis. However, the exon1 of the gene remains hypermethylated even in the adult neural progenitors and in cells differentiated from
the progenitors, i.e. neurons, astrocytes and oligodendrocytes. The methyl-CpG binding proteins (MBDs) bind to methylated DNA and suppress the target gene expression. They are strongly expressed only in
neurons in the nervous system and the cells do not respond to astrocyteinducing signals to express GFAP. In contrast, by using Cre-recombinase
fate tracing, we show here that oligodendrocytes, in which MBDs are not
expressed, expressed GFAP upon stimulation with the astrocyte-inducing cytokines. Overexpression of MeCP2, one of the MBD family proteins, in oligodendrocytes inhibited the GFAP expression by the
cytokines, implicating MBDs as key molecules to restrict the transdifferentiation of neural cells. It is well known that astrocytes increase dramtically in number after insult to the nervous systems. Taking the above
results into consideration, oligodendrocytes could be a source of the
newly generated astrocytes in damaged nervous systems in vivo.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Current topics of stem cell biology—focusing on neural stem cells
Molecular mechanism of the reversion of
oligodendrocyte precursor cells.
Roles of the bHLH genes Hes1/Hes3/Hes5
in neural development
Kondo, Toru1,2 (1RIKEN CDB, Kobe, Japan; 2Univ. Cambridge,
Kageyama, Ryoichiro (Inst. Virus Res., Kyoto Univ., Kyoto,
Brain Repair Centre, Cambridge, United Kingdom)
There is increasing evidence that some kinds of glial cells in central nervous system (CNS) can behave as multipotent neural stem cells (NSCs)
and generate neurons, astrocytes, and oligodendrocytes in vivo and in
vitro. However it is still unknown how such glial cells acquire multipotentiality. Oligodendrocyte precursor cells, which exist in many area in
CNS, can also behave as multipotent NSC when the cells are exposed to
specific conditions. Recently we have shown that sox2, which is an essential transcription factor in NSCs, is reactivated in the OPC reversion.
We have also shown that in the reversion SWI/SNF chromatin remodeling complex is recruited to an enhancer in the sox2 promoter and lysine
4 and 9 of histone H3 in the enhancer are methylated and acethylated, respectively. We propose that the reversion of OPCs to NSCs depends on
extensive chromatin remodeling, which is in part mediated by SWI/SNF.
Neuroepithelial cells are first generated from the ectoderm, forming the
neural plate. These cells undergo symmetric cell divisions to produce
more neuroepithelial cells. After neural tube formation, they become radial glial cells, which undergo asymmetric cell divisions, forming one
radial glial cell and one neuron (or a neuronal precursor) from each cell
division. After production of neurons, radial glial cells finally give rise
to glial cells such as astrocytes. Thus, neural stem cells change their
characteristics of morphology and competency over time during development. We found that inactivation of the bHLH genes Hes1 and Hes5,
known Notch effectors, and additional inactivation of Hes3 extensively
accelerate cell differentiation and cause a wide range of defects in brain
formation. In Hes-deficient embryos, initially formed neuroepithelial
cells are not properly maintained, and radial glial cells are prematurely
differentiated into neurons and depleted without generation of late-born
cells. Furthermore, loss of radial glia disrupts the inner and outer barriers
of the neural tube, disorganizing the histogenesis. We also found that the
boundary structures such as the isthmus and the zona limitans intrathalamica are not maintained and that the boundary cells are differentiated
into neurons and lose the organizer activity. Thus, Hes genes are essential for generation of brain structures of appropriate size, shape and cell
arrangement by controlling the timing of neural stem cell differentiation
and by maintaining the boundaries with the organizer activity.
Fate regulation of mouse telencephalic
neural precursor cells
Gotoh, Yukiko (Inst. Mol. Cell. Biosci. Univ. of Tokyo, Tokyo,
Cortical neural precursor cells (NPCs) sequentially undergo expansion,
neurogenic and gliogenic phases during development, although the underlying mechanisms are poorly understood. We have recently shown
that Wnt signaling instructively induces neuronal differentiation of
NPCs. Importantly, Wnt signaling does so only in midgestation stage
(neurogenic phase) of NPCs but not in early embryonic stage (expansion
phase) or in perinatal stage (gliogenic phase) of NPCs. In early embryonic stage, Wnt signaling rather promotes proliferation of NPCs. Here I
will discuss possible mechanisms that might account for these stage-dependent responses. Likewise, STAT3-activating ligands induce astrocytic differentiation in late (gliogenic phase) but not in early (expansion and
neurogenic phases) NPCs. These stage-dependent responses of NPCs
might play a central role in determining the timing of differentiation and
the size of final population of each differentiated cell type.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Vascular endothelial cells and blood rheology
Stretch-induced Ca increase in endothelial
Naruse, Keiji (Okayama Univ. Grad. Sch. of Med., Dent. and
Pharmaceut. Sci., Okayama, Japan)
Vascular endothelial cells and blood
Human umbilical endothelial cells (HUVECs) show various responses
including morphological changes and protein expressions in response to
mechanical stretch. Our previous studies revealed that intracellular Ca
increase in response to mechanical stretch via Ca permeable stretch-activated (SA) channel activation is critical in HUVECs cultured on an
elastic PDMS (polydimethylsiloxane) membrane. Since recent reports
suggest that the transient receptor potential (TRP) channels may form
the SA channel, we investigated the involvement of TRPV2 in the
stretch-induced Ca increase in HUVECs. Human TRPV2 was isolate
from a HUVEC cDNA library. Heterologous expression of the human
TRPV2 in COS7 cells resulted in the stretch-induced Ca increase and injection of a TRPV2-specific siRNA in HUVECs abolished the stretchinduced Ca increase.These observations indicate that TRPV2 plays a
critical role in the stretch-induced Ca increase in HUVECs.
Purinoceptor-mediated blood flow sensing
mechanism in endothelial cells
Maintenace of the Blood fluidity
Ando, Joji; Yamamoto, Kimiko (Grad. Sch. Med. Univ. Tokyo,
KOJIMA, TETSUHITO (Nagoya University School of Health
Sciences, Nagoya, Japan)
Vascular endothelial cells (ECs) alter their morphology, function, and
gene expression in response to shear stress generated by blood flow.
However, the molecular mechanism of shear stress sensing by ECs has
not been clarified. We investigated the mechanism from the aspect of
calcium (Ca) signaling. Human pulmonary artery ECs (HPAECs) loaded
with the Ca indicator Indo-1/AM were exposed to laminar flow and
changes in intracellular Ca concentrations were monitored. A stepwise
increase in flow rate elicited a corresponding stepwise-increase in Ca
concentrations. Apyrase or EGTA completely abolished the flow-induced increase in Ca concentrations, indicating that ATP and influx of
extracellular Ca are essential for the Ca responses. Flow increased the release of ATP from HPAECs in a shear stress-dependent manner.
HPAECs predominantly express a subtype of ATP-operated cation channel P2X4, and antisense oligonucleotides targeted to P2X4 abolished the
flow-induced Ca influx. Pulmonary microvascular ECs cultured from
P2X4-deficient mice showed no flow-induced Ca influx and nitric oxide
production. Human embryonic kidney 293 cells became sensitive to
flow and show flow-induced Ca influx when transfected with P2X4 cDNA. Flow-induced dilation of skeletal muscle arterioles was markedly
suppressed in P2X4-deficient mice. P2X4-deficient mice had higher systolic blood pressure values than wild-type mice. Thus, ECs transduce the
signal of shear stress into Ca influx via P2X4, and that the purinoceptormediated blood flow-sensing plays important roles in endothelial NO
production and vascular tone control.
Hemostasis is a physiologic mechanism that maintains blood in a fluid
state within the circulation. The blood-coagulation cascade has the ability to transduce a small initiating stimulus into a large fibrin clot, which
is mediated by cellular components and soluble plasma proteins. The potentially explosive nature of this cascade is counterbalanced by natural
anticoagulant mechanisms. The maintenance of adequate blood flow and
the regulation of cell-surface activity control the local accumulation of
activated blood-clotting enzymes and complexes. Antithrombin is a
plasma protein that inhibits the blood serine proteases of the intrinsic and
common coagulation pathways. Heparin-like molecules, heparan sulfate
proteoglycans, are closely associated with endothelial cells and enhance
the action of circulating antithrombin. In this session, the endothelial
heparan sulfate and antithrombin system, which plays an important role
in the natural hemostatic balance to maintain the blood fluidity, will be
discussed through the data from the congenital deficient mouse-models,
i.e. KO mice.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Mechanisms of sensory processing under pathological pain conditions
Regulatory mechanism of fibrinolysis in
the vasculature by vascular endothelial
Urano, Tetsumei; Suzuki, Yuko; Ihara, Hayato; Mogami,
Hideo (Hamamatsu Univ. Sch. of Med., Hamamatsu)
Tissue plasminogen activator (t-PA), the primary PA in vasculature, is
synthesized and secreted from vascular endothelial cells (VECs). Besides other serine proteases involved in both coagulation and fibrinolysis, t-PA has unique characteristics of possessing physiological activity
as a single chain form and being secreted as an active form. In blood
there also exist plasminogen activator inhibitor type 1 (PAI-1), a member
of serine protease inhibitor superfamily (SERPINS), which inhibits the
activity of both single chain- and two chain- forms of t-PA by forming
an equimolar high molecular weight complex. We have reported that total fibrinolytic activity in plasma is regulated by the balance between
these two molecules, showing that increase in PAI-1 level under either
physiological or pathological conditions suppresses fibrinolytic activity,
whereas the enhanced t-PA secretion accelerates fibrinolysis. Recently,
we have studied the dynamics of t-PA secretion from its containing granules in VECs using total internal reflection fluorescence microscopy
(TIRFM). We obtained results suggesting that secreted t-PA by regulatory exocytosis stays on the membrane of VECs for certain period of time,
and expresses its specific activity on VECs. PAI-1 appeared to modify
the dynamics of t-PA secretion, and thus fibrinolytic activity on
VECs.Showing these resuls, we discuss how fibrinolytic activity is regulated by t-PA and PAI-1 both in plasma and on VECs. We also want to
discuss the physiological relevance of this regulatory mechanism which
is naturally modified by many physiological stimuli
Mechanisms of sensory processing
under pathological pain conditions
Mechanism of Platelet Thrombus
Formation under Blood Flow Condition.
BDNF derived from microglia involving
neuropathic pain
Goto, Shinya; Tamura, Noriko; Ishida, Hideyuki
Inoue, Kazuhide; Tsuda, Makoto (Grad. Sch.Pharma.Sci,
(Department of Medicine, Tokai University School of Medicine)
Kyushu Univ, Fukuoka, Japan)
Introduction. Atherothrombosis, including myocardial infarction and
ischemic stroke, is a leading cause of death in the modern world. Platelet,
forming thrombi at site of ruptured or disrupted atherosclerotic plaque,
play crucial role in the onset of atherothrombosis. Mechanism of platelet
thrombus formation under blood flow condition may not be the same as
platelet aggregation under static conditions. Method. Whole blood, containing platelets rendered fluorescent by addition of mepacrine or calcium sensitive dye of Fluo-3, was perfused on the immobilized collagen
fibrils at various shear rate conditions. Two-dimensional and three-dimensional growth of platelet thrombi on the collagen fibrils were detected by epi-fluorescent video-microscpy and ultra-fast laser confocal
miscroscope equipped with piezo-motor control unit, respectively, Real
time visualization of intra-cytoplasmic calcium ion concentration was
also achieved with the use of laser confocal microscopy. Results. Unlike
platelet aggregation under static condition, platelet thrombus growth on
the collagen fibrils under blood flow conditions was markedly inhibited
by blocking von Willebrand factor binding with glycoprotein Ibα, ADP
binding with P2Y12 ADP receptor, and so on. Our results also revealed
that cyclic increase in intracytoplsmic calcium ion concentration was
abolished when P2Y12 was blocked. Collagen and thrombin also play
important roles in the growth of platelet thrombi. Conclusion.
Mechanism of platelet thrombus formation under blood flow conditions
are different from that of platelet aggregation under static condition.
Microglia play an important role as immune cells in the central nervous
system. Recently, accumulating evidences indicate the important role of
ATP receptors of activated microglia in the neuropathic pain. Neuropathic pain is often a consequence of nerve injury through surgery, bone
compression, cancer, diabetes or infection. The expression of P2X4 receptor is enhanced in spinal microglia after peripheral nerve injury model, and blocking pharmacologically and suppressing molecularly P2X4
receptors produce a reduction of the neuropathic pain (Tsuda et al. Nature 424, 778-783, 2003). Several cytokines such as interleukin-6 and tumor necrosis factor in the dorsal horn are also increased after nerve
lesion and have been implicated in contributing to nerve-injury pain.
ATP can activate MAPK leading to the release of bioactive substances
including cytokines from microglia (Shigemoto-Mogami et al., J Neurochem 78, 1339-1349, 2001; Suzuki et al., J Neurosci 24, 1-7, 2004).
Thus, diffusible factors released from activated microglia by the stimulation of purinergic receptors may have an important role in the development of neuropathic pain (Tsuda, M., Inoue, K., & Salter, M.W. Trend
Neurosci 28, 101-107, 2005). I will discuss the mechanism of P2X4evoked allodynia with an effect of a neurotrophic factor from activated
microglia based on the latest our findings (Nature, in press).
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Mechanisms of sensory processing under pathological pain conditions
Role of BDNF in inflammatiory
hyperalgesia and sprouting
Yoshimura, Megumu; Matayoshi, Satoru (Grad. Sch. Med.
Sci. Kyushu Univ. Fukuoka, Japan)
Brain-derived neurotrophic factor (BDNF) is known to involved in the
development of spinal plasticity underlying inflammation-induced hyperalgesia. An injection of complete Freund adjuvant (CFA) into rat plantar
surface produced hyperalgesia, which was significantly attenuated by intraperitoneal administration of anti-BDNF antiserum performed a day
before and just after CFA. In vivo patch-clamp recordings from the spinal substantia gelatinosa (SG) neurons of the inflamed rats demonstrated
a marked enhancement of excitatory synaptic responses to noxious and
non-noxious stimuli, suggesting an increase in the activity-dependent
synthesis and release of BDNF in the SG. In the spinal slice preparations,
BDNF, but not nerve growth factor (NGF) or neurotrophin-3 (NT-3), acted presynaptically to increase frequency of miniature EPSCs in SG neurons of the inflamed, but not naive rats, through an activation of
lidocaine-sensitive, TTX-resistant sodium channels. This effect was observed in slices of the inflamed rat only 2-4 days after CFA injection. On
the other hand, the number of monosynaptic A-beta afferent inputs to the
SG significantly increased a week after the onset of the inflammation,
and this increase was significantly suppressed by treatment with antiBDNF antiserum. These findings, taken together, suggest that BDNF,
which is considered to be released from the sensitized primary afferents,
increases the excitability of SG neurons through its action on the presynaptic terminals, and may thereafter trigger plastic changes in the spinal
sensory transmission to develop hyperalgesia/allodynia during inflammation.
Changes in brain dynamics by chronic
pain: Molecular mechanisms of the
suppression of morphine-induced
rewarding effects and the aggravated
Narita, Minoru; Niikura, Keiichi; Kuzumaki, Naoko;
Suzuki, Tsutomu (Dept. Toxicol., Hoshi Univ. Sch. Pharm.
Pharmaceut. Sci., Tokyo, Japan)
It has been widely recognized that chronic pain could cause physiological changes at supraspinal levels. Here, we found that chronic pain
caused a dramatic down-regulation of µ-opioid receptor function to
activate its coupling with G-proteins of ventral tegmental area (VTA),
and produced of the suppression of morphine-induced rewarding effect.
Using the fluoro-gold (FG) microinjection into the VTA, numerous FGlabeled cells were detected in the lateral preoptic nucleus (LPO) and
dorsolateral hypothalamus (DMH) of nerve-ligated rats. Subpopulations
of β-endorphin-positive fibers in the LPO and DMH were co-labeled by
FG. Furthermore, we found that chronic pain caused a dramatic downregulation of cortical δ-opioid receptor function to activate its coupling
with G-proteins, which is associated with the increased δ-opioid receptor
phospholylation, and produced anxiety-like behaviors in mice, as
characterized by both the light-dark and elevated plus-maze tests. These
data provide direct evidence that the endogenous opioid-containing
neuron projecting from the pain processing regions may be continuously
activated by nerve ligation, resulting in the long-lasting down-regulation
of µ- or δ-opioid receptors. This phenomenon may lead to the
suppression of the morphine-induced rewarding effect and emotional
disorders including aggravated anxiety under chronic pain-like state.
Molecular mechanisms for neuropathic
pain - lysophosphatidic acid as the initiator
Ueda, Hiroshi (Nagasaki University Graduate School of
Biomedical Sciences,Nagasaki,Japan)
Here I report the initiation mechanisms underlying neuropathic pain
(NP) following nerve damages. In this experiment, we used partial sciatic nerve ligation (PSNL) to induce NP in mice. The abnormal chronic
pain including hyperalgesia and tactile allodynia was observed 3 days after the PSNL and lasted for more than 2 weeks. We found many nociceptive fiber-specific changes in protein and/or gene expression of
molecules involved in pain transmission. They include the novel expression of Ca channel alpha2-delta1 subunit in myelinated fiber DRG neurons, which may account for hyperalgesia. At the same time, we found
the loss of function in unmyelinated fiber-mediated pain transmission.
They include the down-regulation of substance P in dorsal horn of spinal
cord. In addition to these phenotypic changes in the expression, we observed marked demyelination and remyelination in dorsal root, a phenomenon which is closely related to allodynia. All these changes were
abolished in lysophosphatidic acid (LPA) receptor (LPA1) knock-out
mice, and mimicked by the intrathecal single injection of LPA. In ex vivo
culture system of dorsal root, the addition of LPA caused demyelination
and sprouting/remyelination, which were accompanied with down-regulation of myelin basic protein expression. We also found that LPA causes
microglia activation in the spinal cord. I will discuss how LPA plays the
initiation role in many molecular events observed in PSNL-induced NP.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Behavioral analysis of genetically modified mice in the postgenomic era [Workshop Organized by Program Organizing
Comprehensive behavioral test battery for
genetically engineered mice: A proposal to
use it as a "hub" for large-scale
Miyakawa, Tsuyoshi (Grad. Sch. Med. Univ. Kyoto, Kyoto,
Behavioral analysis of genetically
modified mice in the postgenomic
era [Workshop Organized by
Program Organizing Committee]
Identification of the functions of the genes at system level is one of the
greatest challenges in the post genome-sequence era. To reveal functional significance of the genes expressed in the brain, we have been utilizing
a comprehensive behavioral test battery on various kinds of geneticallyengineered mice. The behavioral test battery covers a broad range of various behavioral domains, such as learning and memory, sensory-motor
functions, emotions and motivation. During the past two years, we have
assessed behaviors of more than 30 different strains of mutant mice and,
surprisingly, we found at least one significant behavioral abnormality in
more than 90% of those strains. This fact not only demonstrates that our
strategy is useful in elucidating the functions of the genes expressed in
the brain but also implies that a large part of the genes expressed in the
brain may have some functions. In this workshop, I'd like to introduce
some examples of our work using the test battery on mutant mice and to
make a proposal to use it as a "hub" for large-scale neuroscience. Establishing a collaborative network among experts in the field would be necessary for greater success of this approach. The potential issues related
to implementing such collaborative network or consortium will be also
Behavioral analyses of genetically
manipulated mice for synaptic plasticityrelated genes
Kojima, Nobuhiko; Shirao, Tomoaki (Dept. of Neurobiol. and
Behav., Gunma Univ. Grad. Sch. of Med., Maebashi, Japan)
To understand molecular mechanisms of learning and memory is one of
major topics in neuroscience research. To identify genetic components
underlying synaptic plasticity and memory processes, I have been so far
focusing on analyzing behavioral phenotypes, especially learned fear, of
several strains of genetically manipulated mice for plasticity-related
genes. One example is Fyn-overexpressing mice exhibiting hyper-tyrosine-phosphorylation of the NMDA receptor (NR). These mice
showed NR activity-dependent impairment of fear conditioning, suggesting that this tyrosine kinase is a key molecule that controls conditioned fear through NR phosphorylation. Another is knockout (KO)
mice for drebrin A, a F-actin binding protein in the dendritic spines.
Context-dependent fear conditioning and MK-801-induced hyperlocomotion were changed in these KO mice. These results suggest that drebrin A has a pivotal role in the regulatory mechanism of NR function.
The mutant mice for ICER, a CRE-binding transcriptional repressor,
also showed the phenotype on fear conditioning. In ICER-overexpressing mice, long-term retention of fear memory was impaired, while the
short-term memory remained intact. ICER-KO mice conversely showed
a better performance in the retention of conditioned fear. These results
suggest that ICER acts as a negative regulator for memory consolidation.
Thus, these three examples demonstrate that the learned fear is a good
target of behavioral analysis of mutant mice for genes being critical for
synaptic plasticity and it should be included in the behavioral test battery.
Studies of emotional and socio-sexual
behaviors in genetically modified mice
Ogawa, Sonoko (Lab. Behav. Neuroendo., Dept. Kansei Behav.
Brain Sci., Grad. Sch. Comprehen. Human Sci., Univ. Tsukuba,
Tsukuba, Japan)
We have been studying neuroendocrinological bases of emotional and
socio-sexual behaviors. Our studies using two types of estrogen receptor
knockout mice during the last ten years have proven that the knockout
mouse model is a powerful tool to delineate the relationship between
genes, hormones, and behaviors. Since our main research interest, as behavioral scientists, is to understand brain mechanisms of emotional and
socio-sexual behaviors, we have been focusing on developing test paradigms that are able to measure the behaviors of interest most appropriately and reliably. On the other hand, progress in molecular biology has
enabled production of enormous numbers of genetically modified animals. As a result, interest and demand for analyses of behaviors as one
of the phenotypes of these animals, have rapidly grown for molecular biologists. In this talk, I will first overview our findings in behavioral characteristics of estrogen receptor knockout mice and then discuss the
problems in standardization of behavioral testing paradigms. Particularly, I will talk about differences between "how to measure" and "what is
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Behavioral analysis of genetically modified mice in the postgenomic era [Workshop Organized by Program Organizing
Mouse behavior analysis support service
at RIKEN Brain Science Institute (BSI)
Transponder-based approaches to
behavioral phenotyping in mice
Yamada, Kazuyuki (ATDG/RRC. BSI. RIKEN, Saitama, Japan)
Lipp, Hans-Peter (Institute of Anatomy, University of Zurich,
Research Resources Center (RRC) in RIKEN Brain Science Institute
(BSI) provides the mouse behavior analysis support service. As a base of
experimentation, RRC provides the common laboratories for mouse behavior analysis in the laboratory animal facility in BSI. This laboratory
area consists of 12 separated rooms, and one or several kinds of devices
for the behavioral experiment are set up in each room. BSI researchers
can use these rooms and devices freely following a guidance. The mouse
behavior analysis support service section offers user guidance on use,
maintains, develops and updates the laboratories and the equipped experimental apparatuses. Another important part of this service is consultation. We help to plan for behavior experiments, paradigms and/or
methods, data analysis and so on. Furthermore, a primary behavioral
screening of mutant mice based on a "behavioral test battery" is currently
provided on a trial basis. In this workshop, the advantage and disadvantage of the mouse behavior analysis support service at BSI will be discussed from the point of view of standardization of the mouse behavior
analysis at a large-scale research facility like BSI.
Zurich, Swiss Confederation)
Extensive behavioral testing of mice including common laboratory
strains and genetically modified lines has revealed that many measures
of hippocampus-dependent memory and learning are confounded by reactions of mice towards handling and test conditions. Moreover, very little is known about cognitive activity of mice in their natural
environment. Therefore, we have developed transponder-based techniques permitting to assess learning and cognition in social settings, first
in outdoor pens and later in large cage units called "Intellicage". Data
presented will show how hippocampal malfunction is manifested in the
water maze, under outdoor conditions and in Intellicages, and how transponder-based technology can be used for fully automated assessment of
memory and learning in large number of mice.The main conclusions for
high-throughput phenotyping are:- Mutation, strain and hippocampal lesion effects can easily be discovered within a social group - testing single
mice in isolation is methodologically not necessary.- Automated testing
is 10 to 40 times more economic than manual testing.- Comparability
across labs is improved and standardization is much facilitated.- Transponder-based automated systems can be used for both, screening by
laypersons and sophisticated analysis by behavioral experts.Since all
testing is done without human interference by computers, different
groups can easily share data over the web. This will enable researchers
to perform comparative analysis of animals having undergone identical
test protocols in different locations. Supported by Swiss National Science Foundation and NCCR "Neural Plasticity and Repair".
G-substrate, a putative downstream
component of NO-cGMP-PKG cascade,
plays a important role in cerebellardependent long-term memory
Endo, Shogo (Initial ResearchProject, Okinawa Institute of
Science and Technology, Okinawa, Japan)
Cerebellum plays an important role in non-declarative memories such as
motor memories. Cellular substrate for the cerebellar-dependent memory is long-term depression (LTD). NO-cGMP-PKG (cGMP-dependent
protein kinase) pathway is shown to be involved in the induction of cerebellar LTD. NO absorbing reagent prevents cerebellar-dependent learning such as the adaptation of perturbed locomotion and VOR adaptation.
Furthermore, each component of NO-soluble guanylate cyclase-cGMPPKG pathway has been shown to be essential for the induction of LTD.
However, the downstream component of PKG was not identified.
Recently we molecularly cloned and characterized G-substrate,
localized specifically in cerebellar Purkinje cells, as a downstream
component of PKG. Further, we have generated a mice lacking Gsubstrate gene. Homozygous mice are vital as expected from the
restricted localization of G-substrate. The behavioral analyses were
conducted on the G-substrate gene knockout mice. Significant
difference was not observed between the control and G-substrate
knockout mice in general behaviors. However, a specific impairment
was observed in long-term horizontal optokinetic response (HOKR), a
cerebellar-dependent memory, without any impairment in short-term
HOKR. In addition, we observed G-substrate, with shuttles between
nuclear and cytosol of Purkinje cells. G-substrate may have a role in
transcription and translation in the nuclear that is essential for the longterm memory.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Basic approach for therapy of failing heart models
Molecular cloning of cardiac troponin I
Suzuki, Hideaki; Takeda, Nobuakira (Jikei Univ. Sch. Med.,
Tokyo, Japan)
Basic approach for therapy of
failing heart models
Background and Objectives: Laminin, which is a major component of
the extracellular matrix, is known to increase in the myocytes in the diabetic heart and dilated cardiomyopathy. The laminin gamma 1 chain
promoter contains a transcriptional element denoted bcn-1 that is inducible and active (Suzuki H, et al. J Biol Chem 271:1996). To elucidate the
molecular pathogenesis of the increase of laminin in the cardiac muscle
cells of cardiomyopathy, we have carried out the yeast one-hybrid screen
using bcn-1 as a bait, and cloned Smarce 1r protein as a molecular partner which interacts with bcn-1 protein. Next, we repeated two-hybrid
screen using Smarce 1r as a bait and cloned MLF11P as a partner (Suzuki
H, Exp Clin Cardiol 9:2004).Methods: The yeast two-hybrid screen
analysis was carried out again using MLF11P protein (amino-acids 1318) as a bait. A human heart cDNA library was screened by the yeast
mating method for overnight culture.Results: We isolated two final positive clones encoded the same protein, which is an alternative-RNA
splicing form of the human cardiac troponin I (TnI) protein and we call
this as a spliced form of TNI (STNI). The mRNA expression pattern of
STNI is heart-specific. The STNI shares several sequence similarities
with the human cardiac TNI but lacks troponin T binding protein.Conclusions: We report the heart-specific segment of the human cardiac
troponin I isoform which lacks troponin C binding portion. These results
suggest that STNI might be involved in the molecular pathogenesis of
the increase of laminin in the cardiomyopathy.
Translocation and cleavage of myocardial
dystrophin as a common pathway to
advanced heart failure: a scheme for the
progression of cardiac dysfunction and the
novel treatment
A mouse knock-in model of dilated
cardiomyopathy associated with δK210
mutation in cardiac troponin T and its
potential pharmacotherapy
Toyo-oka, T.1; Kumagai, H.1; Kawada, T.2; Nakazawa,
M.3; Takeo, S.4; Ozawa, K.5 (1TUBERO, Sendai, Japan;
Univ.; 3Niigata Univ.; 4Tokyo Pharmac.Univ.; 5Jichi
The progression mechanism from cardiac dysfunction to advanced heart
failure (HF) including dilated cardiomyopathy (DCM) should be clarified to establish a novel treatment. We have identified a gene mutation
in δ-sarcoglycan (SG) that causes hereditary DCM in both animals
(Sakamoto et al., PNAS, 1997) and humans (Tsubata et al., JCI, 2000).
TO-2 hamsters with hereditary DCM show age-dependent cleavage and
translocation of myocardial dystrophin (Dys) from sarcolemma (SL) to
myoplasm, enhanced SL permeability in situ, and a close relation
between Dys loss and hemodynamics. Dys disruption is not an
epiphenomenon but directly causes advanced HF, because in vivo
transfer of the missing gene to degrading cardiomyocytes ameliorated all
of the pathological features and improved the disease prognosis
(Kawada et al., PNAS, 2002). Furthermore, acute HF after isoproterenol
toxicity and chronic HF after coronary ligation in rats both timedependently cause Dys disruption in the degrading myocardium
(Takahashi et al., CVRes., 2005). Dys cleavage was also detected in
human hearts from patients with DCM of unidentified etiology,
supporting a scheme of vicious cycle consisting of SL instability, Dys
cleavage, and translocation of Dys from the SL to the myoplasm (Toyooka et al., PNAS, 2004), irrespective of an acute or chronic process and
a hereditary or acquired origin. Activation of endogenous calpain will be
discussed for the Dys disruption.
Morimoto, Sachio (Kyushu Univ. Grad. Sch. Med., Fukuoka,
Dilated cardiomyopathy (DCM) is characterized by cardiac dilation and
systolic dysfunction, which often leads to severe heart failure and sudden
death. However, little is known about the pathogenic mechanism for
DCM, and no therapeutical method is established at present except for
cardiac transplantation. We created a knock-in mouse model of DCM
caused by a deletion mutation δK210 in cardiac troponin T and explored
its molecular pathogenic process and potential pharmacotherapy. Mutant
mice developed enlarged hearts with bi-ventricular dilation and systolic
dysfunction and suffered sudden death frequently, recapitulating human
DCM with this mutation. Skinned cardiac muscle fibers from mutant
mice showed a decreased Ca2+ sensitivity of force generation,
confirming our previous hypothesis that decreased contractility of
cardiac muscle is a primary pathogenic mechanism of this mutation.
Surprisingly, however, intact cardiac muscle fibers from mutant mice
showed no significant reduction in isometric force per cross-sectional
area. Analyses of Fura-2 loaded cardiomyocytes revealed that this was
due to an increase in the amplitude of intracellular Ca2+ transient.
Biochemical analyses, including DNA microarray, strongly suggested
that Ca2+ transient was increased through down-regulation of a specific
isoform of phosphodiesterase (PDE4B) and associated increase in
cAMP in cardiomyocytes of mutant mice, which could compensate for
the decreased myofilament Ca2+ sensitivity but at the same time would
increase the risk for arrhythmia leading to sudden death due to a Ca2+
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Pacemaker mechanism of the rhythmic activity of cell and tissue
Gene therapy by SERCA2a for failing
hearts in spontaneous type II DM model
Neuronal mechanisms of respiratory
rhythm generation
Sakata, Susumu1; Nakajima-Takenaka, Chikako1;
Hajjar, Roger2; Takaki, Miyako1 (1Nara Med. Univ.,
School of Med., Tokyo, Japan)
Kashihara, Japan; 2Mass. General Hosp., Harvard Med. Sch.,
Boston, USA)
OLETF rat is a model of spontaneous non-insulin-dependent diabetes
mellitus (DM), accompanying diastolic dysfunction associated with abnormal Ca2+ handling and decrease in sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) expression. The aim of this study was to examine
whether SERCA2a gene transfer can restore left ventricular (LV) function in this DM in terms of LV mechanical work and energetics. DM rats
were randomized to receive adenovirus carrying SERCA 2a gene
(DM+Serca) or β-galactosidase (DM+Gal), or saline (DM+Sa) by
catheter-based (cross-clamping) technique. LV mechanoenergetics was
assessed in cross-circulated whole heart preparations 3 days after the
infection. In DM, end-systolic pressure (ESP0.1) was low and enddiastolic pressure (EDP0.1) was high at 0.1 ml intraballoon water. In
DM+Serca, however, ESP0.1 increased and EDP0.1 decreased. LV
relaxation rate in DM+Serca was faster than that of DM+Gal/Sa groups.
Oxygen cost of LV contractility in DM was higher than that of normal,
indicating energy wasting in Ca2+ handling during E-C coupling. The O2
cost of LV contractility decreased to non-DM rat level in DM+Serca,
although it remained high in DM+Gal and DM+Sa. These results
indicate that SERCA2a overexpression by SERCA2a gene transfer
improves not only LV mechanics but also energetics in DM rat hearts.
Onimaru, Hiroshi; Homma, Ikuo (Dept. Physiol., Showa Univ.
Is respiratory rhythm generated by pace-maker neurons or by network
mechanisms? Although this longtime and important question has not
been solved yet, recent studies provide evidence supporting pace-maker
theory at least under some experimental conditions and clarified characteristics of respiratory pace-maker neurons. In respiratory rhythm generation, a term of pace-maker neurons indicates neurons that possess
cellular property intrinsically producing rhythmic burst (but not tonic)
activity. In pace-maker theory, it is hypothesized that intrinsic burst generating neurons are embedded in a network composed of neuronal population with same active phase and contribute to produce basic
synchronized rhythm, whereas inhibitory synaptic connections are not
necessary for the rhythm generation. At least two respiration-related
rhythm generators have been suggested to exist in the medulla and to
produce intrinsic periodic burst activity. One is an inspiratory (Insp) neuronal network (i.e., Insp rhythm generator), which is localized predominantly in the pre-Boetzinger complex of the ventrolateral medulla.
Another is a pre-inspiratory (Pre-I) neuronal network (i.e., Pre-I rhythm
generator), which generates activity prior to Insp bursts and is located in
the more rostral ventrolateral medulla including the para-facial respiratory group (pFRG). We have shown that pFRG-Pre-I neurons serve as a
primary rhythm generator, triggering inspiratory burst periodically in the
brainstem-spinal cord preparation from newborn rat. In this presentation,
we focus on mainly burst generating properties of Pre-I neurons and the
ionic and synaptic mechanisms.
Role of interstitial cells in generating
spontaneous activity of smooth muscles
Hashitani, Hikaru; Kito, Yoshihiko; Suzuki, Hikaru
(Dept.Regulatory Cell Physiol., Grad.Sch.Med.Sci., Nagoya City
Univ., Nagoya, Japan)
Pacemaker mechanism of the
rhythmic activity of cell and tissue
Many smooth muscles exhibit spontaneous electrical and mechanical activity. This has been considered to "myogenic" activity for many years,
however, it is in fact generated by specialized cells, namely interstitial
cells of Cajal (ICC). In the gastrointestinal tract, a network of myenteric
ICC (ICC-MY) generates pacemaker potentials to initiate spontaneous
electrical activity. Intramuscular ICC (ICC-IM) augment the depolarizations by generating ongoing unitary potentials (UPs). However, corporal
ICC-IM may be dominant pacemaker as they create a greatest frequency
in spontaneous activity. ICC-like cells have been found in other smooth
muscle organs and may play a similar role. In the urethra, ICC-like cells
generate spontaneous transient depolarizations (STDs), which sum to
activate L-type Ca channels. UPs and STDs result from the opening of
Ca-activated Cl– channels. UPs solely depend on inositol 1, 4, 5-trisphosphate (InsP3)-dependent Ca release, whilst the generation of STDs requires both InsP3- and ryanodine-receptors. In corpus cavernosum (CC),
where smooth muscle cells are capable of generating spontaneous depolarizations, ICC-like cells express cyclooxygenase 2 and spontaneously
produce prostaglandins to reinforce spontaneous activity. Therefore, although ICC or ICC-like cells in different regions or tissues share many
similarities, they also have a significant diversity. Interaction between
ICC and smooth muscle cells result in a further heterogeneity of spontaneous activity, and thus develops characteristics of individual smooth
muscle organs.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Regulation of feeding and metabolism: molecular and physiological mechanisms
In silico analysis of the cardiac
Sarai, Nobuaki1; Matsuoka, Satoshi2; Noma, Akinori2
(1Nano-Medicine Merger Edu. Unit, Grad. Sch. Med., Kyoto Univ.,
Kyoto, Japan; 2Physiol. and Biophys., Grad. Sch. Med., Kyoto Univ.,
Kyoto, Japan)
Computers have become the necessities of life science with improvements of information technologies. Thus, Systems Biology, whose aim
is to describe the whole biological interactions as a mathematical model
and understand the whole mechanisms by simulation, has been emerged.
We have started developing a comprehensive cardiac model (Kyoto
model), and a cardiac sinoatrial node pacemaker model is developed
with the same set of equations as the ventricular cell model. The model
successfully reconstructs the experimental action potentials at various
concentrations of external Ca2+ and K+, and reaches steady-state and the
sensitivity analyses can be applied to a variety set of parameters. Increasing the amplitude of L-type Ca2+ current (ICaL) prolongs the duration of
the action potential and thereby slightly decreases the spontaneous rate.
On the other hand, a negative voltage shift of ICaL gating by a few mV
markedly increases the spontaneous rate. When the amplitude of sustained inward current is increased, the spontaneous rate is increased irrespective of the ICaL amplitude. When the spontaneous activity is
stopped by decreasing ICaL amplitude, the resting potential is around -35
mV over 1-15 mM [K+]o because of the presence of the background nonselective cation current. The unique role of individual voltage- and timedependent ion channels is clearly demonstrated and distinguished from
that of the background current by calculating an instantaneous equilibrium potential during the course of the spontaneous activity.
Regulation of feeding and
metabolism: molecular and
physiological mechanisms
Circadian rhythms are controlled by Argvasopressin in the suprachiasmatic
Control of appetite and metabolism by
olfactory stimulation: Involvements of
histaminergic nerve and biological clock
Isobe, Yoshiaki; Nishino, Hitoo (Grad. Sch. Med. Nagoya City
Nagai, Katsuya (Institute for Protein Research. Osaka
University. Suita, Osaka, Japan)
Univ. Nagoya, Japan)
Many physiological functions show circadian rhythms controlled by suprachiasmatic nucleus (SCN) in mammals. From the SCN, ca. 24 hr
rhythmicity accompanying the time of day information (phase) is conveyed to the other area of the brain and peripheral organs. Arg-vasopressin (AVP) containing neuron is one of the output neuron from the
SCN. VIP and glutamate increased AVP release from the SCN, meanwhile suppressed by GABA (via GABAa receptor) and melatonin (MT2
receptor). The rPer2 mRNA products (CCG protein) promote the
rBmal1 mRNA increase, which concerns the AVP mRNA transcription.
In the SCN, before and after the 8-hr advance of the LD cycle, AVP
mRNA and AVP peptide in the SCN remain coupled with time. After the
light pulse (800 lux, 15 min at ZT 22), AVP mRNA increased in the
SCN, but not in the paraventricular nucleus (PVN). Following the melatonin injection (1 mg/kg, i.p.), AVP content in the SCN decreased at both
light and dark period, while no changes were observed in the PVN. In the
SCN, rPer2 mRNA increased at both light and dark period. In the PVN,
rPer2 decreased and increased at light and dark period, respectively.
While in the pineal gland, rPer2 mRNA increased at 180 min after the
melatonin injection. The melatonin-AVP reciprocal system indicates that
PVN is temporally coupled with the SCN clock activity. The AVP and
melatonin on the locomotor activity and body temperature rhythms are
interesting theme. Body temperature decrease caused by the melatonin
application (i.p.) during the night is considered to be a decrease of AVP
in the SCN. Functional significance of AVP in the SCN would be discussed.
Recently, we observed that olfactory stimulation with scent of grapefruit
essential oil caused excitations of sympathetic nerves innervating the
epididymal (white) adipose tissure, interscapular (brown) adipose tissue
and adrenal gland and inhibition of a parasympathetic nerve innervating
the stomach. In contrast, olfactory stimulation with scent of lavender essential oil inhibited sympathetic nerves innervating the epididymal adipose tissue, interscapular adipose tissue and adrenal gland and excitated
a parasympathetic nerve innervating the stomach. These findings suggest that the scent stimulation with grapefruit oil elevates lipolysis, thermogenesis, adrenaline secretion, thus, the blood glucose and the blood
pressure, and suppression of food intake, and that the scent stimulation
with lavender oil induced opposite responses. In the experiments examined this, we found evidences suggesting that these are the cases. Moreover, we observed that bilateral electrolytic lesions of the hypothalamic
suprachiasmatic nucleus (SCN), a master circadian clock, and histaminergic blockers eliminated these responses. That is, a histamine H3-antagonist, thioperamide, abolished the effects of lavender oil and a histamine
H1-receptor-antagonist, diphenhydramine, suppressed the effects of
grapefruit oil. Anosmic treatment with zinc sulfate eliminated all of the
changes due to olfactory stimulations. These findings suggest that olfactory stimulations with scents of grapefruit and lavender oils affect appetite and metabolism via the functions of the SCN and histaminergic
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Regulation of feeding and metabolism: molecular and physiological mechanisms
Regulation of glucose and energy
metabolism by PACAP
Nakata, Masanori; Yada, Toshihiko (Dept. of Physiology,
Division of Integrative Physiology, Jichi Medical University,
Kawachi, Tochigi, Japan)
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a ubiquitous neuropeptide in the central and peripheral nervous systems. Previously we reported that PACAP potentiates both insulin release from
pancreatic β-cells and insulin action in adipocytes, contributing to
reduction of blood glucose and energy storage. PACAP38 is localized in
pancreatic islets and serves as an endogenous amplifier of glucoseinduced insulin secretion via VPAC2 and PAC1 subtypes of PACAP
receptors. In an adipocyte cell-line, 3T3-L1 cells, PACAP enhances
insulin-stimulated glucose uptake via PAC1 receptors and promotes
adipocyte differentiation. In contrast, PACAP stimulates secretion of
glucagon and catecholamine and glucose output from the liver, causing
elevation of blood glucose. Thus, PACAP regulates the glucose and
energy metabolism at multiple processes in several tissues. In this
symposium, we present three novel effects of PACAP in the metabolism.
(1) The action of PACAP to protect islet cells against lipotoxicity and
glucotoxicity. (2) PACAP knock out mice exhibits decreased fat mass
and increased insulin sensitivity, suggesting a role of PACAP to
facilitate adiposity and decrease insulin sensitivity. (3) PACAP
promotes feeding behavior by activating neuropeptide Y neurons in the
hypothalamic arcuate nucleus, a feeding center.Based on these well
known and newly identified metabolic effects of PACAP, we discuss a
possible therapeutic use of PACAP receptor subtype-specific agonists
and/or antagonists in the treatment of metabolic syndrome.
Adipocytokines and metabolic regulation
Ogawa, Yoshihiro (Dept. Mol. Med. Metab., Med. Res. Inst.,
Tokyo Med. Dent. Univ., Tokyo, Japan)
Weight gain is associated with infiltration of fat by macrophages, suggesting they are an important source of inflammation in obese adipose
tissue. We have recently developed an in vitro co-culture system composed of adipocytes and macrophages and examined the molecular
mechanism whereby these cells communicate. Co-culture of differentiated 3T3-L1 adipocytes and macrophage cell line RAW264 results in
marked up-regulation of pro-inflammatory cytokines such as MCP-1
and TNF-α and down-regulation of anti-inflammatory cytokine
adiponectin. Such inflammatory changes are induced by the co-culture
without direct contact, suggesting the role of soluble factors. A
neutralizing antibody to TNF-α, which occurs mostly in macrophages,
inhibits the inflammatory changes in 3T3-L1, suggesting that TNF-α is
a major macrophage-derived mediator of inflammation in adipocytes.
Conversely, FFAs may be an important adipocyte-derived mediator of
inflammation in macrophages because the production of TNF-α in
RAW264 is markedly increased by palmitate, a major FFA released
from 3T3-L1. The inflammatory changes in the co-culture are
augmented by use of either hypertrophied 3T3-L1 or adipose stromal
vascular fraction obtained from obese ob/ob mice.
We postulate that a paracrine loop involving FFAs and TNF-α between
adipocytes and macrophages establishes a vicious cycle that aggravates
inflammatory changes in the adipose tissue. This study suggests the
pathophysiologic implication of the intimate crosstalk between
adipocytes and macrophages in the development of inflammatory
changes in obese adipose tissue and thus the metabolic syndrome.
The Brain-Adipose axis and
Obesity:Identification of nesfatin that
causes anorexia
Shimizu, Hiroyuki; Mori, Masatomo (Department of Medicine
and Molecular Science,Gunma Univ. ,Grad.Sch.Med,
Many molecules compose the brain-adipose axis that controls appetite.
Using a subtraction cloning assay, we searched genes which were activated by a PPAR-gamma activator and identified nesfatin, a secreted
protein of unknown function, which was expressed in the appetite-control hypothalamic nuclei and adipose in rats. Intracerebroventricular
(icv) injection of nesfatin caused a dose-dependent decrease in food intake, and icv injection of its antibody stimulated feeding. The structure
of nesfatin possesses several cleavage sites that may undergo processing
by prohormone convertase (PC), and nesfatin was co-localized with PC2 and PC-3. Western blot analysis demonstrated the presence of nesfatin1 in the hypothalamic extract. Icv injection of nesfatin-1, but not nesfatin-2 or -3, produced satiety, and injection of an antibody neutralizing
nesfatin-1 stimulated feeding. Chronic icv injection of nesfatin-1 reduced body weight, and rats gained body weight after chronic icv administration of an antisense morpholino-oligonucleotide against the nesfatin
gene. The present data provide evidence that nesfatin is a novel, secreted
anorexigenic molecule in the hypothalamus.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Molecular dynamics in cardiac function [YFI (Young Foreign Investigator) Symposium]
Molecular Dynamics in Cardiac Function
Fujita, Hideo1; Yamashita, Hiroshi1; Sugiura, Seiryo2
(1Univ. of Tokyo Hosp., Tokyo, Japan; 2Grad. Sch. Frontier Sci.,
Univ. of Tokyo, Tokyo, Japan)
Molecular dynamics in cardiac
function [YFI (Young Foreign
Investigator) Symposium]
Clinical trials have demonstrated the adverse long-term effect of inotropic drugs for the treatment of heart failure. Because most of these drugs
exert their effects via the increase of intracellular calcium concentration,
people have sought a new class of agents working independently of the
calcium handling mechanism. We have studied the function of cardiac
myosins with various molecular structures to elucidate their role in diseased conditions, but, at the same time, such studies suggested the possible new mechanisms for modulating cardiac contractility at the
crossbridge level. Studies on mutant myosins implicated in familial hypertrophic cardiomyopathy, replacement of a single amino-acid located
in the C-terminus thus being far from the functional domains responsible
for ATP hydrolysis or actin-binding introduced severe functional defect.
On the other hand, myosin light chains seem to modulate crossbridge kinetics without changing ATPase activity of myosin. These calcium-independent (downstream) mechanisms for the modulation of cardiac
contractility will be discussed with additional observations.
An X-ray Diffraction Study on Mouse
Cardiac Cross-Bridge Function in vivo :
Effects of Adrenergic β-stimulation
Transmural heterogeneity of crossbridge
dynamics and lattice spacing in isolated rat
Yagi, Naoto1; Toh, Ryuji2; Shinohara, Masakazu2;
Takaya, Tomofumi2; Yamashita, Tomoya2; Masuda,
Shigeru2; Kawashima, Seinosuke2; Yokoyama,
Mitsuhiro2 (1SPring-8/JASRI; 2Kobe Univ. Grad. Sch. Med.)
Shimizu, Juichiro1; Mohri, Satoshi2; Miyasaka,
Takehiro2; Okuyama, Hiroshi3; Toyota, Hiroko3;
Tsujioka, Katsuhiko3; Takaki, Miyako1; Kajiya,
Fumihiko4; Yagi, Naoto5 (1Dept. of Physiol. II, Nara Med. Univ.
In order to investigate how β-stimulation affects the contractility of
cardiac muscle, x-ray diffraction from cardiac muscle in the left
ventricular free wall of a mouse heart was recorded in vivo. This is the
first x-ray diffraction study on a heart in a living body. After the R-wave
in electrocardiograms, the ratio of the intensities of the equatorial (1,0)
and (1,1) reflections decreased for about 50 msec from a diastolic value
of 2.1 to a minimum of 0.8, and then recovered. The spacing of the (1,0)
lattice planes increased for about 90 msec from a diastolic value of 37.2
nm to a maximum of 39.1 nm, and then returned to the diastolic level,
corresponding to about 10% stretch of sarcomere. Stimulation of βadrenergic receptor by dobutamine (20 µg/kg/min) accelerated both the
decrease in the intensity ratio, which reached a smaller systolic value,
and the increase in the lattice spacing. However, the intensity ratio and
spacing at the end-diastole were unchanged. The recovery of the lattice
spacing during relaxation was also accelerated. The mass transfer to the
thin filaments at systole in a β-stimulated heart was close to the peak
value in twitch of frog skeletal muscle at 4 °C, showing that the majority
of cross-bridges have been recruited with few in reserve.
Sch. of Medicine, Kashihara, Japan; 2Okayama Univ. Grad. Sch. of
Med, Dent, and Pharm., Okayama, Japan; 3Dept. of Physiol.
Kawasaki Med. Sch., Kurashiki, Japan; 4Dept. of Med. Eng.
Kawasaki Med. Sch, Kurashiki, Japan; 5Japan Synchrotron
Radiation Research Institute, Sayo-gun, Hyogo, Japan)
To analyze the fundamental mechanism of the Frank-Starling's law, we
studied the left ventricular (LV) transmural difference in the time course
of crossbridge dynamics (CBD) and the end-diastolic myosin filament
lattice spacing (EML) in the isolated crystalloid-perfused isovolumically
contracting rat hearts (n=11) paced at 2 Hz using X-ray diffraction at
SPring-8. We recorded x-ray diffraction patterns of the epicardial (EPI)
and deeper (DEEP) myocardium region of the LV free wall along with
LV pressure (LVP) at the end-diastolic pressure of either 0 or 20 mmHg
by adjusting LV volume. We analyzed transmural CBD from the X-ray
diffraction patterns according to the transmural variations of regional
myofilament orientation. The developments of CBD of both EPI and
DEEP and normalized LVP were synchronous during contraction, but
not during relaxation. The CBD decay was significantly faster in DEEP
than EPI. The LVV increase significantly potentiated LVP development
and more prolonged the CBD decay in EPI than DEEP associated with
more reduction of EML in EPI than DEEP. From studies on CBD and
EML, we were able to propose a possible underlying mechanism for the
Frank-Starling's law in the whole heart.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: The new evolution of molecular imaging—To spy spatiotemporal mechanism on cells [Workshop Organized by Program
Organizing Committee]
Effects of Length-Dependent Changes and
Ischaemia on Cardiac Cross-Bridge
Single-molecule analyses of the
interactions of neurotrophins and the
receptors on the growth cone
Pearson, James T.1; Shirai, Mikiyasu2; Schwenke, Daryl
O.3; Tsuchimochi, Hirotsugu4; Suga, Hiroyuki3; Yagi,
Naoto5 (1Physiology, Monash University, Melbourne, Australia;
Tani, Tomomi (Res. Inst. Electronic Sci., Hokkaido Univ.,
Hokkaido, Sapporo, Japan)
Intl. University, Kurose 555-0036, Japan; 3Natl.
Cardiovasc. Ctr. Res. Inst., Suita 565-8565, Japan; 4Yamaguchi
University, Ube 755-8505, Japan; 5SPring-8/JASRI, Harima 6795198, Japan)
We demonstrate using synchrotron x-ray radiation that length-dependent
mechanisms influence cross-bridge cycling in in situ rat hearts, and then
describe the effects of ischaemia-reperfusion on contractility. All experiments were performed in real time using synchrotron radiation at
Spring-8 and x-ray diffraction techniques. Mass transfer of myosin to actin during contraction was inferred from the change in diffraction intensity ratio (intensity of 1,0 reflection / 1,1 reflection) derived from the
myosin-actin filaments in fibres. Sustained volume loading by infusion
of sodium lactate (40-60 ml/h for <5 min) evoked a rightward shift in left
ventricular volume and significant decreases in epicardial myosin spacing, consistent with sarcomere stretching. Increases in stroke volume
were correlated with increases in mass transfer and myosin spacing
change. Local comparisons of intensity ratio and myosin spacing cycles
during ischaemia-reperfusion indicate that 1) mass transfer in the damaged area was reduced by moderate ischaemia, and sometimes unsynchronised, 2) compensatory increases in contractility were detected in
non-ischaemic areas (and during reperfusion), and 3) myosin spacing increases were reduced and delayed or reversed under severe ischaemia.
These findings suggest that non-functional fibres are stretched under
high cardiac output during acute ischaemia.
The growth cone is a motile structure located at the distal tip of the nerve
fiber. The 2.5S nerve growth factor (NGF) promotes the axonal growth
and the survival of sensory neurons by reacting with their growth cones.
We observed the behavior of single molecules of NGF on the growth
cone of sensory neurons by using a fluorescent NGF, Cy3-NGF. Upon
the application of 0.4 nM of Cy3-NGF, the growth cones responded
within one minute of adding the stimulus by expanding their lamellipodia. Only 40 molecules of Cy3-NGF, which occupied less than 5% of the
estimated total binding sites on a single growth cone, were required to
initiate the motile responses. After binding to the receptor, Cy3-NGF
displayed lateral diffusion on the membrane of the growth cones. The behavior of Cy3-NGF was shifted to a one-directional rearward movement
toward the central region of the growth cone. The one-directional movement of Cy3-NGF displayed the same rate as the rearward flow of actin
and the movements could be stopped by the application of the potent inhibitor of actin polymerization, latrunculin B. Molecules of Cy3-NGF
were internalized in the vicinity of the central region of the growth cone
during this rearward trafficking, as Cy3-NGF remained in the growth
cone after the removal of Cy3-NGF from the receptors on the surface of
growth cones. These results suggested that actin-driven trafficking of the
NGF-receptor complex is an essential step to the accumulation and endocytosis of NGF at the growth cone.
Molecular imaging and functional analysis
of RNG105: an RNA-binding protein
responsible for regulation of local
translation in neurons
Shiina, Nobuyuki1,2; Shinkura, Kazumi1; Tokunaga,
Makio1,2,3 (1National Institute of Genetics, Shizuoka, Japan; 2The
Graduated University for Advanced Studies, Shizuoka, Japan;
3RIKEN, RCAI, Kanagawa, Japan)
The new evolution of molecular
imaging—To spy spatiotemporal
mechanism on cells [Workshop
Organized by Program Organizing
Local translation in neuronal dendrites plays a key role in activity-dependent synaptic modifications, and is needed for long-term synaptic
plasticity. RNA granules, which consist of clusters of ribosomes and
RNAs, are responsible for transport of mRNAs to the dendrites and local
translational control.
We identified RNG105 (RNA granule protein 105) as a novel
component of the RNA granules in dendrites of hippocampal and
neocortex neurons. The RNG105-localizing RNA granules contain
mRNAs, the translational products of which play key roles in synaptic
plasticity. RNG105 is an RNA-binding protein and has ability to repress
translation both in vitro and in vivo. Time-laps fluorescence imaging
revealed that dissociation of RNG105 from the RNA granules is induced
by BDNF (brain-derived neurotrophic factor) stimulation. In contrast,
even after the BDNF stimulation, ribosomes remain in/near the RNA
granules. The RNG105 dissociation is concomitant with the induction of
local translation of the mRNAs located in the RNA granules. These
findings suggest that RNG105 is a translational repressor in the RNA
granules and becomes dissociated from the granules by synaptic
stimulation, which cancels the translational repression of the mRNAs in
the RNA granules.
We also want to show our recent progress in the study of RNG105
knockout mice and identification of RNG105-associated components of
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: New frontiers of research in synaptic plasticity—cellular and molecular mechanisms underlying reorganization of synapses in
the cerebellum and visual cortex
Single-molecule imaging of biomolecular
Funatsu, Takashi (Grad. Sch. Pharm. Sci. Univ. Tokyo, Tokyo,
Single-molecule imaging is a useful technique for analyzing functions
and interactions of protein molecules. I will show some instances how
this technique is applied to the biological studies. The first example is the
study of chaperonin assisted protein folding. GroEL mediates the folding
of nascent or denatured proteins in the E.coli collaborating with co-chaperonin GroES. We visualized GroEL-GroES interaction at the single
molecule level [1]. Release of GroES from GroEL occurred after a rag
period (~3s), that was not recognized in previous bulk-phase studies.
Furthermore, we succeeded in observing the refolding of denatured GFP
in the GroEL-GroES complex, and found that GFP could not start to refold for 3s after GroES binding. This observation suggests the presence
of a new kinetic intermediate "cis ATP*-complex" in the GroEL-GroES
reaction pathway. It is important for the efficient encapsulation of nonnative protein into the GroEL cavity. The second example is the transport of mRNA within a living cell. Fluorescently labeled mRNA was
injected into the nuclei of living cells and was visualized by fluorescence
microscopy. The injected mRNA molecule were in equilibrium of two
states, Brownian motion with diffusion coefficients of 0.2 µm2/s and
corralled in a restricted area for 20 s. These results suggested that mRNA
travels from the site of synthesis to nuclear pore by diffusion process.
New frontiers of research in
synaptic plasticity—cellular and
molecular mechanisms underlying
reorganization of synapses in the
cerebellum and visual cortex
[1] Taguchi, H., T. Ueno, H. Tadakuma, M. Yoshida, and T. Funatsu
(2001) Nature Biotechnol. 19: 861-865.
[2] Ueno T., H. Taguchi, H. Tadakuma, M. Yoshida and T. Funatsu
(2004) Molecular Cell, 14: 423-434.
Visualization of microtubule dynamics in
Mimori-Kiyosue, Yuko (KAN Research Institute, Kyoto, Japan)
Microtubule, a polarized hollow tube having plus and minus ends, is
highly dynamic structure repeating growth and shortening, especially at
its plus end. In cells, the parameters of microtubule dynamics are spatiotemporally regulated by a number of microtubule-binding proteins that
stabilize or destabilize microtubules, and thereby asymmetrical microtubule networks are generated.
The generation of a polarized microtubule organization is critically
important for proper cellular functions, such as cell division and
migration. To explain how microtubules set up and make contacts with
cellular structures, a "search-and-capture" mechanism has been
proposed, in which the microtubule plus ends dynamically search for and
capture specific sites, such as mitotic kinetochores and cell cortex. To
date, several classes of proteins called "microtubule plus-end-tracking
proteins" or "+TIPs" have been shown to be associated with "growing"
microtubule plus ends in a wide range of organisms from fungi to
humans to play critical roles in the "search-and-capture" mechanism.
Some of +TIPs highlight every growing microtubule plus ends, while
some of them accumulate at the microtubule-capturing structure created
at the specialized sites, and the complex formation of these molecules
serves a link between the microtubule plus end and variety of cellular
In this talk, I will introduce the tools to visualize microtubule dynamics
in living cells and overview our current understanding of the +TIPs.
Activity-dependent maintenance of
climbing fiber synaptic function in the
Kano, Masanobu1,2; Kakizawa, Sho3; Miyazaki,
Taisuke4; Yanagihara, Dai5; Iino, Masamitsu3;
Watanabe, Masahiko4 (1Grad. Sch. Med. Osaka Univ. Suita,
Osaka, Japan; 2Grad. Sch. Med. Sci. Kanazawa Univ. Kanazawa,
Japan; 3Grad. Sch. Med. Univ. Tokyo, Tokyo, Japan; 4Grad. Sch.
Med. Hokkaido Univ. Sapporo, Japan; 5Grad. Sch. Arts and Sci.
Univ. Tokyo, Tokyo, Japan)
Synapses undergo activity-dependent changes not only during development but also in adulthood. Mechanisms underlying these dynamic
changes have been studied intensively. However, it is largely unknown
how the strength of once matured synapse is maintained stably in the
brain. We show that significant weakening in the strength of excitatory
climbing fiber (CF) to Purkinje cell (PC) synapse followed chronic inhibition of neuronal activity by tetrodotoxin or persistent blockade of
postsynaptic AMPA receptors by their selective antagonist, NBQX.
These treatments reduced glutamate concentration transients at synaptic
clefts and decreased the frequency of quantal excitatory postsynaptic
current (EPSC). In contrast, neither the amplitude of quantal EPSC nor
the release probability was changed. Our morphological examination
demonstrates selective reduction of CF innervation at PC shaft dendrites
after NBQX-treatment. We thus conclude that in the mature cerebellum,
AMPA receptor-mediated neuronal activity in PCs maintains CF's functional release sites and its innervation of PC shaft dendrites.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: New frontiers of research in synaptic plasticity—cellular and molecular mechanisms underlying reorganization of synapses in
the cerebellum and visual cortex
Analysis of functions of parallel fiberPurkinje cell synapses by using virus
vector-mediated gene transfer in young
adult cerebellum
Orientation map reorganization induced by
persistent exposure to a dynamic or
stationary single orientation
Kohda, Kazuhisa; Kakegawa, Wataru; Yuzaki,
Michisuke (Dept. of Physiol., Keio Univ. Sch. Med.)
BASI, Wako, Saitama, Japan)
Parallel fibers (PFs), axons of cerebellar granule cells, transmit multimodal sensory information via mossy fiber inputs originating from the pontine nuclei. Each PF forms synapses with multiple Purkinje cells (PCs)
whose plasticity is believed to play cardinal roles in motor learning. In
addition, PF-PC synapses were reported to be dynamically reorganized
in an activity-dependent manner even in adulthood. Several lines of evidence have suggested that the δ2 glutamate receptor (GluRδ2) is one of
the key molecules that regulate functions of PF-PC synapses; GluRδ2null mice show ataxia and loss of long-term depression (LTD), a putative
cellular model of cerebellar information storage. In addition, GluRδ2null mice display half the number of PF-PC synapses. Despite their
importance, the mechanisms by which GluRδ2 participates in PF-PC
synaptic functions have been elusive, mainly because it is not activated
by glutamate analogs. To gain insight into GluRδ2's mechanisms, we
developed a Sindbis virus vector that express a wild-type GluRδ2. By
introducing this virus vector into GluRδ2-null cerebellum in vivo, we
could rescue several abnormal phenotypes, such as impaired LTD and
enhanced paired-pulse facilitation of excitatory postsynaptic currents at
PF-PCs synapses. This virus-based method has several advantages over
non-viral conventional gene expression methods and transgenic
techniques. Based on studies expressing a mutant GluRδ2, in which
several functional domains were mutated, GluRδ2's mechanisms in
controlling PF-PC synapses will be discussed.
Tanaka, Shigeru; Tani, Toshiki; Ribot, Jérôme (RIKEN
To examine the plastic changes in orientation selectivity of developing
visual cortex, we manipulated visual experience of kittens for 1-7 weeks
under a freely moving condition, mounting cylindrical-lens-fitted goggles to present uni-axially elongated images of their environments, or
spherical-lens-fitted goggles to present a stationary black-and-white oriented grating. We performed intrinsic signal optical imaging from those
animals to reconstruct cortical representation of orientation preferences.
For exposure to either a dynamic or stationary single orientation through
the goggles, the extreme over-representation of exposed orientation was
found in the visual cortex immediately after 1-2 weeks of continuous
goggle rearing. For kittens exposed to a dynamic single orientation for a
longer time, reorganized orientation maps were preserved although the
degree of over-representation was somehow reduced. However, for kittens persistently exposed to a stationary oriented grating for long time,
the over-representation effect almost disappeared or in some cases the
orientation maps paradoxically exhibited the over-representation of the
orientation orthogonal to the exposed orientation. It is suggested that the
consolidation of reorganized orientation maps requires the experience of
moving visual stimuli with behavioral relevance.
Rapid reorganization of synaptic input in
visual cortex by serine proteases
Mataga, Nobuko; Hensch, Takao (Neuronal Circuit
Development, Brain Science Institute, RIKEN)
Loss of responsiveness to an eye deprived of vision is mediated by rapid
functional disconnection followed by slow axonal rearrangement within
kitten and rodent visual cortex (Antonini and Stryker, 1993; Antonini et
al., 1999). We hypothesized that extracellular serine proteases (tissuetype plasminogen activator (tPA)-plasmin system) act to degrade celladhesion molecules or extracellular matrix proteins to permit earlier synaptic remodeling dependent on visual experience. Indeed, functional ocular dominance (OD) plasticity was reversibly impaired in tPA knockout
mice (KO) (Mataga et al, PNAS, 2002). Moreover, we identified a robust
anatomical change in layer 2/3 of mouse visual cortex by brief monocular deprivation (MD) (Mataga et. al., Neuron, 2004). Protrusions on the
apical dendrite of pyramidal cells increased steadily in number with
postnatal age, but were rapidly and transiently lost after MD only during
the physiological critical period (CP). Targeted disruption of tPA or its
upstream regulation by glutamic acid decarboxylase (GAD65) reversibly prevented MD-induced spine pruning in vivo. The tPA-plasmin system may, thus, rapidly couple physiological perturbation of sensory
input to early structural rearrangement of synaptic input.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: Social brain and physiology:Å@Neural mechanism for predictive environmental cognition
Self and other representation in the parietal
cortex based on sense of body
Murata, Akira (Dept.Physiol, Sch. Med., Kinki Univ. Osakasayama, Japan)
Social brain and physiology: Neural
mechanism for predictive
environmental cognition
Recently, it is claimed that automatic simulation of inner state in other's
brain by observation of action is very important neural mechanism for
social interaction. The idea is on the lines of simulation theory. Mirror
neurons, which were found in the ventral premotor cortex and inferior
parietal cortex of the monkey, are considered to be neuronal correlates of
this simulating mechanism. On the other hand, self-other representation
in the brain should be necessary in social interaction. Recent imaging experiments suggest that motor control system may be involved in recognition of agency of action or ownership of one's own body parts. We
speculate the mirror neurons in the parietal cortex of the monkey would
be also involved in monitoring own body action. Actually, we found that
some neurons related to the hand manipulation task in the parietal cortex
responded to the movie of own hand action. These neurons were also active during observation of other's hand action. Further, we also found
that this visual response was less active in the delayed feedback than in
the real time. These results suggest that matching between efference
copy and sensory feedback (visual and somatosensory) may occur in the
parietal cortex. This may be neural bases of self-other distinction. Further, I will also present visual-somatosensory bimodal neurons have visual receptive field on the corresponding other's body parts. I would like
to discuss functional property of inferior parietal cortex for the self-other
distinction and matching.
Brain activity during Social Interaction
between Japanese monkeys
Fujii, Naotaka; Hihara, Sayaka; Iriki, Atsushi (Laboratory
Ethological approach to understand the
behavioral transmissions in social
for Symbolic Cognitive Development, BSI, RIKEN, Japan)
Kikusui, Takefumi (Grad. Sch. Agr. Univ. Tokyo, Tokyo, Japan)
We are surrounded by rich environment which include varieties of social
properties. Our brains are handling such information for choosing and
performing behaviors. Social meaning of each behavior is always changing time to time so that the brain has to take much effort to extract social
parameters mined in the environment. Although we are highly social animal, we know very little about how the brain is manipulating such social
parameters. There is almost no neurophysiological study focused on social brain functions. Therefore we tried to see how social parameters are
affecting on brain function at single neuronal level by using Japanese
monkeys known for their complex social behaviors.
Social stimuli during the neonatal and juvenile periods are known to affect various aspects of physiological and behavioral development in rodents. For example, maternal behavior and partner preference in the
adulthood are determined by the social environments in which the animals are reared. These phenomena are so called "non-genomic transmissions". In this study, we investigated the long-lasting influences of
earlier weaning on adulthood behavioral traits in rats and mice. Subjects
were weaned from their mothers one week earlier than the normal weaning period. To assess anxiety levels at ages 8 weeks and 22 weeks, both
early weaned and control mice pups were subjected to several behavioral
tests, and it was found that the early-weaned animals had a sustained increase in anxiety levels compared to the control groups. Social behavioral tests performed in other sets of mice revealed that early weaned pups
engaged in more fights under several conditions, including co-housing
them with other mice, although neither isolation-induced aggression nor
territorial aggression differed from normally weaned mice. In addition,
early weaned females repressed maternal behavior. Similar results were
obtained in rats as well. Concurrently, neurochemicals that are responsible for the behavioral and endocrine responses to stress were affected by
the early weaning manipulations. These results suggest that the absence
of mother-pup interactions during the last several days of pre-weaning
period may lead to a persistent increase in anxiety and aggression during
adulthood in rodents.
Two Japanese Macaques were used. We implanted multiple electrodes
chronically (24 and 30 for each monkey) in prefrontal and parietal
cortices. During the experiment, we altered monkeys' relative spatial
positions so as to manipulate their social spaces. In some cases, their
peri-personal spaces were made either overlapped or separated. Their
behaviors were monitored by using motion capture technique and video.
These techniques allowed monkeys relatively free movement during
We found that monkeys altered their behaviors dramatically simply by
changing social structure between them. While monkeys were showing
social interaction, prefrontal and parietal neurons showed varieties of
response patterns depending on social context. Prefrontal neurons
tended to respond earlier and parietal responses followed, suggesting
two areas are managing different aspects of social functions in the brain.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: What we learn about the physiological roles of Ca2+/calmodulin-dependent protein kinases (CaMKs) from genetically
Neuropsychological study for abnormal
social interaction
Kato, Motoichiro (Department of Neuropsychiatry, Keio
University School of Medcine, Tokyo, Japan)
Interpersonal activity is essential in making humans the uniquely social
beings that we are. The underlying neural grounds of the social interaction are the fusiform region, amygdala, superior temporal sulcus, orbitofrontal and dorsolateral prefrontal cortex, which are of great interest to
neuroscientists. To anticipate possible futures and coordinate thought
and action for achieving desired outcomes, the prefrontal lobes play a
pivotal role. The dorsolateral prefrontal cortex is essential to guide the
behavior by thought and language, while the orbital and ventromedial regions have been considered to be the neural correlates for affective evaluation of the consequences of our action. The brain region that is
implicated in gaze processing, STS, has repeatedly been activated when
viewing gaze in the normal brain. We have presented a case, MJ, in a recent report, with a circumscribed lesion in the right superior temporal
gyrus, due to a cerebrovascular accident, who manifested a puzzling difficulty in obtaining eye-contact. As the STG comprises a part of the STS,
we investigated her ability in processing gaze. Indeed, MJ demonstrated
a unique impairment in discriminating gaze direction, which is the first
neuropsychological evidence that establishes STS as a gaze processor, so
often implicated in animals and human neuroimaging studies.
Comprehensive behavioral analysis of
Ca2+/calmodulin-dependent protein kinase
4 knockout mouce
Tanda, Koichi1,2; Yamasaki, Nobuyuki1; Toyama,
Keiko1; Sakagami, Hiroyuki3; Miyakawa, Tsuyoshi1
(1Grad. Sch. Med. Univ. Kyoto, Kyoto, Japan; 2Kyoto. Pref. Univ.
Med, Kyoto, Japan; 3Grad. Sch. Med. Univ. Tohoku, Sendai, Japan)
Ca2+/calmodulin-dependent protein kinase 4(CaMK4)is a protein kinase
that activates the transcription factor, cAMP response- element binding
protein (CREB). CaMK4 has been hypothesized to play a significant role
in synaptic plasticity and in learning and memory. However, functions of
CaMK4 in a variety of behaviors, e.g., motor function, nociception, fear,
anxiety, depression, learning and so on, have not yet been fully elucidated. To gain more insight into behavioral significance of CaMK4, we subjected CaMK4 -/- mice to a battery of behavioral tests, including
neurological screen, light/dark transition, open field, elevated plus maze,
social interaction, rotarod, hot plate, prepulse inhibition, Porsolt forced
swim, 8-armed radial maze, Barnes maze, fear conditioning, latent inhibition, and passive avoidance tests.
CaMK4 -/- mice exhibited increased social interaction in home cage.
They did not display any deficit in spatial reference memory and
working memory tests, but had mild performance deficit in fear
conditioning tests. These results indicated selective and specific
involvement of CaMK4 in regulating emotional behaviors.
Assessments of depression and the
sensitivity to antidepressants in calcium/
calmodulin-dependent protein kinase IVknockout mice
Kasahara, Jiro1; Kohji, Fukunaga1; Hiroyuki, Sakagami2
(1Dept. Pharmacol. Grad. Sch. Pharmaceu. Sci. Tohoku Univ.
Sendai, Japan; 2Dept. Histol. Grad. Sch. Med. Tohoku Univ. Sendai,
What we learn about the
physiological roles of Ca2+/
calmodulin-dependent protein
kinases (CaMKs) from genetically
engineered mice
Calcium/calmodulin-dependent protein kinase IV (CaMKIV) is expressed abundantly in the nuclei of neurons and thought to regulate gene
expressions mediated by the transcriptional factors such as CREB. We
have been characterized CaMKIV comparing with other subtypes CaMKI and CaMKII in the hippocampal neurons. The basal CaMKIV activity is kept low by constant inactivation by associated protein
phosphatase 2A, and the activation is transient because calcineurin inactivate CaMKIV after the neuronal stimuli, while the activities of CaMKI
and CaMKII are sustained. In other words, CaMKIV is hard to be
switched on and is easy to be switched off. Based on this fact, we thought
the functions of CaMKIV are reflected in the animals after the chronic
stimulation rather than the acute one. Recently, we found that chronic
treatments of the rats with antidepressants increased CaMKIV activity
and CREB phosphorylation in the prefrontal cortex and the hippocampus, suggesting the importance of CaMKIV in the effects of antidepressants. These results led us to perform the behavioral assessments of
anxiety, depression and the sensitivity to antidepressants in CaMKIVknockout mice by some experimental paradigms including the forced
swim test, the tail suspension test and the novelty-suppressed feeding
test. From the results of these experiments, it was suggested that
CaMKIV is involved in some of the depression-related behaviors and the
sensitivities to antidepressants.
J. Physiol. Sci., Vol. 56, Suppl., 2006
SYMPOSIA: What we learn about the physiological roles of Ca2+/calmodulin-dependent protein kinases (CaMKs) from genetically
Comprehensive brain-behavior
phenotyping of Ca2+/calmodulindependent protein kinase IIα heterozygous
knockout mice
The role of Ca2+/calmodulin-dependent
protein kinase II (CaMKII) in neuronal
activity revealed by inactivated CaMKIIα
knock-in mouse
Yamasaki, Nobuyuki1,2; Miyakawa, Tsuyoshi1
Yamagata, Yoko1,2 (1Natl. Inst. for Physiol. Sci., Okazaki,
Japan; 2SOKENDAI, Okazaki, Japan)
(1Grad.Sch.Med.Univ.Kyoto, Kyoto, Japan;
2Grad.Sch.Med.Univ.Kyoto, Kyoto, Japan)
Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous
serine/threonine protein kinase that is abundant in the brain as a major
constituent of the postsynaptic density and critically involved in synaptic
plasticity, learning and memory. Several behavioral abnormalities of
CaMKIIα mutant mice were reported, but systematic assessments of
CaMKIIα mutant mice have not been well conducted. To analyze the
behavioral effects of CaMKIIα deficiency, we subjected CaMKIIα
heterozygous knockout mice to a comprehensive behavioral test battery.
The mutant mice showed increased locomotor activity, markedly
decreased anxiety, decreased depression-related behavior, increased
offensiveness, selective and severe spatial working memory deficit, and
dramatic periodic change of locomotor activity in home cage. To
identify the mechanism underlying the behavioral abnormalities of
CaMKIIα mutant mice, gene expression analysis and biochemical
analysis of the brain of the mutant mice were conducted. The potential
involvement of CaMKIIα in pathogenesis/pathophysiology of
psychiatric disorders, such as schizophrenia, bipolar disorder, and
personality disorders, will be discussed.
Ca2+/calmodulin-dependent protein kinase II (CaMKII) is one of the
most abundant protein kinase in the central nervous system and a key
mediator of intracellular Ca2+ in response to various stimuli. CaMKII is
involved in many neuronal functions including the regulation of neuronal activity. Previous studies reported episodes of epileptic seizure in
CaMKIIα knock-out mice and manifestation of epileptic discharges in
cultured neurons treated with anti-sense oligonucleotide to CaMKIIα.
However, which protein function of CaMKII, i.e., protein kinase
activity, calmodulin-binding capacity or multimeric structure interacting
with other proteins, is responsible for stabilizing neuronal activity
remains to be elucidated yet. To clarify specifically the role of protein
kinase activity of CaMKII, we engineered knock-in mice with the
inactivated α subunit of CaMKII by replacing Lys-42 with Arg-42.
CaMKIIα protein level was unchanged, but CaMKII activity was
specifically decreased in these mutant mice. Spontaneous death rate was
higher, and pentylenetetrazole injection resulted in higher seizureinduced mortality in homozygous mutants. Spontaneous seizure was
sporadically observed in homozygous and heterozygous mutants, but
rarely in wild type controls. Cytochrome oxidase staining revealed
decreased neuronal activity in nucleus accumbens in homozygous
mutants. These results indicate that protein kinase activity of CaMKII,
i.e., protein phosphorylation by CaMKII, is important for maintaining
basic and normal neuronal activity in vivo.
Increase in anxiety and aggression in
transgenic mice overexpressing αCaMKII
in forebrain.
Furuichi, Takahiro1; Suzuki, Akinobu1; Kajii, Yasushi2;
Kida, Satoshi1 (1Tokyo. Univ. Agricul., Tokyo, Japan; 2Mitsubisi
Pharma Corporation, Tokyo, Japan)
Previous studies have shown that αCalcium/Calmodulin dependent
protein kinase II(αCaMKII) plays important roles in not only learning
and memory but also aggressive and fear response in mice. To further
understand roles of αCaMKII in brain function, we have generated
transgenic mice overexpressing αCaMKII in forebrain. Interestingly,
these mutant mice showed increase in anxiety in open field and elevated
zero maze tests and increase in offensive aggression in resident-intruder
test. Increase in anxiety observed in these transgenic mice suggests that
expression level of CaMKII positively correlates with expression of
anxiety related behavior. We next examined effects of administration of
selective serotonin reuptake inhibitor (SSRI) on anxiety related behavior
observed in these mutant mice. Treatment of these transgenic mice with
SSRI suppressed anxiety-related behavior in both tests. These results
raise the possibility that these mutant mice is a mouse model of anxiety
disorder, that allows to develop therapeutic drugs . In addition, we have
examined the expression profile of these mutant mice with or without the
treatment of SSRI and tried to find out the anxiety-related genes.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Oral Presentations
ORAL: Cellular & molecular physiology
Stimulation of the gastric H,K-ATPase
activity by CLC-5 Cl– channel
Takahashi, Yuji1; Ohira, Yuta1; Tabuchi, Yoshiaki2; Ikari,
Akira3; Sakamoto, Hisato4; Naito, Ichiro5; Manabe,
Koji5; Uchida, Shinichi6; Sasaki, Sei6; Asano, Shinji7;
Morii, Magotoshi1; Takeguchi, Noriaki1; Sakai, Hideki1
(1Fac. Pharmaceu. Sci., Univ. Toyama, Toyama, Japan; 2Life Sci.
Res. Ctr., Univ. Toyama, Toyama.; 3Sch. Pharmaceu. Sci., Univ.
Shizuoka, Shizuoka.; 4Sch. Med., Kitasato Univ., Sagamihara.;
5Shigei Med. Res. Inst., Okayama.; 6Grad. Sch., Tokyo Med. Dent.
Univ., Tokyo.; 7Col. Info. Sci. & Eng., Ritsumeikan Univ., Shiga.)
Cellular & molecular physiology
Dikic, Ivan; Hoeller, Daniela; Bienko, Magda; Crosetto,
Nicola; Zapart, Gregorz; Haglund, Kaisa (Institute of
Biochemistry II, Goethe University, Frankfurt, Federal Republic of
The attachment of a ubiquitin (Ub) to a substrate serves as an important
regulatory modification implicated in receptor endocytosis, virus budding, gene transcription, DNA repair and replication, etc. The discovery
of Ub-binding domains (UBDs) has indicated how Ub can regulate such
distinct cellular functions. We have recently cloned two novel Ub-binding domains named UBM (Ub binding motif) and UBZ (Ub binding Zn
finger), which are evolutionarily conserved in Y-family translesion polymerases (pols). These domains are required for binding of pols to Ub,
their accumulation in replication factories and interaction with monoubiquitinated PCNA. In addition, Ub-binding domains of Y-family polymerases play essential roles for in vivo translesion synthesis, which is
the major pathway by which mammalian cells replicate across DNA lesions. Interestingly, novel Ub-binding domains are found in several other
proteins implicated in regulation of DNA repair and replication.In addition to binding Ub, several UBDs promote monoubiquitination of host
proteins. Biochemical, biophysical and mathematical evidence support
the concept whereby monoubiquitination of the Ub binding proteins facilitates intramolecular interactions with the UBDs, thus preventing
them from binding in trans to ubiquitinated targets. Monoubiquitination
of Ub-binding proteins thus represents a regulatory mechanism that inhibits their capacity to bind to and control functions of ubiquitinated targets in vivo.
In the stomach, protons are actively secreted by the gastric H,K-ATPase,
but it is unclear what molecule contributes to the secretion of Cl–. We
have previously shown that CLC-2 may not be responsible for the Cl–
transport in the gastric acid secretion. However, we found that CLC-5
was expressed in the gastric parietal cells. Here, we examined the interaction between CLC-5 and the gastric H,K-ATPase. We constructed a
tetracycline-regulated expression system of CLC-5 in the HEK293 cells
stably expressing the gastric H,K-ATPase. The H,K-ATPase activity and
the 86Rb+ transporting activity were examined by using SCH 28080, a
K+-competitive inhibitor of the H,K-ATPase. Expression of CLC-5 in
the HEK293 cells significantly increased the H,K-ATPase activity by
24.1 ± 8.9% (n = 6) and 86Rb+ transporting activity by 28.2 ± 5.9% (n =
5). The expression level of H,K-ATPase in the plasma membrane was
not affected by CLC-5 in the HEK293 cells. Furthermore, we found that
expression of CLC-5 significantly up-regulated the phosphorylation
level of H,K-ATPase by 48 ± 18% (n = 4). These results suggest that
CLC-5 may be a modulatory subunit of the gastric H,K-ATPase.
Change in intracellular chloride
concentration caused by regulatory
volume decrease is the primary hypotonic
signal in renal epithelial A6 cells
Miyazaki, Hiroaki; Niisato, Naomi; Marunaka, Yoshinori
(Mol. Cell Physiol., Grad. Sch. of Med. Sci., Kyoto Pref. Univ. of
Med., Kyoto, Japan)
Our recent study indicates that hypotonicity-induced decreases in intracellular Cl– concentration ([Cl–]i) could act as a signal to regulate Na+ reabsorption through changes in αENaC mRNA expression in renal
epithelial A6 cells. This result suggests that the change of [Cl–]i is one of
the important signals to cell function. However, currently reported
techniques for the measurement of [Cl–]i by using halide-specific
fluorescent dyes lack sufficient sensitivity and accuracy. One reason for
problems in the use of these dyes for measurement of [Cl–]i during
hypotonicity-induced regulatory volume decrease (RVD) is a change of
intracellular dye concentration during RVD, since a fluorescent intensity
of these indicators depends not only on [Cl–]i but also on intracellular
concentration of dyes. In this study, we have developed a new method
for measuring [Cl–]i by using a cell analyzer Quanta. This flow
cytometer can simultaneously measure the exact cell volume by Coulter
principle and the fluorescent intensity. The concentration of Cl– in A6
cells diminished during RVD by 72% (from 47.3 mM to 13.3 mM). This
reduction of [Cl–]i was blocked by inhibition of RVD with quinine (K+
channel blocker) or NPPB (Cl– channel blocker). These results suggest
that a change in external osmolality is converted into the change in [Cl–
]i, and that the change of [Cl–]i is the primary hypotonic signal in A6
cells. This work was supported by Grants-in-Aid from JSPS (17390057,
17590191 and 17790154).
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Cellular & molecular physiology
P2Y receptor-induced increase in
sensitivity of adipogenic hormones to
The important role of Fyn tyrosine kinase
on the Ca2+-independent contraction of
vascular smooth muscle
Omatsu-Kanbe, Mariko; Fujii, Yusuke; Matsuura,
Hiroshi (Shiga Univ. Med. Sci. Otsu, Shiga, Japan)
Guo, Fengling1,2; Kawamichi, Hozumi1; Kishi, Hiroko1,2;
Miao, Junying1; Miwa, Saori1; Kajiya, Katsuko1; Xu,
Dan1,2; Kobayashi, Sei1,2 (1Dept. Mol. Physiol. Sch. Med,
The effect of extracellular ATP on adipogenesis was investigated using
3T3-L1 cell line. Incubation of cells with ATP (1-100 µM) for 5 min
induced membrane ruffling and migration (chemokinesis). In this cell
line, growth arrest is required before initiation of differentiation, and
growth-arrested post-confluent cells can be converted to adipocytes by
the presence of the adipogenic hormones dexamethasone, 3-isobutyl-1methylxanthine and insulin. On the other hand, those hormones alone do
not trigger differentiation in proliferating cells. ATP did not induce
differentiation when applied alone to either proliferating or postconfluent cells. In contrast, proliferating cells (density<50%)
preincubated with ATP for 5 min and subsequently given the adipogenic
hormones in the continued presence of ATP underwent adipocyte
differentiation mediated through phospholipase C-coupled P2Y
receptors. These adipocytes were found to show very similar
characteristics, including morphology and intracellular triglyceride
accumulation, compared with adipocytes differentiated from postconfluent preadipocytes with those adipogenic hormones. When
proliferating cells were preincubated with ATP prior to the addition of
the adipogenic hormones, gene expression of adipose protein 2 was
markedly increased in 6 days, while the expression level stayed very low
without ATP pretreatment. These results suggest that extracellular ATP
renders preadipocytes responsive to the adipogenic hormones during the
growing phase.
Yamaguchi Univ. Ube, Japan; 2Grad. Sch. Med. Yamguchi Univ.
Ube, Japan)
Rho-kinase (ROK) is being regarded as the critical signaling molecule of
the Ca2+-independent contraction of vascular smooth muscle (VSM). We
recently identified that the sphingosylphosphorylcholine (SPC) induced
Ca2+-independent contraction of VSM by activating ROK and that SPC
activated ROK via activating Src family tyrosine kinase (Src-TK). Since
VSM expresses Fyn and c-Src among Src-TK, we analysed which SrcTK is involved in this SPC/ROK-mediated Ca2+ sensitization. The inhibitors of Src-TK (PP1 and PP2) abolished all the reactions of Ca2+-independent contraction, activation of Src-TK and ROK, and tyrosine
phosphorylation of p60 protein induced by SPC. SPC induced the translocation of Fyn from cytosol to the plasma membrane of VSM cells, but
not that of c-Src. In order to examine directly the ability of Fyn to induce
Ca2+-independent contraction, we made recombinant Fyn proteins using
a baculovirus system. In beta-escin permeabilized VSM, constitutively
active Fyn induced Ca2+-independent contraction which was inhibited
by Y27632, while dominant negative Fyn inhibited the contraction induced by U46619+GTP. These findings suggest that Fyn tyrosine kinase
plays a pivotal role in the SPC-induced and ROK-mediated Ca2+-independent contraction.
The screening of novel inhibitors for Ca2+independent abnormal contraction of
vascular smooth muscle which have
similar inhibitory effects to
eicosapentaenoic acid
Nishimura, Shigehiko1; Kishi, Hiroko1,2; Guo,
Fengling1,2; Morita, Naoki3; Ohgiya, Satoru3;
Hosokawa, Masashi4; Miyashita, Kazuo4; Kawamichi,
Hozumi1; Kajiya, Katsuko1; Xu, Dan1,2; Wang, Chen1;
Kobayashi, Sei1,2 (1Dept. of Mol. Physiol., Yamaguchi Univ. Sch.
of Med., Ube, Japan; 2Mol. Cell. Dig Bioreg. Grad. Sch. Med.
Yamaguchi Univ., Ube, Japan; 3Res. Inst. Gen-Based Biofact., AIST,
Sapporo, Japan; 4Lab. Bioresouces Chem, Marine Biosci., Grad.
Sch. Fish.Sci. Hokkaido Univ., Hakodate, Japan)
We previously identified sphingosylphosphorylcholine (SPC) and Fyn
as upstream signal molecules of Rho-kinase-mediated Ca2+-independent
abnormal contraction of vascular smooth muscle (VSM) and found that
eicosapentaenoic acid (EPA) can selectively inhibit such abnormal
events without affecting Ca2+-dependent normal VSM contraction by
blocking the translocation of Fyn to plasma membrane. Moreover, we reported that EPA was clinically and highly effective in preventing vasospasm after subarachnoid hemorrhage. However, EPA is limited to oral
administration and thus unsuitable for clinically serious patients unable
to ingest orally. We therefore screened novel compounds which could inhibit Ca2+-independent abnormal VSM contraction and could substitute
for EPA. Tension study of VSM showed that several compounds inhibited SPC-induced abnormal VSM contraction, which was comparable to
the effects of EPA. These results suggest that these newly found compounds would be the candidates for novel therapeutic drugs for vasospasm which could substitute for EPA.
Glucagon-Like Peptide 1 Activates Protein
Kinase C in a Ca2+-Dependent Manner in
Insulin-Secreting Cells
Mogami, Hideo1; Suzuki, Yuko1; Urano, Tetsumei1;
Zhang, Hui2; Kojima, Itaru2; Saitoh, Naoaki3 (1Dept.
Physiol., Hamamatu. Univ. Sch. Med., Hamamatsu, Japan; 2IMCR,
Gunma Univ., Gunma,Japan; 3Lab.Mol. Pharm, Biosignal Research
Center, Kobe Univ., Kobe, Japan)
Introduction : GLP-1, a cAMP mobilizing agonist, is an insulinotropic
peptide released from the intestinal L cell in response to a meal. However, the underlying mechanisms of the stimulatory effect of GLP-1 on insulin secretion remain fully elucidated. Aim: The present study was
conducted to examine whether GLP-1 can activate PKCalpah and PKCepsilon in INS-1 cells at a substimulatory concentration of glucose.
Methods: We employed either GFP or DsRed-tagged proteins related to
PKC signaling pathway using epifluorescence microscopy and total internal reflection fluorescence microscopy. Results: We first showed that
GLP-1 translocated endogenous PKCalpah and PKCepsilon from the cytosol to the plasma membrane. Then we assessed the phosphorylation
state of the PKC substrate, myristoylated alanine-rich C kinase substrate
(MARCKS), as a maker of PKC activation. GLP-1 translocated GFPtagged MARCKS from the plasma membrane to the cytosol and the
GLP-1-evoked translocation of MARCKS-GFP was blocked by PKC inhibitors. The above observations were verified in three different ways
using live cell imaging technique.In addition to these results, PKC inhibitors reduced forskolin-induced insulin secretion in INS-1 cells and rat
islet beta cells. Conclusion: GLP-1 can activate PKCalpah and PKCepsilon, and the GLP-1-activated PKCs may contribute considerably to insulin secretion at a substimulatory concentration of glucose.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Cellular & molecular physiology
Ghrelin attenuates glucose-induced insulin
release via inhibition of cyclic AMP
productions in rat islet β-cells
Dezaki, Katsuya1; Sone, Hideyuki1; Kakei, Masafumi2;
Yada, Toshihiko1 (1Dept. Physiol., Jichi Med. Sch., Tochigi,
Japan; 2Dept. Intern. Med., Sch. Med., Akita Univ., Akita, Japan)
Ghrelin, isolated from the human and rat stomach, is the endogenous
ligand for the growth hormone secretagogue receptor (GHS-R). We have
reported that GHS-R was expressed in rat pancreatic islets and that ghrelin suppressed glucose-induced insulin release via activation of voltage-dependent delayed rectifier K+ channels and attenuation of glucoseinduced action potentials, leading to suppression of glucose-induced
Ca2+ signaling in β-cells. In this study, we aimed to determine the
involvement of cyclic AMP productions, another major signalling
pathway for insulin release, in the ghrelin-induced suppression of insulin
release. Both GHS-R blockade and anti-ghrelin antiserum markedly
enhanced 8.3 mM glucose-induced insulin release in rat perfused
pancreas and isolated islets. GHS-R blockade and anti-ghrelin antiserum
also enhanced 8.3 mM glucose-induced cyclic AMP productions in rat
islets. Conversely, exogenous ghrelin (10 nM) suppressed insulin release
and cyclic AMP productions. In the presence of either dibtyryl cyclic
AMP or adenylate cyclase inhibitor MDL12330A, ghrelin failed to
attenuate glucose-induced insulin release. This study suggests that
ghrelin suppresses glucose-induced cyclic AMP production as well as
cytosolic Ca2+ response. These abilities of ghrelin to impede cyclic AMP
and Ca2+ signaling routes at least partly account for the inhibition of
glucose-induced insulin release.
Gao, Ying (Department of Biochemistry & Molecular Biology,
Dalian Medical Universityt, Dalian, People's Republic of China)
Myosin light chain kinase (MLCK) is a multifunctional regulatory protein of smooth muscle contraction. The well-established mode for its
regulation is to phosphorylate the 20kDa myosin light chain (MLC20) to
activate myosin ATPase activity. MLCK exhibits myosin-binding activity in addition to this kinase activity. The myosin-binding activity also
stimulates myosin ATPase activity without phosphorylating MLC20. In
order to study this non-kinase activity of MLCK and its active site. We
engineered two MLCK fragments, one contained the myosin-binding
domain but devoid of a catalytic domain and another further deleted a
calmodulin (CaM) domain. The former fragment stimulated myosin ATPase activity by Vmax= 5.53 ± 0.63- fold with Km = 4.22 ± 0.586µM (n
= 4). Similar stimulation figures were obtained by measuring the
ATPase activity of HMM and S1. We failed to observe the stimulation
with the latter fragment. Similar stimulating effect were obtained by
measuring the ATPase activity of phosphorylated myosin, HMM and
S1. Binding of the fragment to both HMM and S1 was also verified,
indicating that the fragment exerts stimulation through the myosin
heads. We conclude that the non-kinase stimulation of MLCK are
involved in the mode for activation of myosin. The CaM domain is one
of the active site for non-kinase activity of MLCK fragment.
Development of cardiac fibrosis in
sphingosine kinase 1 transgenic mice
Takuwa, Noriko1; Ohkura, Sei-Ichiroh1,2; Oyama,
Osamu1,3; Takashima, Shin-Ichiroh1,2; Sugimoto,
Naotoshi1; Takuwa, Yoh1 (1Dept. of Physiol., Grad. Sch. Med.
Kanazawa Univ. Kanazawa, Japan; 2Dept. of Cancer Gene Therapy,
Grad. Sch. Med., Kanazawa Univ.; 3Dept. of Blood Pathoinformatics, Grad. Sch. Med., Kanazawa Univ.)
Sphingosine-1-phosphate (S1P) is a plasma lysophospholipid with diverse activities, and is released in a large amount from activated platelets. Our laboratory as well as others have identified the existence of the
G protein-coupled S1P receptor family, which include ubiquitously expressed S1P1, AGR16/Edg5/S1P2 and S1P3. In an attempt to get more
insight into roles of S1P in vivo, we have generated transgenic (TG)
mice that overexpress a major S1P synthetic enzyme sphingosine kinase
1 (SPHK1) in diverse tissues, with up to several ten fold increases in the
SPHK1 activity. Although previous reports suggested the involvement
of SPHK1 in cell proliferation and transformation, the TG mice show
normal growth and no obvious increase in spontaneous malignancy. Importantly, TG mice with a high but not a low level of SPHK1 expression
in the heart show age-dependent, progressive cardiac fibrosis with development of dilated cardiomyopathy in a limited population. Transgenic
heart tissues show elevated activities in both Rac1 and RhoA small molecular weight G proteins and enhanced superoxide generation in responses to phorbol ester. Treatment of TG mice with an HMG-CoA
reductase inhibitor or an antioxidant N-2-mercaptopropyonylglycine,
but not an angiotensin II type 1 receptor blocker, resulted in alleviation
of cardiac fibrosis. These results provide evidence for a pathophysiological role of SPHK1 and probably S1P.
Myosin light chain kinase stimulates
smooth muscle ATPase activity with its
non-kinase activity by binding to the
myosin heads: a study with proteinengineering
Cellular mechanism of experimental
autoimmune uveoretinitis
Takeda, Sayako1; Yoshida, Ryotaro1; Yamaji, Junko1;
Takahashi, Takeshi1; Inui, Takaki1; Mori, Yoshiaki1; Oku,
Hidehiro2; Ikeda, Tsunehiko2; Kubota, Takahiro1 (1Dept. of
Physiol, Osaka Med. Col. Osaka, Japan; 2Dept. of Ophthalmol,
Osaka Med. Col. Osaka, Japan)
[BACKGROUND] Experimental autoimmune uveoretinitis (EAU) is an
organ-specific autoimmune disease that is induced in animals sensitive
to retinal antigens. The recruitment of leukocytes is crucial for ocular inflammation in EAU, whereas target cells and effector cells have not yet
been clearly characterized.[PURPOSE] Isolation and characterization of
target cells in EAU[METHODS] EAU was induced in B10 mice by immunization with 50 µg human interphotoreceptor retinoid binding
protein peptide 161-180 in emulsion with CFA supplemented with 3.5
mg/ml M. tuberculosis(1:1,vol/vol). Disease severity was assessed
clinically by funduscopic examination. Retinal cells and ocular
infiltrating cells were obtained by enzyme digestion from the eyecups of
normal and EAU mice,respectively, and were separated into different
types of cells by Percoll density gradient centrifugation. To assess which
types of cells were target and effector cells in EAU, we determined
cytotoxic activity of infiltrating cells against retinal cells by 51Cr release
assay.[RESULTS] Two kinds of retinal cells were isolated as 51Cr
incorporationg cells. They appeared to be retinal pigment epithelial
(RPE) cells and monocytic cells judging from their morphological
features under an electron microscope. The identification of effector
cells responsible for EAU is under investigation in our
laboratory.[CONCLUSIONS] We isolated RPE cells and monocytic
cells as monodispersed growing cells or a candidate for target cells in
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Cellular & molecular physiology
Localization of the short-chain fatty acid
receptor, GPR43, in the rat intestine
Karaki, Shin-ichiro1; Mitsui, Retsu1; Ono, Shigeyuki1,2;
Kuwahara, Atsukazu1 (1Lab. of Physiology, Inst for
Environmental Sciences, University of Shizuoka, 52-1 Yada, Surugaku, Shizuoka, Japan; 2Kao Corporation, 1-3-2 Bunka, Sumida-ku,
Tokyo, Japan)
Chloride ions play important roles on the
cell death caused by the plasma membrane
permeability increase.
Hazama, Akihiro1; Miyake, Masao1; Saito, Akiko1;
Suzuki, Daiji1; Wada, Ikuo2 (1Dept. Physiol. Fukushima Med.
Univ. Fukushima, Japan; 2Dept. Cell Science, Fukushima Med.
Univ. Fukushima, Japan)
Short-chain fatty acids (SCFAs), including acetate, propionate and butyrate, are the products of bacterial fermentation from indigestible dietary fibers in the large intestine. SCFAs have been known to play a
variety of physiological and pathophysiological roles for intestine. We
have recently reported the mechanism of SCFA-induced responses to the
motility of the rat distal colon (Ono et al. Jpn J Physiol 65: 69-76, 2004;
Mitsui et al. Neurogastroenterol Motil 17: 585-594, 2005). These results
suggest that SCFAs are sensed at mucosa and modulate the colonic motility through the enteric nervous system as a neural reflex. However, the
mechanism of the sense of SCFA is currently unknown. In 2003, two orphan G protein-coupled receptors, GPR41 and GPR43, have been identified as the SCFA receptors (Brown et al. J Biol Chem 278: 1131211319, 2003; Le Poul et al. J Biol Chem 278: 25481-25489, 2003). The
present study shows the localization of GPR43 in the rat intestine by RTPCR, Western blotting and immunohistochemistry. The results of the
present study indicate that GPR43 is expressed by enteroendocrine cells
and mucosal mast cells in the rat intestine.
Too much increase of the plasma membrane permeability causes necrotic death in the many types of cells. Detail mechanisms of this type of cell
death are still unclear. We use amphotericin B (amB) as the membrane
pore formation agent and investigate the ionic dependency on the cell
death caused by amB. First we apply propidium iodede (PI) to the HeLa
cells together with 10 µg/ml amB and observe the staining nucleus by PI
using the fluoresent microscope. 2 hours after apllication of amB, we
could observe the PI signal in the nucleus, indicating that the large pores
were formed after the application of amB. These pores should be larger
than the pores formed by amB because PI did not enter into the cells just
after amB application. Cl– replacement by gluconate or Cl– channel
blocker, DIDS (0.5 mM) inhibited the staining of nulueus after amB
application. Next we stained the lysosomes in the HeLa cells by FLlabelled pepstatin A. In the control condition, fluorescent dots were
observed around the nucleus and such dots gradually disappered after
amB application. Low Cl– condition or DIDS application inhibited the
disappearance of the fluorescent dots by amB. These results suggest that
the Cl– ions can enter into the cell after amB application and those ions
may couse the disruption of lysosome, which enhances the membrane
permeability increase by the attacking membrane proteins by the
lysosomal enzymes.
Catecholamine-induced apoptosis in
cultured striatal neurons in 2 weeks old
Suzuki, Sayaka; Tsunematsu, Takashi; Minamisawa,
Susumu; Iwatsubo, Kousaku; Nakamura, Fumi; Otsu,
Koji; Ishikawa, Yoshihiro (Dept. of Physiol. Yokohama-City
Role of plasma membrane Ca2+-ATPase
(PMCA) in contraction-relaxation
processes of the bladder using PMCA gene
Ishida, Yukisato; Liu, Li; Okunade, Gbolahan; Shull,
Gary; Paul, Richard (Univ. Cincinnati Coll. Med., Cincinnati,
Univ. Grad Sch. of Med., Yokohama,Japan)
Ohio, USA)
[Background] cAMP is produced from ATP by adenylylcyclase. Despite
various G-protein coupled receptor (GPCR) signals share cAMP as second messenger, physiologicaleffects of stimulation may not be identical
amongreceptors. In addition, the role of cAMP in developing apoptosis
is not well understood in neuronal cells, especially during development.
[Method] We first examinedthe effects of stimulating two GPCR on neuronal cell apoptosis using neonatal striatal neurons. Then, weinvestigated developmental changes of such effects in striatal neurons obtained
from 2 week old mice by a newly developed culture technique in our laboratory. Cultured striatal cells were incubated with isoproterenol or
dopamine, then, apoptosis was evaluated. [Result] In neonatal neurons,
neither isoproterenol nor dopamine stimulation induced apoptosis. In
contrast, in neuronsfrom 2 week old mice, TUNEL staining and DNA
fragmentation ELISA revealed that dopaminergic receptor stimulation
significantly increased the number of apoptotic cells.Western blot analysis revealed that only isoproterenol stimulation increased phosphorylation of Akt and MAP kinase. In contrast, both isoproterenol and
dopamine stimulation increased cAMP. Accordingly, only dopamine
stimulation induced cellular apoptosis while isoproterenoldid not, presumably due to cytoprotective effect through Akt and/or MAP kinase activation. [Conclusion] The role of cAMP in developing neuronal
apoptosis differs among GPCRas well as in developmental stages.
Role of PMCA in smooth muscle contractility was investigated using the
bladder isolated from PMCA gene manipulated mice: PMCA4 null mutant (Pmca4–/-) and PMCA1 and PMCA4 double gene targeted (Pmca1+/
-4–/-) mice. Western blot shows the loss of PMCA4, a major isoform, but
not PMCA1, sarco-endoplasmic reticulum ATPase (SERCA) and Na+/
Ca2+ exchanger (NCX), in the muscle layer preparation of Pmca4–/- and
Pmca1+/-4–/-. Half-times of contraction and relaxation upon treatment
with 80 mM KCl were determined. Surprisingly, half-times of contraction in Pmca4–/- and Pmca1+/-4–/- muscles tended to be prolonged, when
compared with that in WT muscle. Relaxation half-times were also prolonged in the gene manipulated muscles, as expected. On the other hand,
inhibition of SERCA or NCX marginally shortened the contraction halftime and prolonged the relaxation half-time in muscles of all tested genotypes. Using relaxation half times, the contribution of PMCA to relaxation was calculated to be 25%, SERCA 20% and NCX 70%. PMCA and
SERCA appeared to function additively, but the function of NCX might
overlap with those of other components. FuraPE3 signal shows that the
basal level of [Ca2+]i slightly increased in Pmca1+/-4–/- muscle. In summary, the gene manipulation of PMCA indicates that PMCA, in addition
to SERCA and NCX plays a role in both excitation-contraction coupling
and Ca2+-extrusion-relaxation relationship, i.e., Ca2+ homeostasis, of the
bladder smooth muscle.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Transport across cell membrane
Study of the role of Fyn in the
formation of stress fibers and filopodia-like
protrusions in NIH3T3 fibroblasts
Xu, Dan1,2; Kishi, Hiroko1,2; Kawamichi, Hozumi1;
Kajiya, Katsuko1; Kobayashi, Sei1,2 (1Dept. Mol. Physiol.,
Cell. Dig. Bioreg.
Yamaguchi Univ. Sch. Med., Ube, Japan;
Grad. Sch. Med. Yamaguchi Univ, Ube, Japan)
We previously showed that Fyn, a member of Src family tyrosine kinase
(Src-TK), was involved in the activation of Rho-kinase (ROK) to develop Ca2+ sensitization of vascular smooth muscle contraction induced by
sphingosylphosphorylcholine (SPC). Recently we found that SPC can
stimulate the formation of stress fibers and filopodia-like protrusions in
NIH3T3 fibroblasts through Src-TK/ROK-dependent and independent
pathway, respectively. Then, we further investigated the role of Fyn in
the SPC-induced formation of stress fibers and filopodia-like protrusions
with RNA interference method. Three different Fyn siRNAs were designed and transfected into NIH3T3 cells using Lipofectamine 2000. As
control, a non-silencing siRNA and a positive MAPK1 control siRNA
were transfected in parallel. The non-silencing AF 488-labeled siRNA
was used to monitor transfection efficiency. Our results showed that
transfection efficiency was high above 90% and the down-regulation of
Fyn expression was confirmed in western blot with concentration-dependency and incubation time-dependency. SPC-induced stress fiber
formation was partially inhibited by Fyn siRNAs, but not by other control siRNAs. The formation of filopodia-like protrusions induced by
SPC was not affected by Fyn siRNAs. These findings suggest that Fyn
plays a role in SPC-induced stress fiber formation, but not in the formation of filopodia-like protrusions in NIH3T3 cells.
Effects of osmolarity change on fluid
secretion by the perfused submandibular
glands in normal and low-AQP5 rats
Murakami, Masataka1; Murdiastuti, Kuwartarini2; Hosoi,
Kazuo3; Hill, Adrian E4 (1Nano-Struc Physiol, NIPS, NINS,
Okazaki, Japan; 2Periodontol, Fac Dent, Gadjah Mada Univ,
Jogjakarta, Indonesia; 3Mol Oral Physiol, Inst Healthbiosci, Univ
Tokushima Grad Sch, Tokushima, Japan; 4Physiological
Laboratory, Cambridge Univ, Cambridge, UK)
Osmolarity changes created by sucrose during the perfusion of isolated
rat submandibular glands (SMG) in vitro alter secretion rates much more
than predicted by the osmotic theory of fluid production. However, these
are in accord with a theory involving AQP5 feedback control of paracellular fluid transfer (Hill & Shachar-Hill 2002). The changes in transport
rate can be predicted with parameters determined earlier for this gland
(Murakami et al. 2001) and a model of the SMG system is presented. Experiments were performed with SMG from genetically selected rats that
have very low levels of AQP5 as determined by Western blotting (Murdiastuti, K. et al. 2002). The fluid secretion rates after osmolarity changes were those expected for the osmotic theory. We suggest that control of
paracellular flow has been lost in these low AQP5 rats which have reverted to osmotic fluid production. Retrograde injection of Hg ions into
the duct partially inhibited AQP5, leading to a concentration-dependent
reduction in flow rates. However, reduction of fluid secretion after osmolarity changes was still close to that of normal rats. The results suggest the involvement of a feedback loop including AQP5 and
paracellular fluid transport.
Ethanol alters anion composition of the
fluid secreted by guinea-pig pancreatic
Ishiguro, Hiroshi1; Yamamoto, Akiko1; Ko, Shigeru2;
Kondo, Takaharu1; Naruse, Satoru2 (1Human Nutrition,
Nagoya Univ. Grad. Sch. Med., Nagoya, Japan; 2Gastroenterology,
Nagoya Univ. Grad. Sch. Med., Nagoya, Japan)
Transport across cell membrane
We reported that 1 mM of ethanol (relevant to blood level after social
drinking) potentiated secretin-stimulated fluid secretion in interlobular
ducts isolated from guinea-pig pancreas. In this study, we examined anion composition of the secreted fluid during the hypersecretion. Isolated
ducts were cultured overnight, during which time both ends of the ducts
sealed. The lumen was punctured and injected with solutions containing
BCECF-dextran or ABQ-detxtran (plus Cl-NERF-dextran as ratiometric
reference). Luminal pH (pHL) and luminal Cl– ([Cl–]L) were estimated
by microfluorometry. Images of luminal fluorescence were obtained at
1-min intervals and the rate of fluid secretion was calculated from the increment of luminal volume. During stimulation with secretin (10 nM),
the rate of fluid secretion was 2.28±0.14 nl min–1 mm–2 (per unit area of
epithelium, n=5, mean±SE), pHL increased due to HCO3– secretion, and
[Cl–]L steadily decreased. The net Cl– transport calculated from [Cl–]L
and luminal volume was nearly zero. Ethanol (1 mM) increased secretinstimulated fluid secretion to 4.11±0.22. pHL slightly decreased and [Cl–
]L was stable in the presence of ethanol. The net Cl– transport was
0.265±0.042 nEq min–1 mm–2. The increase of fluid secretion by ethanol
was almost equivalent to fluid secretion accompanying Cl– efflux.
Ethanol induced Cl– secretion during secretin stimulation without
affecting HCO3– secretion, which resulted in the increase of Cl– of
secreted fluid from zero to about 70 mM.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Transport across cell membrane
Functional characterization of L1156F
CFTR: a newly identified mutation in
Japanese patients with chronic
Ko, Shigeru1; Zeng, Weizhong2; Fujiki, Kotoyo1;
Nakakuki, Miyuki1; Ishiguro, Hiroshi1; Yamamoto,
Akiko1; Yoshikawa, Toshiyuki1; Kondo, Takaharu1; Goto,
Hidemi1; Muallem, Shmuel2; Naruse, Satoru1 (1Nagoya
Univ. Grad. Sch. Med. Nagoya, Japan; 2Dep. Physiol. UT
Southwestern Med. Ctr at Dallas, Dallas, USA)
Cystic fibrosis transmembrane conductance regulator (CFTR) is a
cAMP-regulated chloride channel that plays an important role in bicarbonate transport in the exocrine pancreas. Accumulating evidence suggests that CFTR mutations are associated with a subset of patients with
chronic pancreatitis. We have identified a new point mutation (L1156F)
in exon 18 in patients with chronic pancreatitis. In order to elucidate a
disease associated with this mutation, we examined the function of
L1156F-CFTR. The mutation was introduced in pCMV-wild type (WT)
CFTR plasmid by site directed mutagenesis. Chloride channel activity
was measured in HEK293 cells expressing either WT or L1156F-CFTR
by whole cell current recording. The amount of CFTR proteins expressed was analyzed by immuno-blotting using an anti-CFTR antibody.
When stimulated with 10µM forskolin, WT and L1156F generated a
chloride current of 1534±72 and 476±45 pA (n=5), respectively. The
introduction of the L1156F mutation did not affect the expression of
CFTR protein compared with the WT. In conclusion the L1156F
mutation reduces the CFTR chloride current by 69%. The lack of lung
and intestinal symptoms and the chronic pancreatitis in these patients
further highlight that low activity of CFTR is sufficient for normal lung
and intestinal function and the particular susceptibility of the pancreas to
mutations in CFTR.
Properties of a novel splicing variant of
ATP1AL1, a human non-gastric proton
Sakai, Hideki; Ohira, Yuta; Anze, Taketo; Takahashi,
Yuji; Morii, Magotoshi; Takeguchi, Noriaki (Dept.
Pharmaceu. Physiol., Fac. Pharmaceu. Sci., Univ. Toyama, Toyama,
Rat non-gastric H+,K+-ATPase is highly expressed in the distal colon and
may be associated with K+ conservation in the colon. But physiological
function of human non-gastric H+,K+-ATPase (ATP1AL1) has not been
clarified yet. Here, we have cloned a normal type (NT) and a novel splicing valiant deleting exon4 (∆exon4) of ATP1AL1. Then, the stable cell
lines expressing gastric H+,K+-ATPase β-subunit (gHKβ) were
transfected with the pcDNA4/His-ATP1AL1 cDNA (NT or δexon4)
construct. The activities of the K+-dependent ATPase and the 86Rb+
uptake of ATP1AL1 were estimated by subtracting 1 mM ouabainsensitive activity from 5 µM ouabain-sensitive activity. These activities
of ATP1AL1 were also measured by using 100 µM SCH 28080, an
inhibitor of gastric H+,K+-ATPase. We found that δexon4 is expressed
in the plasma membrane of the cells. Similar to the case for NT, the
gHKβ was required for expression of δexon4. We found that δexon4 has
no activities of the K+-dependent ATPase and the 86Rb+ uptake. When
NT and δexon4 were co-transfected into the stable cell lines expressing
gHKβ, the 86Rb+ uptake activity was significantly lower than that in the
cells transfected with NT alone. Apparently, δexon4 had no effect on the
level of expression of NT in the cells. These results suggest that δexon4
exerts a dominant negative effect on NT.
Expression of K+-Cl– cotransporters in
gastric parietal cells
Fujii, Takuto; Itomi, Yasuo; Takahashi, Yuji; Morii,
Magotoshi; Takeguchi, Noriaki; Sakai, Hideki (Dep.
Pharm. Physiol., Fac. Pharm. Sci., Univ. Toyama, Toyama, Japan)
For gastric acid (HCl) secretion, protons are actively secreted by H+,K+ATPase in the apical membrane of gastric parietal cells, but it has not
been established what molecule contributes to Cl– secretion. K+-Cl–
cotransporter (KCC) plays a significant role in epithelial transport and
cell volume regulation. Four KCC isoforms have been cloned. KCC1, 3,
4 are widely expressed and KCC2 is neuron specific. However, the expression of KCC isoforms in stomach has not been reported. Here we examined whether the KCC isoforms are expressed in gastric parietal cells.
Western blot analysis showed that KCC3 and KCC4 were expressed in
isolated gastric mucosa of rats and mice. Immunohistochemistry in the
isolated gastric mucosa showed that KCC3 was expressed in the basolateral membrane and KCC4 was expressed in the apical membrane of the
gastric parietal cells. Interestingly, KCC3 and KCC4 were abundantly
expressed in the parietal cells located at luminal segment of the gland.
Because luminal segment parietal cells are much more active in HCl secretion than those of the basal segment, KCC3 and KCC4 may be involved in the mechanism of HCl secretion. We constructed the T-REx
system for KCC3 in the LLC-PK1 cells. Tetracycline-induced expression of KCC3 protein significantly increased ouabain-sensitive Na +,K+ATPase activity.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Heart & circulation
Axial Stretch Acutely Increases Ca2+ Spark
Rate in Rat Ventricular Myocytes
Iribe, Gentaro1; Ward, Christopher2; Lederer,
Jonathan2; Kohl, Peter1 (1University of Oxford, Oxford, UK;
Heart & circulation
The slow response of myocardial contractility to stretch has been linked
to a nitric oxide mediated stretch-induced increase in ryanodine receptor
Ca2+ release, revealed as an elevated diastolic Ca2+ spark rate in rat ventricular myocytes exposed to 10% stretch for 10 min (Vila Petroff et al.
2001). Here, we investigate whether stretch-induced changes in Ca2+
spark rate may occur more rapidly. Isolated rat ventricular myocytes
were exposed to 10% axial stretch using computer-controlled piezo-manipulated carbon fibres, attached to the centre and one end of a cell. Controlled and reversible stretch was applied selectively to a half-cell only,
allowing the non-stretched part to serve as control. Diastolic spark rate
was studied using a Zeiss 510 system and software detection of signal
deviation from background by > 2 S.D. Within 10 s, axial stretch transiently increased Ca2+ spark rate by 31 ± 6.5% (n = 8, p < 0.05), followed
by return to background levels within 1 min. The response was not
blunted by 1 mM L-NAME (nitric oxide synthase inhibitor; n = 7). We
conclude that: i) axial stretch acutely raises diastolic Ca2+ spark rate in
rat ventricular myocytes; ii) underlying mechanisms differ from those
involved in the slow response to stretch. This study is supported by the
British Heart Foundation and Eisai Co., Ltd.
of Maryland, Baltimore, MD, USA)
Inward rectifier K+ current IK1 and cardiac
repolarization: a simulation study using the
Kyoto model
β-adrenergic stimulation does not enhance
Na/Ca exchange current in guinea-pig,
mouse and rat ventricular myocytes
Ishihara, Keiko1; Asakura, Keiichi2; Sarai, Nobuaki3;
Matsuoka, Satoshi3; Noma, Akinori3 (1Dept. Physiol.,
Matsuoka, Satoshi1; Lin, Xue2; Jo, Hikari2; Sakakibara,
Yutaka2; Tambara, Keiichi2; Kim, Bongju1; Komeda,
Masashi2 (1Dept. Physiol. and Biophys., Grad. Sch. Med., Kyoto
Facult. Med., Saga Univ., Saga, Japan; 2Nippon Shinyaku, Co., Ltd.,
Kyoto, Japan; 3Dept. Physiol. and Biophys., Grad. Sch. Med., Kyoto.
Univ., Kyoto, Japan)
IK1 is known to be responsible for the late rapid repolarization phase of
action potential in cardiac ventricular myocytes, yet its role in the slow
repolarization phase remains unclear. The amplitude of IK1 is determined
by voltage-dependent block of the channel by internal spermine (SPM)
and Mg2+. During repolarization, the release from the Mg2+ block in the
presence of SPM induces a significant transient component of IK1 in
voltage-clamp experiments. We developed a new model of the IK1 channel, which includes the high- and low-affinity modes of blocks by SPM
and by Mg2+ (Yan & Ishihara, J. Physiol. 563, 2005), and examined the
participation of this dynamic gating of IK1 channel to action potential repolarization by incorporating it into the Kyoto cardiac ventricular cell
model. The model shows that the Mg2+-induced transient component of
IK1 appears during the slow phase of repolarization. Thus, changes in the
IK1 density significantly alter the action potential duration (APD), as has
been demonstrated in experiments. A decrease in the Mg2+ concentration
or an increase in the SPM concentration prolongs APD by reducing the
transient component of IK1. Under this pathological condition, if the rapid component of delayed rectifier K+ current is blocked, APD is markedly prolonged. This model study predicts that the internal spermine and
Mg2+ are important factors affecting the occurrence of early afterdepolarization and arrhythmia.
Univ., Kyoto, Japan,; 2Dept. Cardiovascular Surgery, Grad. Sch.
Med., Kyoto Univ., Kyoto, Japan,)
The effect of β-adrenergic stimulation on cardiac Na/Ca exchange
current (INCX) has been controversial. Recently, 25 - 400% increase by
isoproterenol of INCX was reported. To reexamine this effect, we
measured INCX in voltage-clamped guinea-pig, mouse and rat ventricular
cells. When INCX was defined as a 5 mM Ni2+-sensitive current in guineapig ventricular myocytes, 1 µM isoproterenol apparently augmented
INCX by about 32%. However, this increase was probably due to
contamination of the cAMP-dependent Cl– current (ICFTR), because Ni2+
inhibited the activation of ICFTR by 1 µM isoproterenol, with a halfmaximum concentration of 0.5 mM under the conditions where INCX was
suppressed. 5 or 10 mM Ni2+ did not inhibit ICFTR activated by 10 µM
forskolin, an activator of adenylate cyclase, suggesting that Ni2+ acted
upstream of adenylate cyclase in the β-adrenergic signaling pathway.
Furthermore, in a low Cl– bath solution, 1 µM isoproterenol did not
significantly alter the amplitude of Ni2+-sensitive INCX at +50 mV, which
was close to the reversal potential of ICFTR. No change in INCX amplitude
was induced by 10 µM forskolin. When INCX was activated by external
Ca2+, it was not significantly affected by 1 µM isoproterenol in guineapig, mouse or rat ventricular cells. We concluded that β-adrenergic
stimulation does not have significant effects on INCX in guinea-pig,
mouse or rat ventricular myocytes.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Heart & circulation
Lysosomal degradation may accelerate
down-reguration of cardiac gap junction
protein Connexin-43 (Cx43) via protein
kinase C (PKC)-mediated hyperphosphorylation
Hai, Lin1; Ogawa, Koichi2; Imanaga, Issei3; Inoue, Ryuji1
(1Dept. Physiol., Fukuoka-univ. Sch. Med., Fukuoka, Japan; 2Dept.
Anat., Fukuoka-univ. Sch. Med., Fukuoka, Japan; 3General Res.
Cent. Med. Sci., Fukuoka-univ. Sch. Med., Fukuoka, Japan)
It is well accepted that gap junctions (GJs) play a pivotal role in the intercellular spread of electrical flows between cardiac myocytes. Dysfunction of GJs can thus be one of major arrhythmogenic factors. In this
study, we have obtained the evidence that in both type I and II diabetic
hearts, myocardial intercellular communication through GJs is impaired
via PKC-mediated hyper-phosphorylation of the GJ protein Cx43. Western blot and immunohistochemical analyses of the PKC-activated diabetic hearts indicated that the expression level of Cx43 is significantly
reduced compared with control hearts, with altered distribution of sparse
or sporadic pattern at the intercalated disk. These alterations were ameliorated by treatment with lysosomal inhibitors as well as PKC inhibitors, but could not be prevented by the proteosmal inhibitor ALLN.
These results strongly suggest that facilitated lysosomal degradation of
Cx43 via PKC-mediated hyper-phosphorylation may underlie the downregulation of Cx43 protein in rat diabetic hearts. This mechanism may in
part account for the reported vulnerability of rat diabetic hearts to ventricular fibrillation.
Essential role of phosphoinositide 3kinase class IIα in Ca2+-dependent Rho
GTPase activation and contraction in
vascular smooth muscle cells.
Yoshioka, Kazuaki; Azam, Mohammed Ali; Miyazawa,
Hidekazu; Takuwa, Noriko; Sugimoto, Naotoshi;
Takuwa, Yoh (Kanazawa Univ. Med., Ishikawa, Japan)
We previously demonstrated that excitatory agonists such as noradrenaline (NA) and membrane depolarization induce Ca2+-dependent activation of Rho GTPase in vascular smooth muscle (VSM) cells, resulting in
inhibition of myosin phosphatase (MP) through the mechanisms involving Rho kinase-mediated phosphorylation of its regulatory subunit
MYPT1/MBS. We found that phosphoinositide 3-kinase class IIα
(PI3K-C2α) plays an essential role in NA-induced Rho activation and
contraction in differentiated VSM primary cultured-cells. In the present
study we show that ionomycin, a Ca2+ ionophore, induced contraction
with stimulated phosphorylation of MYPT1. Ionomycin-induced
MYPT1 phosphorylation and contraction was inhibited by the Rho
kinase inhibitor Y-27632. Silencing PI3K-C2α, but not PI3K p110α,
expression by small interfering RNA (siRNA) in differentiated VSM
cells inhibited ionomycin-induced phosphorylation of MYPT1,
consequent reinforcement of 20-kDa myosin light chain (MLC)
phosphorylation and contraction. Consistent with this, the PI3K
inhibitors Wortmannin and LY294002 inhibited both MLC
phosphorylation and contraction. These findings indicate an essential
role of PI3K-C2α in Ca2+-dependent, Rho/Rho kinase-mediated
negative control of MP and VSM contraction.
New function of creatine kinase in
Takahashi, Eiji (Department of Physiology, Yamagata University
School of Medicine, Yamagata, Japan)
Present study was designed to demonstrate intracellular radial gradients
of myoglobin oxygen saturation and ATP in single cardiomyocytes with
elevated oxygen demand. Intracellular ATP concentration was indirectly
assessed from intracellular free Mg 2+ concentration using a Mg2+ sensitive ratiometric fluorescent dye mag-fura-2 (MF2). A bright field fluorescence microscopy was newly manufactured for simultaneous
measurements of myoglobin light absorption (spectrophotometry) and
MF2 fluorescence (fluorometry). Uncoupler of oxidative phosphorylation (1 µM CCCP) was used to increase oxygen flux. While significant
intracellular gradients of myoglobin oxygen saturation was
demonstrated in CCCP treated cells at physiological extracellular Po2,
no significant heterogeneity was found in MF2 fluorescence. In contrast,
in cardiomyocytes treated with 0.5 mM iodoacetamide (a blocker of
creatine kinase) in addition to CCCP, gradients of MF2 fluorescence
from the sarcolemma to the cell core (indicating radial gradients of ATP)
were demonstrated. Such MF2 fluorescence gradients were not
demonstrated when extracellular oxygen concentration was elevated to
20%. From these results, it is concluded that significant gradients of ATP
may be produced in the isolated single cardiomyocyte when
mitochondrial oxygen consumption is moderately elevated at
physiological extracellular Po2. However, such potential ATP
heterogeneities are effectively buffered in the normal cardiomyocyte by
ATP supplementations from creatine phosphate. Thus, present results
propose a new function of creatine kinase in the myocardium. This study
was supported by JSPS KAKEN #15390061.
Identification of dynamic and static
characteristics of a baroreflex system
using a neural cascade
Kawada, Toru; Kamiya, Atsunori; Shishido, Toshiaki;
Sugimachi, Masaru (Dept. of Cardiovasc. Dynamics, Adv. Med.
Eng. Cntr, Natl. Cardiovasc. Cntr. Res. Inst., Osaka, Japan)
Background: Identification of dynamic and static characteristics of a
given biological system promotes the understanding of the system behavior under a variety of circumstances. Purpose: To estimate the dynamic and static characteristics of a baroreflex neural arc from pressure
input to efferent sympathetic nerve activity, we developed a system identification method using a neural cascade. Method: A "neuron" used in
a neural network can represent the dynamic linear element followed by
a nonlinear transfer function. By connecting two neurons in series, we
can represent a system comprised of dynamic-linear (L1), static-nonlinear (NL), and dynamic-linear (L2) subsystems. Because the contamination of noise to the observed output resulted in biased estimates of the
system characteristics, we added an iterative noise cancellation procedure where the noise was estimated by an autoregressive model. Results: In a simulation study, the neural cascade effectively identified the
dynamic and static characteristics of an L1-NL-L2 system. The baroreflex neural arc is known to have derivative characteristics followed by a
sigmoidal nonlinearity. When applied to the actual input-output data of
the baroreflex neural arc obtained from rabbits, the neural cascade could
identify the derivative characteristics followed by the sigmoidal nonlinearity. Conclusion: The neural cascade proposed in the present study
may provide a useful method to simultaneously identify the dynamic and
static characteristics of a biological system.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Respiration
Antioxidative effect of estrogen in
hypertensive Dahl salt-sensitive rats
Fujii, Shigemoto; Zhang, Ling; Kosaka, Hiroaki (Dept.
Cardiovasc. Physiol. Facult. Med. Kagawa Univ., Kagawa, Japan)
Estrogen deficiency in the menopause is associated with an increased
cardiovascular risk. Endogenous estrogen has been suggested to exert
vasoprotective effects through decreasing vascular oxidative stress. To
investigate the mechanism of the decreasing in oxidative stress by estrogen, we examined superoxide production and antioxidant enzyme expression in aorta in ovariectomized Dahl salt-sensitive (DS) rats. Female
DS rats (8 weeks old) were ovariectomized (OVX group) or sham-operated (sham group). Estrogen pellets were subcutaneously implanted in
ovariectomized rats for estrogen treatment (OVX + E group). After 4
weeks of salt-loading (8% NaCl diet), blood pressure was increased in
OVX group compared with sham and OVX + E groups. Superoxide production in aortic ring was higher in OVX group than in sham and OVX
+ E groups. Increase in superoxide production was abolished by pretreatment with diphenyleneiodonium, a NADPH oxidase inhibitor. Expression of mRNA of p22phox, a NADPH oxidase subunit, increased in
aorta from OVX group compared with sham group. In contrast to
p22phox, mRNA expressions of antioxidant enzymes extracellular superoxide dismutase (ecSOD) and glutathione peroxidase (GPX) were
decreased in OVX rats. Expression levels of p22phox, ecSOD and GPX
in OVX + E rats were not different from that in sham group. These data
suggest that estrogen deficiency in ovariectomized DS rats enhances oxidative stress through increased NADPH oxidase expression and decreased antioxidant enzymes, and promotes vascular injury by saltloading.
Age-dependent roles of an ATP-sensitive
potassium channel Kir6.2 in the hypoxic
ventilatory response in the mouse
Oyamada, Yoshitaka1; Nakatani, Michie2; Harada,
Naoko2; Ishizaka, Akitoshi2; Okada, Yasumasa3 (1Dept. of
Respiratory Med., Tokyo Medical Center, National Hospital
Organization, Tokyo, Japan; 2Dept. of Pulmonary Med., School of
Med., Keio Univ., Tokyo, Japan; 3Dept. of Med., Keio Univ.
Tsukigase Rehabilitation Center, Izu city, Japan)
Acute hypoxia elicits a biphasic ventilatory response, initial augmentation and subsequent depression. Although oxygen-sensitive channels of
type I cells in the carotid body are considered to be involved in the initial
augmentation, the underlying cellular mechanism for the subsequent depression, hypoxic ventilatory decline (HVD), has not been fully elucidated. The purpose of the present study is to examine the role of an ATPsensitive potassium channel, Kir6.2, in the hypoxic ventilatory response
including HVD in the mouse. We serially measured minute ventilation
volume (Ve) of the Kir6.2-knockout mouse (Kir6.2-/-: n = 5) exposed to
hypoxia (12% O2 in N2: 10min) in an unanesthetized unrestrained state
by whole body plethysmography in the 2nd and 4th postnatal weeks.
Percent changes from the baseline Ve in the room air were calculated and
compared with that in the C57BL/6 mouse (n = 10). In the 2nd postnatal
week, there was no difference in the hypoxic ventilatory response between the C57BL/6 and Kir6.2-/- mice. Meanwhile, in the 4th week, the
initial augmentation lasted longer, and HVD was much weaker in the
Kir6.2-/- than in the C57BL/6. It is concluded that Kir6.2 is involved in
the hypoxic ventilatory response including HVD in an age-dependent
manner in the mouse.
Changes in electroencephalogram and
cerebral blood flow during frequent yawns
in human
Seki, Yoshinari; Fumoto, Masaki; Nakatani, Yasushi; Yu,
Xinjun; Nakasato, Akane; Kambayashi, Eri; Kikuchi,
Hiromi; Sato-Suzuki, Ikuko; Arita, Hideho (Dept. Physiol.,
Toho Univ. School of Medicine, Ota-ku, Tokyo, Japan)
Our previous studies showed that frequent yawns can be evoked by microinjection of L-glutamate into the paraventricular nucleus (PVN) in an
anesthetized, spontaneously breathing rat. The yawning response was
characterized by an arousal shift in the ECoG to lower voltage and faster
rhythms. We focused the present study upon frequent yawns in human.
Yawns occurred once a few minutes for more than 5 minutes in this
study. We monitored electroencephalogram (EEG) and cerebral blood
flow (CBF) using near-infrared spectroscopy (NIRS). We found a shift
in the EEG to lower voltage and faster rhythms and an increase of CBF
in ventromedial prefrontal cortex (PFC) during and immediately after
yawning responses. These results suggest that the activation of ventromedial PFC may be associated with an arousal/yawning response in human.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Respiration
Metabolic changes in cerebral cortex and
spectral analysis of EEG during long-term
breath holding in professional divers
Fumoto, Masaki; Seki, Yoshinari; Nakasato, Akane;
Nakatani, Yasushi; Kikuchi, Hiromi; Yu, Xinjun;
Kambayashi, Eri; Sato-Suzuki, Ikuko; Arita, Hideho
(Dept. Physiol., Toho Univ. School of Medicine, Ota-ku, Tokyo)
Factors affecting expired minute volumes
of low molecular weight compounds
Shimouchi, Akito; Okina, Masako; Hayashi, Hiroko;
Shimouchi, Supika; Mori, Mariko; Ukai, Kazutoshi (Dept.
Etiology and Pathogenesis, National Cardiovascular Center
Research Institute, Suita, Osaka, Japan)
We investigated metabolic changes in prefrontal and parietal cortices
and EEG changes during a long-term breath holding (BH) in professional divers. He/she performed BH for 2 to 7 minutes following a preparatory period of approximately 5 minutes. Such BH procedures were
repeated 3 times in this study. We measured metabolic changes in cerebral cortex using near-infrared spectroscopy (NIRS) and made spectral
analysis of EEG before, during and after BH. Concentration of deoxygenated hemoglobin (deoxyHb) showed a gradual increase during BH in
both prefrontal and parietal cortices. There is a linear relationship between the maximal level of deoxyHb and the duration of BH. In contrast,
concentration of oxygenated hemoglobin (oxyHb) of BH decreased
along with prolonged duration of BH. Voluntary abdominal breathing
(VAB) was performed during the preparatory period before BH. Spectral
analysis of EEG was focused on the high-frequency alpha (HF-alpha)
band (10-13Hz) in this study. The higher power of HF-alpha band was
observed before and immediately after the onset of BH, and thereafter
the HF-alpha power exhibited a gradual decrease until the end of BH.
Since previous study showed that VAB produced increase in HF-alpha
power and urinary 5-HT level (fumoto et al, 2004), we suggest that the
higher HF-alpha power evoked before BH may contribute to the tolerance to cerebral hypoxia during BH.
Chemical compounds in breath originate mainly from volatile metabolic
products in health and diseases. Among them, hydrogen and methane are
believed to represent the conditions of gastro-intestinal systems in the
presence of bacterial flora. On the other hands, carbon monoxide and nitric oxide are well-known bioactive chemicals to play important roles in
physiological and pathophysiological conditions. Because matrix analysis based on simultaneous measurements of exhaled chemicals with such
low mass numbers may give us important information of physical conditions, we conducted the following experiment.Nine hundreds and forty
two subjects aged from 20 to 88 years old volunteered for the present
study. After tooth brushing and gargling, fasted subjects breathed purified artificial air via a mouthpiece in a sitting position for 12 min, during
which exhaled air was collected via one-way valve into the Douglas bag.
Minute ventilation volume and concentrations of carbon dioxide and oxygen in exhaled air were monitored by respiratory metabolic analyzer
with the chemical sensors. Concentrations of hydrogen, methane, carbon
monoxide and nitric oxide in the bag were analyzed by gas chromatography and chemiluninescence methods. We will present the summarized
results of the relationships between minute expired volumes of these
compounds and such physical conditions as age, gender, laboratory data,
scores of health-related questionnaires, diseases and disorders by using
the multivariate analysis.
Ventilatory long-term facilitation following
intermittent hypoxia is state-dependent in
Acute effects of thixotropy conditioning of
inspiratory muscles on end-expiratory
chest wall volume
NAKAMURA, AKIRA; Wenninger, JM; Olson, JR., EB;
Bisgard, GE; Mitchell, GS (Dept Comp Biosci, Univ Wisconsin-
Izumizaki, Masahiko; Iwase, Michiko; Ohshima,
Yasuyoshi; Homma, Ikuo (Showa Univ. Sch. Med., Tokyo,
Madison, Madison, WI, USA)
Ventilatory long-term facilitation (vLTF) following acute intermittent
hypoxia (AIH) has been reported to variable extent in unanesthetized
rats. However, none of these studies reported sleep-state, a critical variable in many physiological functions. We hypothesized that vLTF would
be preferentially expressed in sleeping vs. awake Lewis rats following
AIH. The sleep-wake state of unrestrained rats was determined from implanted EEG and nuchal EMG electrodes. Tidal volume (VT), frequency
(f), minute ventilation (VE) and CO2 production (VCO2) were determined in unanaesthetized male Lewis rats via plethysmography before,
during and after AIH (five, 5-min exposures, 10.5% O2; 5-min normoxic
intervals) or acute sustained hypoxia (25-min exposures, 10.5% O2,
ASH). VE, VT and f in quiet wakefulness (QW) or NREM sleep were
normalized to its own baseline value during the corresponding state during baseline, pre-hypoxia conditions. LTF was observed in VE after
AIH, but not ASH. Following AIH in NREM, VE gradually increased
and reached maximum level at 20 min post-hypoxia, remaining at that
level for at least 60 min (26.6±5.2% baseline). The main contributor to
vLTF was VT (13.5±2.3%), with a lesser increase in f (7.4±1.7%). The
corresponding increase in VE/VCO2 was 35.5±2.4% baseline. In QW,
significant vLTF was not observed. The duration, magnitude, and
pattern in vLTF in NREM were similar to phrenic LTF in anesthetized
rats. In conclusion, vLTF is highly state and pattern sensitive in
unanesthetized rats. (Supported by NIH HL65383, HL07654,
Thixotropy is a passive property of the skeletal muscle that depends on
the muscle's immediate history of contraction and length change. We
showed that inspiratory muscle thixotropy affects the end-expiratory position of the rib cage. The present study aimed to test whether changes in
end-expiratory chest wall volume (Vcw) occurs after thixotropic inspiratory muscle conditioning in normal subjects (n = 32). We first examined
effects of the conditioning on end-expiratory Vcw of succeeding five
breath cycles with respiratory induction plethysmography. Subjects participated in the conditioning at three different Vcw (60% inspiratory capacity [IC] + end-expiratory Vcw of baseline breathing [EEB], EEB, and
residual volume [RV]) giving one of two levels of inspiratory effort (no
effort or maximal inspiratory effort) with airway closure in the sitting
position. End-expiratory Vcw increased after conditioning at 60% IC +
EEB and decreased after conditioning at RV. We then measured the time
course of changes in spirometrically determined IC, which confirmed
thixotropic changes in end-expiratory Vcw. A decrease in the IC was
found at 60 s after conditioning performed at the higher volume. However, the decrease disappeared until 180 s. Conditioning at the lower volume was followed by an increase in the IC, which was maintained even
after 180 s. Furthermore, the thixotropic inflation/deflation of the chest
wall was proved by the helium-dilution FRC and esophageal pressure
measurements. In conclusion, thixotropy conditioning of inspiratory
muscles changes end-expiratory Vcw.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Blood
Genetic or pharmacological ablation of
orexin attenuated and supplementation
ameliorated hypercapnic chemoreflex
Kinetics of tissue plasminogen activator
(tPA) exocytosis from endothelial cells and
its modulation by PA inhibitor-1 (PAI-1).
Deng, Ben-Shiang1; Nakamura, Akira2; Yanagisawa,
Masashi3; Fukuda, Yasuichiro2; Kuwaki, Tomoyuki1,2
Suzuki, Yuko; Ihara, Hayato; Mogami, Hideo; Urano,
Tetsumei (Hamamatsu Univ. Sch. of Med. Hamamatsu,)
(1Dept. Molec. Integ. Physiol., Chiba Univ. Grad. Sch. Med., Chiba
260-8670, Japan; 2Dept. Autonom. Physiol., Chiba Univ. Grad. Sch.
Med., Chiba 260-8670, Japan; 3Dept. Molec. Genetics, Univ. Texas
SWMC, Dallas TX, USA)
We examined whether the chemoreceptor reflex in prepro-orexin knockout mice was blunted or not, and if so, whether supplementation of exogenous orexin restore the abnormality. In addition, we studied whether
pharmacological blockade of orexin in the wild-type mice resulted in a
similar abnormality. A cannula for intracerebroventricular injection to
the lateral ventricle was implanted to the isoflurane-anesthetized mice
together with electrodes for recording electroencephalogram and electromyogram. Ventilation was recorded by whole body plethysmography
after recovery period of at least 7 days. After recording of baseline
breathing for 1 hr, orexin-A, -B, an orexin receptor antagonist, or vehicle
was intracerebroventriculary injected and hypercapnic or hypoxic gas
mixture was introduced into the recording chamber for 10 min. Data
were examined for only awake period because sleeping distorts
chemoreflex sensitivity. Hypercapnic ventilatory responses but not hypoxic responses were attenuated in orexin knockout mice as compared
to those in the wild-type littermates. Similar abnormality was reproduced in wild-type mice treated with orexin antagonist. Intracerebroventricular injection of orexin partially restored the hypercapnic
chemoreflex in the mutant mice. Our findings suggest that orexin plays
a crucial role for CO2-sensitivity at least during awake periods.
Background: Vascular endothelial cells (VECs) contribute to keep the
patency of vasculature through the regulated expression and secretion of
various molecules having either anti-coagulatory or high fibrinolytic activity. tPA, the primary PA in the vasculature, is secreted from VECs as
an active form and express fibrinolytic activity in blood. In blood there
also exist its specific inhibitor of PAI-1. Though the impaired tPA secretion as well as the elevated plasma concentration of PAI-1 are considered
to be risk factors for thrombosis, precise mechanism underlying in tPA
secretion is not clarified. Here, we analyzed the dynamics of tPA secretion from its containing granules and its modulation by PAI-1 using total
internal reflection fluorescence microscopy(TIRFM). Method: An established cell-line of VECs was cultured and transfected with tPA-GFP.
The dynamics of tPA-GFP secretory granules near the plasma membrane
was analyzed by TIRFM. Results: 1) The dynamics of tPA-GFP granules
including its opening and tPA-GFP secretion were successfully monitored by TIRFM. 2) Once tPA-GFP granules open, they kept open and
tPA-GFP was released slowly. The secreted tPA-GFP was detected as
tPA-GFP-PAI-1 complex in cultured medium. 3) The velocity of tPA release was facilitated by supplementary added PAI-1, which resulted in
the increase in tPA-PAI-1 complex in supernatant. Conclusion: tPA-GFP
is beneficial tool to investigate its exocytotic dynamics. PAI-1 seems to
facilitate tPA release as an inactive complex-form, which suppresses fibrinolytic activity on VECs.
The colony-forming cell assay for human
hematopoietic progenitor cells harvested
by a novel continuous-flow cell separation
Shiono, Hiroyuki1; Okada, Tadashi1; Ito, Yoichiro2 (1Aichi
Med. Univ. Sch. Med. Nagakute, Japan; 2National Institutes of
Health, Bethesda, USA)
A novel cell separation method which can continuously separate the
cells according to their densities has been developed for application to
the transfusion medicine. In the past the performance of this method was
examined on separation of human buffy coat, peripheral blood and cocultured cell suspensions. Flow cytometry analysis on separation of human buffy coat revealed that CD34-positive cells, which were assumed
to be hematopoietic progenitor cells, were distributed around density =
1.065. Recently, the colony-forming cell assay was performed on human
hematopoietic progenitor cells separated from peripheral blood by the
present method. Five polymer media with densities of 1.060, 1.065,
1.070, 1.075 and 1.080, prepared with sterile isotonic Percoll media and
PBS, were used for the separation, and the fractionated cells were cultured in a methylcellulose-based medium containing hSCF, hGM-CSF,
hIL-3 and hEPO. Colony-forming unit-erythroid (CFU-E) were appeared on day 7. Burst-forming unit-erythroid (BFU-E), CFU-GM
(granulocyte, macrophage) and CFU-GEMM (granulocyte, erythroid,
macrophage, megakaryocyte) were observed on day 14. BFU-E, CFUGM and CFU-GEMM were colonies that were found in the fourth fraction (density = 1.075), among those no CD-34 positive cells being detected by flow cytometry analysis. These results suggest that the method
might enable to harvest many types of human hematopoietic progenitor
cells according to minute differences in their densities.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Gastrointestinal functions
The interaction of synthetic peptide
derived from staphylokinase with
Low ghrelin contents in the blood and the
stomach of CCK-A and B receptor gene
knockout mice
Okada, Kiyotaka1; Ueshima, Shigeru1,2; Okamoto,
Chikako1; Kawao, Naoyuki1; Kawada, Shyuhei1;
Matsuo, Osamu1 (1Dept. of Physiol. Kinki Univ. Sch. of Med.;
Sakurai, Chihiro1,2; Kanai, Setsuko1; Ohta, Minoru1;
Uematsu, Hiroshi2; Miyasaka, Kyouko1
of Food Sci. and Nutri. Kinki Univ. Sch. of Agri.)
Staphylokinase (SAK) expresses plasminogen activator (PA) activity by
forming a complex with plasmin. In this report, the interaction of nonadecapeptide derived from staphylokinase with plasminogen, was investigated. The effects of synthetic peptides on plasminogen activation were
estimated by using a chromogenic substrate assay and 125I-labeled plasma clot lysis assay. The binding of peptides to Glu-plasminogen was estimated by using IAsys resonant mirror biosensor. The synthetic
nonadecapeptide (SAK22-40) corresponding to Glu22-Leu40 of SAK
amino acid sequence did not show any PA activity in the presence of
plasmin. However, SAK22-40 enhanced Glu-plasminogen activation by
t-PA. SAK22-40 bound to plasminogen in a concentration-dependent
manner. Although this binding ability was not inhibited in the presence
of anti-K1-K3 (plasminogen fragment containing kringle 1 to 3 domains) IgG or anti-K4 (plasminogen fragment containing kringle 4 domain) IgG, it was partially inhibited by anti- mini plasminogen IgG. The
substitution of Lys35 to Ala in SAK22-40 did not show the enhancement
of PA activity by t-PA. The t-PA activity was enhanced in the presence
of cultured endothelial cells, and it was further enhanced by SAK22-40.
These findings indicate that the synthesized nonadecapeptide, SAK2240, binds to B-chain of Glu-plasminogen and enhances PA activity by tPA. The mechanism by which SAK22-40 enhances t-PA activity seems
to be different from that by which COOH-terminal Lys of fibrin does.
(1Dept.Clin.Phusiol.,Tokyo Metro. Inst. Gerontol., Tokyo, Japan;
2Gerodontology, Department of Gerodontology, Division of
Gerontology and Gerodontology, GraduateSchool, Tokyo Medical
and DentalUniversity)
In mammals including humans, a brain-gut hormone, cholecystokinin
(CCK) mediates the satiety effect via CCK-A receptor (R). However, we
generated CCK-AR gene deficient (-/-) mice and found that the daily
food intake, energy expenditure, and gastric emptying did not change
compared with those of wild-type mice. We also generated CCK-BR (-/
-), CCK-AR (-/-) BR (-/-) mice. Daily food intake and ghrelin contents
in the blood and the stomach were investigated. Male mice at 6-8 months
of age were used. Mice, deprived of food for 18 hr with free access to
water, were injected i.p. (0.1ml/mouse) with either vehicle or CCK-8
(0.3, 1.0, and 3.0 n mol/mouse). Thereafter, daily food intake was measured. Additional animals were sacrificed by guillotine, the blood was
obtained, and the stomach was removed to measure ghrelin contents.
Administration of CCK-8S significantly decreased food intake in wildtype and in CCK-BR (-/-) mice. However, no significant inhibition was
observed in CCK-AR (-/-) and in CCK-AR (-/-) BR (-/-) mice. When
mice injected with vehicle were compared, food intake in CCK-AR (-/) BR (-/-) mice was significantly lower than that in wild-type and CCKBR (-/-) mice. Moreover, the ghrelin contents in the blood as well as in
the stomach were significantly lower in CCK-AR (-/-) BR (-/-) mice than
wild-type mice. CCK-Rs may be involved in the regulation of ghrelin
biosynthesis and secretion.
Monocarboxylate transporter 1 plays a role
in short chain fatty acid absorption in
caprine rumen
Kirat, Doaa; Masuoka, Junji; Hayashi, Hideaki; Iwano,
Hidetomo; Yokota, Hiroshi; Taniyama, Hiroyuki; Kato,
Seiyu (Sch. Vet. Med. Rakuno Gakuen Univ. Ebetsu, Japan)
Gastrointestinal functions
Despite the importance of short chain fatty acids (SCFA) in maintaining
the ruminant physiology, the mechanism of SCFA absorption is still not
fully studied. Therefore, this study was carried out to elucidate the mechanism of SCFA transport in the caprine rumen epithelia by investigating
the possible involvement of monocarboxylate transporter 1 (MCT1) as
well as to delineate the precise localization and the level of MCT1 protein along the caprine gastrointestinal tract. To achieve these objectives,
molecular studies including: RT-PCR, Western blotting, and immunohistochemistry as well as functional studies (in vivo and in vitro) were used.
RT-PCR studies revealed the presence of mRNA encoding for MCT1 in
all regions of the caprine gastrointestinal tract. By immunoblotting analysis on membrane protein extract, a 45-kDa-band corresponding to
MCT1 was detected in all parts of the forestomach and abomasum as
well as along the entire length of the intestine. The MCT1 protein level
was found most abundantly in forestomach, at intermediate levels in
large intestine and abomasum, and lower levels in small intestine. MCT1
is immunolocalized mostly to the stratum basale and stratum spinosum
of the caprine rumen epithelia. The MCT1 inhibitor, pCMB exerted a
significant influence on acetate flux in vitro as well as short-chain fatty
acids absorption in vivo. The results obtained provide evidence, for the
first time, for transepithelial transport of SCFA via MCT1 across the caprine ruminal epithelia.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Muscle physiology
The role of bowel fermentation in appetite
Priyadarshika, Hettiarachchi1; Wickremasinghe, A.R.2;
Jayaratne, S.D.1; Frost, Gary3 (1Dept of Physiol, Facl of Med
Sci, Univ of Sri Jayewardenepura, Nugegoda, Sri Lanka; 2Dept of
Community & Family Medicine, Facl of Med, Univ of Kelaniya;
3Dept of Nutrition and Dietetics, Hammersmith Hosp, London)
Disordered eating has been investigated extensively. The large bowel has
been studied less. The objective is to find its influence on appetite. Particularly, the relationship between large bowel fermentation and appetite
is not known. Hundred patients with no or some colons and the age sex
BMI matched controls were given a breakfast followed by lactulose. The
hydrogen breath and appetite levels were determined. Energy intake of
all patient groups was less than their matched controls. At 180 min, in
proctocolectomised group less hunger and higher satiety levels than that
of controls group were observed. The satiety levels of subtotal/hemicolectomised significantly less at 240 and 330 min. The breath hydrogen
level at -30 min and 60 min is positively correlated with the satiety levels
at 120 min proctocolectomy group. The breath hydrogen level -30 min
and 60 min is negatively correlated with the satiety levels at 240 min
subtotal removal of the rectum group. The breath hydrogen level -30 min
is positively correlated with the satiety levels at 180 min, 300 min and
330 min in total rectal removal group. The subjects who have had large
bowel surgery had lower hunger and higher satiety levels than controls
implicated that large bowel plays a role in appetite regulation. The breath
hydrogen influences the satiety in proctocolectomy and total rectal removal positively and negatively in sub/hemi colectomy, and the hunger
only in sub/hemi colectomy in one time point.
Muscle physiology
Stimulatory effect of a congener of beer (Nmethyltyramine) on pancreatic secretion in
conscious rats
Crowding problem in skinned muscle:
muscle compression with organic solutes
of small molecular weight.
Miyasaka, Kyoko; Kanai, Setsuko; Ohta, Minoru (Tokyo
Takemori, Shigeru; Kimura, Masako; Yamaguchi, Maki
Metropolitan Inst. Gerontol. Tokyo, Japan)
(Jikei Umiv. Sch. Med., Tokyo, Japan)
Alcoholic beverages contain several congeners. N-methyltyramine
(NMT) was isolated from beer as a factor to stimulate gastric acid secretion. We examined whether NMT stimulated pancreatic secretion in conscious rats. Methods: Male Wistar rats (330g) were prepared with
cannulae for draining bile and pancreatic juice separately, with two
duodenal cannulae, with a gastric cannula, and with an external jugular
vein cannula. The rats were placed in modified Bollman-type restraint
cages. After the 4-day recovery period, the experiments were conducted
in unanesthetized rats. Different concentrations of NMT solutions (5, 25,
and 50 micro-g/kg/3ml/3min) were infused into the stomach. NMT content in beer was 2mg/L, so that if 50kg body weight man consumes a bottle of beer, 25 micro-g/kg NMT will be ingested. To examine the
mechanism, the effects of proton pump inhibitor, CCK-BR antagonist,
CCK-AR antagonist and atropine were administered prior to the NMT
infusion. The effect of intravenous infusion of NMT (2.5 micro-g/rat)
was also determined. Results: Intragastric administration of NMT significantly increased pancreatic exocrine secretion in a dose dependent manner. Atropine abolished the stimulatory effect of NMT, but others did
not. Intravenous infusion of NMT did not affect pancreatic secretion.
Conclusions: NMT stimulates pancreatic secretion via cholinergic gastro-pancreatic reflex. Therefore, the stimulatory effect of beer on pancreatic secretion was produced by not only ethanol but also a congener,
We found that organic solutes potently compress the myofilament lattice
of skinned skeletal muscle. This compressing effect cannot be ascribed
to the ordinary osmotic compression due to the filtration of the solutes
by the lattice, because some solutes smaller than ATP in molecular
weight could effectively compress the muscle. Another candidate for the
compressing force comes from the entropic aggregating force of the
macromolecules constituting the myofilament lattice. As Asakura-Oosawa theory and its derivatives describe, the aggregation of macromolecules in the presence of smaller particles depends on the exclusion of the
particles from the very vicinity of the macromolecule surface. To estimate the effective exclusion volume for the organic solutes in the myofilament lattice, we examined the compressing effects of a series of
organic molecules from mono- and poly-hydric alcohols. The results
clearly indicated that the compressing efficiency of the alcohols depends
primarily on the number of CH2 group that is not directly attached by the
hydroxyl group. That is, the unitary component for the compressing effect is a single CH2 group. Since the molecular size of the CH2 group is
very close to that of the water molecule (OH2; 0.31 nm3), any macromolecular aggregation force due to the exclusion of solutes would not work,
unless each CH2 group forms a larger complex with surrounding water
molecules or water molecules form a stable cluster larger than the bare
CH2 group. The possibility of the CH2-OH2 complex formation and the
stable water cluster formation will be discussed.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Muscle physiology
Diameter measurements of single skeletal
myofibrils: AFM studies
Miyashiro, Daisuke; Hamazaki, Atsushi; Fujita,
Hirotaka; Akiyama, Nao; Kunioka, Yuki; Yamada,
Takenori (Dept. Phys. Facult. Sci. Tokyo Univ. of Sciencs, Tokyo,
In the present studies, the diameter of single skeletal myofibrils was
measured under various physiological conditions by AFM. Our AFM instrument is not suitable for precise diameter measurements of specimen
having the diameter range of micrometer like single myofibrils. Therefore, we modified our AFM system by incorporating precise piezo systems so as to be useful for the present purpose.
Single myofibrils were prepared by homogenizing glycerinated muscle
fibers of the rabbit psoas muscle. Myofibrils were fixed on the surface of
cover slip coated with aminosilanes. A small glass sphere was adhered
to the tip of AFM cantilever to smoothly touch to the surface of
myofibrils. The piezo stage could be moved in the horizontal and the
vertical directions in the range of 10 µm with the accuracy of ± 2 nm.
The diameter of myofibrils was determined by comparing the distance
moved for the glass sphere fixed to AFM cantilever to touch the top
surface of myofibrils and that to touch the surface of the cover slip. The
error of the diameter measurements was < ± 5 nm.
The diameter of single myofibrils thus obtained was 1.119 ± 0.042 µm
(n=5) in the relaxed state and 1.026 ± 0.039 µm (n=5) in the rigor state.
Thus the diameter of myofibrils in the rigor state was smaller by 8% than
that in the relaxed state. We also further examined the diameter of single
myofibrils under other physiological states. The results thus obtained
will be compared with the data for the lattice spacing of actomyosin
filaments of muscle fibers reported by X-ray diffraction studies.
The effects of temperature on the
generation of pacemaker potentials
recorded from the mouse small intestine.
Kito, Yoshihiko; Suzuki, Hikaru (Dept.Physiol., Medical
School, Nagoya City Univ., Nagoya, Japan)
Interstitial cells of Cajal distributed in myenteric region (ICC-MY) generate spontaneous electrical activity called pacemaker potential in gastrointestinal tract. Pacemaker potentials recorded from mouse small
intestine have two components: rapidly rising primary component and
following plateau component. In the present study the effects of temperature on the generation of pacemaker potentials were studied with conventional microelectrode techniques. Elevation of temperature increased
the frequency and maximum rate of rise (dV/dtmax) of pacemaker potentials and decreased their duration, without affecting the resting membrane potential and amplitude. CPA (3µM), an inhibitor of internal Ca
pump, abolished pacemaker potentials at around 28°C, while CPA only
shortened the duration of pacemaker potentials with the depolarization
of the membrane at around 40°C. KCN (30µM), an inhibitor of
metabolic process in mitochondria, decreased the frequency of
pacemaker potentials without changing the amplitude and dV/dtmax.
These results suggest that the sensitivity to temperature is different
between two components (primary and plateau) of pacemaker potentials.
The primary component may be generated by the activation of
temperature sensitive Ca permeable channels. On the other hand, the
plateau component seems to be generated by temperature insensitive
mechanisms. The results also suggest that mitochondrial function seems
to be uninvolved in the formation of two components of pacemaker
Effects of BDM on the transverse stiffness
of myofibrils and muscle fibers studied by
atomic force microscopy
Tyrosine-kinase-related Mechanism
Confers Smooth Muscle L-type Ca2+
Channel to a Second Open State
Akiyama, Nao; Miyashiro, Daisuke; Kunioka, Yuki;
Yamada, Takenori (Dept. Phys., Facult. Sci., Tokyo Univ. of
Nakayama, Shinsuke; Kamijo, Atsushi; Liu, Hong-Ning;
Kajioka, Shunichi (Nagoya Univ., Grad. Sch.Med.)
Science, Tokyo, Japan)
In contrast to cardiac myocytes, sympathetic stimulation does not largely
enhance L-type Ca2+ channel current in smooth muscle cells. In the
present study, we assessed possible mechanisms underlying this discrepancy, using a whole-cell clamp technique. In guinea-pig detrusor cells,
only L-type Ca2+ channels occur. During depolarizations of large positivity, the conformation of the majority of Ca2+ channels is converted
from the normal (O1) to a second open state (O2), in which Ca2+ channels
do not, or only slowly inactivate during depolarization. This feature of
the O2 state produces U-shaped inactivation. In order to estimate the
population of Ca2+ channels that can be converted to the O2 state, we applied a paired pulse protocol: Test steps with and without preconditioning step (+80 mV, 4s) were alternately applied. Extracellular application
of genistein decreased the amplitudes of both conditioned and unconditioned test inward currents (Acond and Auncond), accompanied by significant reduction of Acond/Auncond, while genistin, an inactive analogue, did
not. Intracellular application of genistein caused similar or more pronounced effects, when ATP was removed from the patch pipette. This result is consistent with the fact that ATP antagonizes the inhibitory effect
of genistein on tyrosine kinase activity. It is concluded that even under
normal conditions smooth muscle L-type Ca2+ channels are already in a
"stimulated mode° due to a tyrosine-kinase-related mechanism(s).
It is generally accepted that the contraction of muscle fiber is reversibly
inhibited by 2,3-butanedione monoxime (BDM). Since the muscle contraction takes place by the interaction between thin actin filaments and
myosin heads of thick filaments, it was suggested that BDM directly inhibits the actin-myosin interaction. But detail mechanisms of the suppression of contraction by BDM are still unknown. In this study, we
examined how BDM affects the transverse stiffness of myofibrils and
muscle fibers by AFM.
Myofibrils and muscle fibers prepared from psoas muscle of rabbit were
used. The transverse stiffness was measured as in our previous studies.
Modified AFM cantilevers having a glass rod attached to the tip of AFM
cantilever were prepared and used for measurements to minimally alter
the actin-myosin lattice structure. The tip of AFM cantilever was
approached to the surface of preparations attached to coverslip, and
force-curves were measured to obtain the transverse stiffness.
The transverse stiffness of myofibrils decreased in the order of rigor state
> contracting state > contracting state (+BDM) > relaxed state.
Remarkably the transverse stiffness of myofibrils was much greater in
contracting state (+BDM) than in relaxed state. For muscle fibers, the
transverse stiffness decreased in the same order as above for myofibrils.
These results indicate that cross-bridges are formed in contracting state
(+BDM), and suggest that the suppression of muscle contraction by
BDM is not simply due to the inhibition of cross-bridge formation.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Ionic channels & receptors
Voltage sensors influence ciguatoxin
effects on Nav1.4 Na+ channels
Yamaoka, Kaoru1; Inoue, Masayuki2; Kondou, Chie1;
Miyazaki, Keisuke2; Hirama, Masahiro2; Seyama, Issei3
(1Dept Physiol, School of Med, Hiroshim Univ, Hiroshima, Japan;
2Dept Chemi, Grad School of Sci, Tohoku University, Sendai, Japan;
3Facult Human Life Sci, Hiroshima Jogakuin Univ, Hiroshima,
Ionic channels & receptors
Molecular regions underlying the voltagedependence of L-type Ca2+ channel CaV1.3
which activates and inactivates at the
lower-voltage relative to CaV1.2
Hidenori2; Tsuru, Hiromichi1; Adachi-Akahane, Satomi1
(1Dept. Pharmacol., Fac. Med., Toho Univ. Tokyo, Japan; 2Lab. Cell
Signaling, Grad. Sch. Pharmaceutical Sci., Univ. Tokyo, Tokyo,
CaV1.2 (α1C) and CaV1.3 (α1D) are pore-forming subunits of cardiac Ltype Ca2+ channels. We have previously shown that CaV1.3 activates and
inactivates at more negative voltages than those of CaV1.2, thus
contributing to the threshold and the duration of the pacemaker action
potential in SA nodal cells. To elucidate molecular regions underlying
the unique voltage-dependence of CaV1.3 in comparison with CaV1.2,
we examined chimeras of CaV1.2 and CaV1.3, and found that repeat II of
CaV1.3 contains a critical domain for the negative shift of its voltagedependence. In the present study, we further localized the critical regions
of the unique voltage-dependence in repeat II of CaV1.3. We introduced
a series of point mutations in CaV1.2 and CaV1.2/CaV1.3 chimera. The
mutant Ca2+ channels were transiently expressed in BHK6 cells and
analyzed in patch-clamp experiments. As a result, F618L at the outside
of three Rs in IIS4 reversed the negative shift of the steady-state
inactivation of CaV1.2/CaV1.3 chimera channel, indicating that F618 is
one of regions underlying the voltage-dependence of CaV1.3. These
results suggest that the difference in a single amino acid in IIS4 between
CaV1.2 and CaV1.3 confers the unique voltage-dependence in cardiac Ltype Ca2+ channels.
The synthetic ciguatoxin, CTX3C has four distinct effects on Na channels:1) to speed up time-to-peak Na current (INa), 2) to suppress peak INa
amplitude, 3) to shift the activation curve in the hyperpolarizing direction, and 4) to delay recovery from "slow inactivation." In this study, we
explored possible sites modulating CTX effects. Mutant channels
N434A, L437A or A438K lacked effect 3 of CTX3C. These sites in
D1S6 selectively modulate activation mechanisms via CTX binding. In
separate experiments, we systematically replaced positively charged
amino acids (Arg or Lys) with a neutral residue (Gln) in each S4 segment
of the four domains. In all domains, neutralization of positive charges
did not suppress the hyperpolarizing shift of the activation curve induced
by the toxin (effect 3). However, CTX3C(3µM) did not suppress INa in
the four mutants with neutralized charges in D2 (lack of effect 2).
Interestingly, some of the D2 mutants even exhibited an increase in INa
in the presence of CTX3C: INa grew progressively larger as the location
of the mutations approached the extracellular face of the membrane.
CTX3C is a membrane-spanning molecule having a length of 30 Å and
possessing a relatively constrained structure. Thus, CTX3C may affect
the voltage sensor by accessing the sodium channel from its exterior
surface through the membrane lipid phase.
Structural and functional correlation of the
pore region of KCNQ3 channel in the brain
Nakamura, Yuki1; Shioya, Takao2; Imanaga, Issei3;
Inoue, Ryuji1; Uehara, Akira1 (1Dept. Physiol., Sch. Med.,
Fukuoka Univ., Fukuoka, Japan; 2Dept. Physiol., Saga Med. Sch.,
Saga, Japan; 3Gen. Res. Cent. Med. Sci., Dept. Physiol., Sch. Med.,
Fukuoka Univ., Fukuoka, Japan)
Heterotetramers of two kinds of KCNQ subunits form voltage-dependent K channels called M channels in the brain neurons. The outward
current from M channels repolarizes the action potential. It is reported
that point mutations in KCNQ3 cause a neuronal channelopathy. In this
study, we primarily examined the functional consequences by mutations.
Whole-cell currents were measured from the mutant KCNQ3 channels
expressed in HEK293 cells. 1-1) Homomeric channels composed of mutant KCNQ3 lacked currents. 1-2) Heteromeric channels composed of
wild-type KCNQ2 and mutant KCNQ3, which were mimicked to the native M channels, were lower in conductance than those composed of
wild-type KCNQ2 and wild-type KCNQ3. 1-3) The activation curve of
heteromeric channels composed of wild-type KCNQ2 and mutant
KCNQ3 was shifted to the right than that composed of wild-type
KCNQ2 and wild-type KCNQ3. We also reconstituted the three-dimensional protein structure of the mutant K channels. 2-1) The selectivity filter of wild-type KCNQ3 channel was structurally supported by chemical
bonds. 2-2) The mutation, however, lead to a loss of chemical bonds.
Such a structural alteration may result in the lower conductance in mutant KCNQ3 channels.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Ionic channels & receptors
Cytokines affect activity of an inwardly
rectifying K channel in cultured human
proximal tubule cells
Localization and function of isoforms of
the Cl- channel-related molecule, CLCA in
ductal cells of rat submandibular gland
Nakamura, Kazuyoshi; Komagiri, You; Kubokawa,
Manabu (Dept. Physiol. II, Sch. Med. Iwate Med. Univ., Morioka,
Yamazaki, Jun1; Okamura, Kazuhiko2; Ishibashi,
Kazubari3; Kitamura, Kenji1 (1Dept. Physiol. Sci. & Mol. Biol.,
Fukuoka Dent. Coll., Fukuoka, Japan; 2Morphol. Biol., Fukuoka
Dent. Coll., Fukuoka, Japan; 3Functional Biosci., Fukuoka Dent.
Coll., Fukuoka, Japan)
An inwardly rectifying K channel with inward conductance of 40 pS is
the most frequently observed K channel in cultured human proximal tubule cells. We have previously reported that the activity of this K channel
was modulated, at least in part, by nitric oxide (NO) which was mainly
derived from inducible NO synthase (iNOS) in these cells. Since cytokines are known to enhance the expression of iNOS mRNA, we explored
the effects of interleukin-1β (IL), tumor necrosis factor-α (TNF) and
interferon-γ (IFN) on the K channel activity, using the patch-clamp
technique and RT-PCR. After 24-h incubation of cells with each
cytokine, iNOS mRNA was significantly increased by IFN (100 U/ml)
but not by IL (10 U/ml) and TNF (200 U/ml). In cell-attached patches
using the IFN-treated cells, a NOS substrate, L-arginine (500 µM)
suppressed channel activity, whereas a NOS inhibitor, L-NAME (100
µM) stimulated it. These observations were in sharp contrast to the case
with control cells where L-arginine was stimulatory and L-NAME was
suppressive. Acute effects of cytokines on channel activity were tested
in cell-attached patches using control cells. Addition of IL to the bath
suppressed channel activity, which was not restored by the subsequent
addition of L-arginine. On the other hand, addition of IFN stimulated
channel activity and this stimulatory effect was not abolished by LNAME. TNF had little effect on channel activity. These results
suggested that IFN affected K channel activity through the NOdependent and -independent pathways whereas the effect of IL was NOdependent.
A molecular entity for Ca2+-dependent Cl–-transport has not been well
characterized in rat salivary glands. We previously identified a new
member of the CLCA family, rCLCA, which we isolated from rat ileum
by a PCR-based strategy. Here, we report that rCLCA isoforms are expressed in the submandibular gland (SMG). The full length of rCLCA
mRNA is 3.3 kb, and the predicted ORF encodes a 903-a.a. protein. The
amino acid sequence of rCLCA protein has 83% homology to murine
CLCA1 and 2. Transient transfection of HEK293 cells with rCLCA
cDNA resulted in a marked Ca2+-dependent Cl–-conductance with an
outward rectification in the I-V relationship. Intense immunostaining
was detected in the striated ducts of SMG, but not in the acinar cells. Immunoblot of the membrane fraction of the SMG yielded N-glycosylated
137- and 90-kDa bands. Interestingly, we also found the expression of a
truncated isoform (60 kDa, presumably 514 a.a.) including the N-terminal 455 a.a. of the full-length form. Comparison of this mRNA with that
of the full-length form revealed that exon 10 is truncated in this isoform.
The full-length of rCLCA is likely to be responsible for modulation of
Ca2+-dependent Cl– transport in the ductal cells, although the function of
the trancated isoform was not defined.
Implication of plasma membrane raft in
modulation of CFTR channel function
Expression and localization of aquaporin-6
in the rat parotid gland
Yoshiro, Sohma1,2; Ai, Tomohiko2; Mori, Yoshiaki1;
Tzyh-Chang, Hwang2; Kubota, Takahiro1 (1Osaka Med.
Matsuki, Miwako1; Dohke, Yoko2; Hashimoto,
Sadamitsu3; Shimono, Masaki3; Satoh, Keitaroh2;
Sugiya, Hiroshi2 (1HRC, Tokyo Dent.Coll. Chiba, Chiba, Japan;
Coll., Takatsuki, Japan; 2DCRC, Univ. of Missouri-Columbia, MO,
Cystic fibrosis transmembrane conductance regulator (CFTR) plays a
central role in transepithelial fluid and electrolyte transport as a Cl–
channel and also as a regulator of other transporters. CFTR channel
function is widely deviated among several different CFTR epithelia
whereas any CFTR subtype has never been identified. Recent evidence
suggests that ion channels can target to membrane lipid raft and associate
with other regulatory proteins. In this study, we have investigated the
role of cholesterol, an important component of lipid raft, in the regulation of CFTR channel function using patch-clamp technique.
In cell-attached patches, cholesterol-depletion by cyclodextrin treatment
inhibited CFTR channel activity Po from 0.35±0.03 to 0.15±0.02 with
decreasing burst duration (Tb) from 2.3±0.3 to 0.31±0.03 msec and
interburst duration (Tib) from 3.4±0.6 to 1.8±0.03 msec (n = 5). On the
contrary, cholesterol-enrichment by treatment with cyclodextrinsolubilized cholesterol slowed the CFTR channel gating with prolonging
Tb to 5.6±1.1 msec and Tib to 10.8±3.5 msec (n = 5). These results
indicate that cholesterol plays a critical role in regulation of CFTR
channel kinetics. We also found that CFTR channels expressed in Hi-5
insect cell line which is known not to synthesize cholesterol exhibited a
similar gating kinetics to those in cholesterol-depleted NIH3T3 cells.
We speculate that CFTR channels localize to lipid raft and are tissuespecifically modulated.
Nihon Univ. Dent. Matsudo Matsudo, Chiba, Japan;
Tokyo Dent. Coll. Chiba, Chiba, Japan)
Objectives: Aquaporins (AQPs) are 6 trans membrane proteins facilitating water transport via a plasma membrane and are involved in water,
urea, glycerol or lipid transport. It has currently demonstrated that AQPs
are located in intracellular organelle and their localization is changed by
cell stimulation. In this study, we investigated AQP6 in the rat parotid
gland.Methods: Plasma membrane and secretory granule fractions were
isolated from the rat parotid glands using parcoll gradient. mRNA expression was determined by RT-PCR. Protein expression was determined by western blotting analysis. Morphological localization was
observed by confocal microscopy and electron microscopy with immunolabelled ultrathin-cryosection. Results and Discussion: In RT-PCR,
262 bp band of AQP6 was detected in parotid acinar cells. In western
blotting analysis using anti-AQP6 antibody, only 29 kDa protein was detected in plasma membrane and secretory granule membrane fractions of
the parotid gland. In confocal microscopy, immunofluorescence with
anti-AQP6 antibody was positive at the apical sites, especially cell-cell
junctional region, in parotid acinar cells. When the glands were stimulated by β-agonist, AQP6 was observed to accumulate at the apical sites.
These results suggest that AQP6 expresses in the apical sites and
secretory granule membrane in the rat parotid gland as a monomeric
form. The change of localization of AQP6 appears to be involved in
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Ionic channels & receptors
Spontaneous Ca Oscillations activate
NFAT in undifferentiated Human
Mesenchymal Stem Cells
Block of the astroglial inward-rectifier
Kir4.1 by the antiarrhythmic agent
Kawano, Seiko1; Otsu, Keishi1; Muto, Yuko1,2;
Yanagida, Eri1; Shoji, Satoshi2; Kuruma, Akinori2 (1Dept.
Lossin, Christoph1; Hibino, Hiroshi1; Ishii, Atsushi2;
Hirose, Shinichi2; Kurachi, Yoshihisa1 (1Dept.
of Cardiovascular Diseases, MRI, Tokyo Medical and Dental
University; 2Brain Science Institute, RIKEN)
Pharmacology, Osaka University, Suita, Japan; 2Department of
Pediatrics, Fukuoka University)
Previously, we have shown that Ca oscillations modulate the activities of
ion channels and the fluctuation of membrane potentials in human mesenchymal stem cells (hMSCs). We have also found an ATP autocrine/
paracrine signaling pathway as the mechanism for Ca oscillations. In this
study we further investigated whether Ca dependent transcription factors
are regulated by Ca oscillations by examining the localization of NFAT
and NF-κB through immunocytochemical experiments. NF-κB was
found to be more abundant in the cytosol than in the nucleus in most
undifferentiated hMSCs (140/151 cells). On the other hand, nuclear
staining of NFAT was detected in more than 80% of hMSCs (40/48
cells). The nuclear translocation of NFAT was also confirmed by
Western blotting of the cytoplasmic and nuclear fractions. These
findings suggest that NFAT is constitutively activated in hMSCs. When
an ATP autocrine and Ca oscillations signaling pathway was blocked,
the NFAT signals in the nucleus were reduced, indicating the
involvement of Ca oscillations in the regulation of NFAT activation.
Using adipogenic techniques we differentiated hMSCs into adipocytes.
In these adipocytes, no spontaneous Ca oscillations were observed (32/
32 cells). In the immunocytochemical experiments, the nuclear staining
of NFAT was not prominent. Thus, we concluded that Ca oscillations
might play an important role in NFAT activation in undifferentiated
hMSCs but not in derived adipocytes.
Neurological research has traditionally emphasized neuronal components of the nervous system. Recent findings suggest, however, that astrocytes harbor greater (patho-)physiological significance than
previously thought. Known to provide mechanical support and chemical
control of the neuronal environment (neurotransmitter clearance, extracellular K+ siphoning, etc.), newer data now additionally tie astrocytes
to pharmaceutical pathways (Nature 433:73) and neurological disorders
such as epilepsy (Nat. Med. 11:973), calling for a clearer molecular description of astrocyte function. The inwardly-rectifying K+ channel
Kir4.1 (KCNJ10) is exclusively expressed in astrocytes in the brain. Due
to its central role in astroglial K+ buffering, Kir4.1 is likely a critical element of astrocyte performance. We examined the effects of various
neuroactive and neurotoxic compounds on human Kir4.1 using heterologous expression and the whole-cell configuration of the patch-clamp
technique and found that the antiarrhythmic/antimalarial agent quinidine
reversibly blocked Kir4.1 current in a time-dependent manner at concentrations slightly above the therapeutic range. Voltages positive to the
Nernstian potential strongly enhanced Kir4.1 block, but frequency and
number of the supplied depolarizing pulses seemed to be of little importance. It is conceivable that electrochemical alterations caused by astroglial ion channel block are in fact responsible for numerous so-far
unexplained neurological effects of commonly administered therapeutic
High water intake compensates for the
capacity to excrete a normal potassium
load in dominant negative Kir7.1
transgenic mice.
Single molecule imaging of stretchactivated BKca channels on the plasma
membrane of cultured cells:
immobilization on focal contacts
Kawahara, Katsumasa1; Suzuki, Yoshiro1; Yasuoka,
Yukiko1; Kawada, Hideaki1; Azuma, Sadahiro2;
Nakajima, Rie3; Hirose, Shigehisa4 (1Dept of Physiol,
Kobayashi, Takeshi1; Takeda, Yoshie1; Naruse, Keiji2;
Sokabe, Masahiro1,3 (1Dep. Physiol., Nagoya Univ. Grad. Sch.
Kitasato Univ Sch Med, Kanagawa, Japan; 2Genetic Engineering
Kitasato Univ Sch Med, Kanagawa, Japan; 3Dept Biosci, Kitasato
Univ Sch Sci, Kanagawa, Japan; 4Dept of Biol Sci, Tokyo Inst Tech,
Kanagawa, Japan)
Recent data have shown that inward rectifier K+ channel Kir7.1, localized in the basolateral membrane of rat distal nephron (JASN 2000; 11;
1987-1994), may be involved in the development of renal K+ excretion
(Kidney Int 2003; 63; 969-975). To further assess the role of Kir7.1 in
kidney K+ excretion, we have generated transgenic (Tg) mice expressing
dominant-negative mutant of Kir7.1 (dnKir7.1 Tg/+). Wild-type (WT)
and dn Kir7.1 Tg mice were placed in metabolic cages and their water
balance and urine osmolality and concentrations of urine electrolytes
(Na+, K+, Cl–) were examined. Both WT and Tg mice fed a K+-free diet
developed hypokalemia (2.4 and 1.7 mEq/l, respectively) at 3 days and
after. Water intake was increased in WT, but not in Tg mice. On the other
hand, Tg mice fed a normal diet demonstrated a significant polydipsia
and polyuria, with a relatively lower urine osmolality as compared to
WT mice. Further, urinary K+ excretion of WT and Tg mice increased in
proportion to that their water increase. There was no significant difference in renal K+ excretion between WT and Tg mice. In conclusion, Tg
mice being insufficient with the basolateral Kir7.1 of the kidney distal
nephron probably drink more water to compensate for the ability to excrete a K+ load.
Med., Nagoya, Japan; 2Dep. Cardiovasc. Physiol., Okayama Univ.
Grad. Sch. Med., Dent. Pharmaceu. Sci., Okayama, Japan; 3ICORP/
SORST, Cell Mechanosensing, JST, Nagoya, Japan)
Mechano-sensitive channels (MSCs) have been implicated to mediate
the mechano-sensation of various types of cells. It has been well established that, in prokaryotes, MSCs can be activated directly by tension in
the membrane. In contrast, the mechanism for eukaryotic MSCs activation has remained unclear. Since isolated eukaryotic MSCs in liposome
could not be activated, it is proposed that eukaryotic MSCs may form
molecular complex with other accessory proteins such as cytoskeletons
for their function. To address this issue, we have investigated lateral mobility of hSAKcaCs (human stretch activated and Ca2+-activated big K
channels), which we have recently identified, in the plasma membrane
of living cells. We have expressed GFP-tagged hSAKcaCs in HeLa cells
and observed individual channels by single fluorophore video imaging
using total internal reflection fluorescence microscopy. Most of hSAKcaCs exhibited simple diffusion (diffusion coefficient, 0.4 µm2/s on
average), but approximately 25% of the channels was almost stationary.
The observation of single channels of hSAKcaC simultaneously with
RFP-tagged paxillin showed that these immobilized channels were
specifically localized on/near focal contacts. These results suggest that
hSAKcaCs may associate with the force-transmitting modules including
adhesion molecules and cytoskeletons to form a mechano-sensing
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Neurons & synaptic functions
Drebrin A is involved in spine morphology
in vivo
Kobayashi, Chiho; Shirao, Tomoaki (Gunma University
Grad. Sch. of Med., Gunma, Japan)
Neurons & synaptic functions
Dendritic spines are the major sites of excitatory synaptic transmission,
and their morphogenesis plays a pivotal role in neuronal development
and plasticity. Recent studies suggest the correlation between spine morphology and spine functions. Diversity in synapse functions is related to
the diversity in spine morphology. As one of the candidate which control
spine morphology, we focused on a major actin-binding protein in the
brain, drebrin A (DA). We hypothesized that spine morphology depends
on DA, and we analyzed spine ultrastructure in terms of DA localization
using immunoelectron microscopy. We have recently reported that only
75% of spines are DA immuno-positive in adult rat cortex. We first analyzed if there was any morphological differences between DA immunopositive spine (DPS) and DA immuno-negative spines (DNS). We found
that DPS were larger in the spine head area and in the post synaptic density (PSD) length. Next, we analyzed if overexpression of DA changed
spine morphology, using DA transgenic mice (Tg) that overexpressed
DA in the forebrain. Similar to wild type mice (WT), DPS were larger
than DNS in Tg; however, spine head area of DPS was significantly
smaller than that of WT. The amount of DA within each spine did not
change between two genotypes. These data suggest that overexpression
of DA leads to smaller DPS with higher concentration of DA. There
were no differences between two genotypes in the ratio of DPS to DNS,
the spine density, and the DA localization within spines. These results indicate that DA is involved in spine morphology in vivo although its molecular mechanism is not yet clarified.
Long lasting spontaneous Ca2+ transients
in the striatal cells.
Osanai, Makoto; Yamada, Naohiro; Oboshi, Fumito;
Yagi, Tetsuya (Dept. Electrical, Electronic and Information
Engineering, Grad. Sch. Engineering, Osaka Univ., Suita, Japan)
The striatum plays an important role in linking cortical activity to basal
ganglia outputs. We conducted the Ca2+ imaging study to investigate the
spontaneous activities of the striatum.
Corticostriatal slices of 10-25 days old Sprague Dawley rats were
stained with Fura-PE3-AM to measure intracellular Ca2+ concentration
([Ca2+]i) using a cooled-CCD imaging system.
Long lasting spontaneous [Ca2+]i transients, which lasted up to about 250
s, were observed. Most cells exhibited irregular frequencies, but some
exhibited oscillatory feature. A pairwise correlation analysis revealed
that some cells appear to belong to a correlated network. Administration
of TTX or of CNQX + AP5 did not block the [Ca2+]i transients.
Therefore, the action potentials and the excitatory synaptic inputs in the
striatal network were not involved in induction of the [Ca2+]i transients.
In contrast, the number of active cells, which exhibited the [Ca2+]i
transients, was greatly reduced by the intracellular Ca2+ store depletor,
thapsigargin, and was reduced little by the administration of the Ca2+free saline. Therefore, the intracellular Ca2+ store is likely to contribute
to the [Ca2+]i transients.
In the mouse, which expressed green fluorescent protein (GFP) in
astrocytes (GFAP-GFP mouse), both the GFP positive cells and the GFP
negative cells exhibited the [Ca2+]i transients. These results suggested
that both astrocytes and neurons may exhibit the long lasting
spontaneous [Ca2+]i transients in the striatum.
Effects of maintenance of body
temperature on hippocampal neural
activity: control of membrane potential
through TRPV4 activation
Shibasaki, Koji1,2; Suzuki, Makoto3; Mizuno, Atsuko3;
Tominaga, Makoto1,2 (1Dep. of Cell Signaling, Okazaki Inst.for
Integrative Bioscience, Okazaki, Japan; 2Dep. of Physiol. Sciences,
The Graduated Univ. for Advanced Studies, Okazaki, Japan; 3Dep.
of Pharmacology, Jichi Medical Univ.)
TRPV4 is one of the member of themo-TRP channels. We screened
brain-TRPV4 mRNA expressions by in situ hybridization, and found
hippocampus strongly expressed TRPV4 in addition to choloid plexus,
where TRPV4 expression has been reported. We speculated that TRPV4
might be an important ion channel to regulate hippocampal functions.
Therefore, we established a new dissociated neural culture system from
neonatal mice hippocampi. TRPV4 protein was localized in soma and
dendrites in the culture. We also examined whether functional-TRPV4
was expressed in hippocampal neurons by a Ca2+ imaging method with
Fura2. The hippocampal neurons responded to the all reported stimulus,
such as heat (>32 °C), hypotonic stimulus and 4αPDD, but no response
was observed in the TRPV4-mutant neurons. We considered that body
temperature activates brain-TRPV4, and the activation might contribute
to slight depolarization of the resting membrane potential (RMP). Next,
we compared the RMP between wild type and TRPV4-mutant neurons at
37 °C, and found the wild type RMP was approximately 5 mV higher
than the TRPV4-mutant RMP. We also performed current-injection
experiments in both neurons, and found that TRPV4-mutant neurons
required much bigger currents to get their firing. We conclude that
TRPV4 is activated by body temperature in hippocampus, and produces
proper environments for their firing.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Neurons & synaptic functions
Cannabinoid CB1 receptor at excitatory
presynaptic site in the hippocampus and
Dopamine-induced potentiation of the
hippocampal mossy fiber synaptic
Kawamura, Yoshinobu1; Fukaya, Masahiro2; Maejima,
Takashi3; Yoshida, Takayuki1; Miura, Eriko2; Watanabe,
Masahiko2; Ohno-Shosaku, Takako4; Kano, Masanobu1
Nippon Medical School, Tokyo, Japan)
(1Grad. Sch. Med. Univ. Osaka, Osaka, Japan; 2Grad. Sch. Med.
Univ. Hokkaido, Hokkaido, Japan; 3NIPS, Okazaki, Japan; 4Grad.
Sch. Med. Univ. Kanazawa, Kanazawa, Japan)
Endocannabinoids mediate short- and long-term suppression of synaptic
transmission. Nature of presynaptic cannabinoid receptor has been investigated for both excitatory and inhibitory synapses in various regions
of the brain. For inhibitory synapses, importance of type 1 cannabinoid
receptor (CB1) is generally accepted. For excitatory synapses, however,
results are controversial. In the present study, we used electrophysiological and immunohistochemical techniques, and examined the type of
cannabinoid receptors functioning at hippocampal and cerebellar excitatory synapses. Using CB1-knockout mice, we demonstrate predominant
contribution of CB1 to excitatory synaptic transmission on CA1 pyramidal neurons in the hippocampus and that on cerebellar Purkinje cells
from climbing fibers and parallel fibers. The presence of CB1 at presynaptic terminal was confirmed by immunohistochemical experiments
with specific antibodies against CB1. In immunoelectron microscopy of
the hippocampus and the cerebellar cortex, densities of CB1-positive
signal in excitatory terminals were much lower than in inhibitory terminals, but clearly higher than the background level. These results clearly
indicate that CB1 is responsible for cannabinoid-dependent suppression
of excitatory transmission in the hippocampus and cerebellum.
Kobayashi, Katsunori; Suzuki, Hidenori (Dept. Pharmacol.,
Dopamine is thought to play important roles in various brain functions
including learning and memory. In the hippocampus, dopamine has been
shown to modulate synaptic transmission and plasticity in the CA1 region. However, it remains largely unknown how dopamine affects synaptic transmission in the CA3 region where forms of synaptic plasticity
distinct from those in the CA1 region have been demonstrated. In the
present study, we show that application of dopamine reversibly potentiates synaptic transmission between the mossy fibers (MFs) and CA3 pyramidal cells. Pharmacological experiments showed that D1-like
receptors mediate the potentiation induced by dopamine. This potentiation was accompanied by a decrease in the magnitude of synaptic facilitation and occluded by application of forskolin, an adenylate cyclase
activator, suggesting the involvement of presynaptic cAMP-dependent
mechanisms. The MF synaptic transmission is kept inhibited by tonic activation of presynaptic adenosine A1 receptors and GABAB receptors. It
is known that dopamine and adenosine counteract each other in some
brain regions and that adenosine receptor antagonists can enhance effects of dopamine receptor agonists. At the MF synapse, application of
an adenosine receptor antagonist alone, or together with a GABAB receptor antagonist, enhanced the synaptic transmission, but suppressed
the dopamine-induced potentiation. These results suggest that dopamine-induced presynaptic potentiation of the MF synaptic transmission is
unmasked by the tonic presynaptic inhibition mainly mediated by adenosine.
Morphological and behavioral analyses of
mice lacking adult form of drebrin
Imaging analysis of associative LTP in rat
hippocampal CA1 region
Hanamura, Kenji1; Kojima, Nobuhiko1; Yamazaki,
Hiroyuki1; Sekino, Yuko2,3; Shirao, Tomoaki1 (1Dept. of
Kojima, Hiroshi1; Yoneyama, Makoto1; Yamazaki,
Yoshiyuki1; Kamijou, Tadanobu1; Tsukada, Minoru2 (1Lab
Neurobiol. and Behav., Gunma Univ. Grad. Sch. of Med., Maebashi,
Japan; 2Div Neuronal Network, Inst. Med. Sci, Univ. Tokyo, Tokyo,
Japan; 3CREST, JST, Kawaguchi, Japan)
for Cellular and Molecular Physiology, Tamagawa University,
Machida-shi, Tokyo, Japan; 2Lab for Learning and Memory
Drebrin A is a neuron-specific actin-binding protein, which is localized
in mature dendritic spines. During synapse formation, embryonic isoform, drebrin E is converted into neuron-specific isoform, drebrin A. We
have demonstrated that suppression of the upregulation of drebrin A attenuated spine formation in vitro. To investigate physiological differences of drebrins E and A, we generated mice, in which isoform conversion
of drebrin did not occur, by targeted disruption of drebrin A specific
exon (DAKO). In these mice total amount of drebrin was not changed
since drebrin E was overexpressed instead of drebrin A. We first analyzed their dendritic spine morphology on apical dendrites of layer V pyramidal cells in somatosensory cortex using rapid Golgi staining. The
number and length of dendritic spine in adult DAKO mice (16-18 week
old) were comparable to that in wild-type mice. We next analyzed behavioral phenotypes in DAKO mice. These mice showed impaired contextdependent fear conditioning, a hippocampal NMDA receptor-dependent
learning task. Our findings indicate that isoform conversion of drebrin is
required for regulation of synaptic function. In contrast, total amount of
drebrin is important for regulation of spine morphology.
Associative LTP by two independent inputs is known to be an important
feature of synaptic plasticity observed in the CA1 of rat hippocampus.
However, it is still unclear what kind of physiological role, especially in
relation with memory and learning mechanisms, it plays and how it is inducted by two pathways. In order to investigate these questions, the specio-temporal pattern of neuronal activities during LTP induction
followed by long lasting later period were studied by using both voltagesensitive dye optical and extracellular electrical recording. Moreover,
the possible contribution of back-propagating action potentials invaded
into dendritic trees to the induction mechanisms of the present LTP was
investigated under the assumption of STDP (spike timing dependent
plasticity) mechanisms. In order to induce associative LTP rat acute slice
preparations stained with Di-4-anepps were stimulated by two independent Schaffer collateral pathways with the stimulation protocol reported
previously; one pathway is stimulated weakly under threshold (single
pulse) while another pathways is strong over the threshold (multiple
pulses). Optical signals were recorded and analyzed by using the photodiode array system (Neuro-plexVI; Redshirt Imaging, USA). The obtained results from the present study are discussed from the view point
of non-linear summation of two independent inputs and its distribution
along the dendrite, together with the possible induction mechanisms obtained from low TTX experiment.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Neurons & synaptic functions
gene expression fingerprint of C. elegans
embryonic motor neurons
Anye, Chi
Che, Sirri
Dolita, Niba
L.3 (1Department of Medicine, Buea University School of Medicine,
Bamenda, Republic of Cameroon; 2Yaoude University , Cameroon;
3Buea University, Cameroon; 4Yaoude University , Cameroon;
5Buea University, Cameroon)
NEURON AND SYNAPTIC FUNCTIONS Differential gene expression specifies the highly diverse cell types that constitute the nervous
system. With its sequenced genome and simple, well-defined neuroanatomy, the nematode C. elegans is a useful model system in which to correlate gene expression with neuron identity. The UNC-4 transcription
factor is expressed in thirteen embryonic motor neurons where it specifies axonal morphology and synaptic function. These cells can be
marked with an unc-4::GFP reporter transgene. Here we describe a powerful strategy, Micro-Array Profiling of C. elegans cells (MAPCeL), and
confirm that this approach provides a comprehensive gene expression
profile of unc-4::GFP motor neurons in vivo.ResultsFluorescence Activated Cell Sorting (FACS) was used to isolate unc-4::GFP neurons from
primary cultures of C. elegans embryonic cells. Microarray experiments
detected 6,217 unique transcripts of which ~1,000 are enriched in unc4::GFP neurons relative to the average nematode embryonic cell. The reliability of these data was validated by the detection of known cell-specific transcripts and by expression in UNC-4 motor neurons of GFP
reporters derived from the enriched data set. In addition to genes involved in neurotransmitter packaging and release, the microarray data
include transcripts for receptors to a remarkably wide variety of signaling molecules.
Regultation of neurotransmitter release by
tomosyn phosphorylation
Mochida, Sumiko1; Baba, Takeshi2; Sakisaka, Toshiro2;
Takai, Yoshimi2 (1Dept. Physiol. Tokyo Med. Univ. Tokyo, Japan;
Mol Biol. Biochem. Grad. Sch. Med.Fac. Med. Osaka Univ.,
Regulation of neurotransmitter release by tomosyn phosphorylation.
Sumiko Mochida1, Takeshi Baba2, Toshiaki Sakisaka2, and Yoshimi
Takai2. 1Depatment of Physiology, Tokyo Medical University, Shinjuku
160-8402, Japan, and 2Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Japan.PKA phosphorylation of tomosyn, a
SNARE regulatory protein, significantly decreased its binding to syntaxin-1 in vitro, and cAMP stimulation increased the phosphorylation of tomosyn and decreased tomosyn binding to syntaxin-1, resulting in
enhanced SNARE complex formation. Overexpression of tomosyn in
cultured superior cervical ganglion neurons inhibited neurotransmitter
release which could be rescued by the introduction of cAMP into the presynaptic neuron. Expression of tomosyn S724A, a PKA unphosphorylated mutant, or knock-down of tomosyn by siRNA introduction also
decreased neurotransmitter release, but was not rescued by cAMP. Under
high frequency stimulation, expression of tomosyn S724A and S724D, a
PKA phosphorylated mimic mutant, increased the EPSP failure rate and
asynchronous EPSPs. These results indicate that tomosyn is a physiologically significant PKA target that controls neurotransmitter release
through the regulation of SNARE complex formation.
Re-evaluation of GABA releasing
hypothesis from hippocampal mossy fiber
GABAergic modulation for TEA-induced
synaptic plasticity in rat hippocampal CA1,
CA3 and dentate gyrus
Kamiya, Haruyuki (Hokkaido Univ. Grad. Sch. Med., Sapporo,
Suzuki, Etsuko; Okada, Takashi (Dept. Psychol., Senshu
Univ., Kanagawa, Japan)
It has been hypothesized that hippocampal mossy fiber terminals of
young rodents release GABA in addition to glutamate. Using whole cellclamp recordings in mouse slice preparations, I re-examined this possibility with paying attentions about the conditions to evoke monosynaptic
GABAergic responses by stimulation of mossy fibers. Strong stimulus to
the stratum granulosum of dentate gyrus or stimulus to the stratum lucidum of CA3 region elicited IPSCs in CA3 neurons in the presence of
glutamate receptor antagonists 10 µM CNQX and 25 µM D-AP5, and
these putative "monosynaptic IPSCs" were abolished by addition of
GABAA receptor antagonist 100 µM picrotoxin. In contrast, weak
stimulus to the stratum granulosum never elicited IPSCs in the presence
of CNQX and D-AP5. The responses to weak and strong stimuli also
displayed differential sensitivity to group II mGluR agonist DCG-IV;
application of 1 µM DCG-IV almost abolished the responses to weak
stimulus and left substantial responses to strong stimulus which were
inhibited by further application of picrotoxin. These results suggested
that strong stimulus to stratum granulosum causes monosynaptic IPSCs
by stimulating inhibitory interneurons in addition to mossy fibers, and
mossy fiber terminals themselves may not release GABA.
Tetraethylammonium (TEA), a K+ channel blocker, reportedly induces
long-term potentiation (LTP) of hippocampal CA1 synaptic responses in
an NMDA receptor-independent manner. However, the characteristics of
TEA-induced plasticity and modulation by inhibitory interneurons at
CA3 and the dentate gyrus (DG) remain unclear. This study recorded
field EPSPs from CA1, CA3 and DG to examine the involvement of
GABAergic modulation in TEA-induced synaptic plasticity at each region. In Schaffer collateral-CA1 synapses, bath application of 25 mM
TEA for 12 min induced LTP in the presence and absence of 100 µM
picrotoxin (PTX), a GABAA receptor blocker, suggesting little
modulation by interneurons in the CA1 region. In CA3, associational
fiber (AF)-CA3 synapses showed TEA-induced LTP regardless of PTX,
but mossy fiber (MF)-CA3 synaptic plasticity were influenced by PTX
application; TEA-induced LTP was detected only in the absence of PTX.
In DG, synaptic plasticity was modulated by GABAergic inputs, but
characteristics differed between lateral perforant path (LPP) and medial
perforant path (MPP). LPP-DG synapses showed TEA-induced LTP in
the presence of PTX, but no changes in the absence of PTX. At MPP-DG
synapses, TEA-induced long-term depression (LTD) was observed in
the absence of PTX, but no changes were seen in the presence of PTX.
This series of results demonstrated that TEA-induced plasticity at
perforant path-DG synapses and MF-CA3 synapses are modulated by
GABAergic inputs, and that the effects of GABAergic modulation on
plasticity are inhibitory (LPP- and MPP-DG) or excitatory (MF-CA3).
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Neurons & synaptic functions
Changes in neuromodulatory effect of
adenosine A1 receptors of piriform cortex
in amygdala kindled rats
Mirnajafi-Zadeh, Javad; Rezvani, Mohammad Ebrahim;
Fathollahi, Yaghoub (Department of Physiology, School of
Medical Sciences, Tarbiat Modares University, Tehran, Iran)
Considering the anticonvulsant effects of adenosine and the anatomical
connections between piriform cortex (PC) and amygdala, in this study,
the effect of kindling implementation on adenosine A1 receptor mediated neuromodulation in PC pyramidal neurons was tested at 24 h and 1
month after amygdala kindling. Field potentials were recorded from layer II of PC following stimulation of the lateral olfactory tract. Obtained
results showed that N6-cyclohexyleadenosine (CHA; i.c.v.; 1, 10 and
100 µM, as a selective A1 agonist) reduced A1 slope (as an index of
EPSP slope) and B1 amplitude (as an index of spike amplitude) of field
potentials in both kindled and non-kindled rats. However, its effects
were more potent at 24 h, but not 1 month after kindling. Pretreatment of
1,3-dimethyl-8-cyclohexylexantine (CPT; i.c.v.; 50 µM, as a selective
A1 antagonist), eliminated effects of CHA (i.c.v.; 10 µM). These results
indicate that A1 receptors of PC have anticonvulsant effects on
amygdala kindled seizures and the efficiency of the A1 receptor
neuromodulation is increased at short- (24 h), but not long-term (1
month) after kindling implementation.
Tsuzuki, Keisuke1; Tsukada, Shota1,2 (1Dept. Neurophysiol.
Gunma Univ. Grad. Sch. Med, Maebashi, Japan; 2Dept. Pediatrics
Gunma Univ. Grad. Sch. Med, Maebashi, Japan)
The genome project elucidates that the whole genome possesses approximately 25,000 genes. Although it is thought that single cells express approximately 10,000 genes in various copy numbers in a cell-type specific
manner, differences of gene expressions inside and among defined celltypes are not examined at a single cell level. Here, we examined mRNA
expressions in acute rat hippocampal slices where the classification of
cell types was most extensively examined. CA1 and CA3 pyramidal
cells, dentate granule (DG) cells and interneurons were identified from
their morphologies and the locations and cellular contents were harvested into patch pipettes. We performed RT-multiplex PCR of 31 genes, including biochemical markers of calcium binding proteins, neuropeptides
and neurotransmitter receptors. Detection rates of each mRNA were examined. Except for correlations linked to glutamatergic and GABAergic
neuronal markers, strong correlations were scarcely found. When cells
were grouped according cell types, however, large differences of detection rates in many genes were observed. χ-square analysis revealed that
37% of tested genes were differently expressed in interneurons and
principal neurons and that 30% were differently expressed in pyramidal
cells and DG cells. Genes differentially expressed between CA1 and
CA3 pyramidal neurons were not found. Dendrogram showing the
distances of cell types was obtained by cumulative differences of
detection rates.
Regulatory role of small molecule Gprotein Arf1 and subsequent
phospholipase D in the K+-current
response to dopamine in the ganglion cells
of Aplysia
Watanabe, Shuji1; Kawasaki, Satoshi1; Kimura,
Shingo1; Fujita, Reiko2; Sasaki, Kazuhiko1 (1Dept. Physiol.
Sch. Med. Iwate Medical Univ. Morioka, Japan; 2Dept. Chem. Sch.
Lib. Arts Iwate Medical Univ. Morioka, Japan)
Small molecule G-protein Arf1 in combination with phospholipase D
(PLD) is essential for intracellular trafficking of the proteins from endoplasmic reticulum to Golgi apparatus. However, it is recently reported
that it also regulate ionic channel activity presumably though recycling
of the receptors and ionic channels at the cytoplasmic membrane. To examine possible involvement of Arf and subsequent PLD in regulation of
receptor-induced responses in neuron, we recorded K+-current response
induced by dopamine (DA) in the ganglion cells of Aplysia under conventional two-electrode voltage clamp. Intracellular application of
brefeldin A, a specific blocker of Arf GEF, significantly depressed the
K+-current response to DA. The DA-induced response was also inhibited
by injection of 2-(4-fluorobenzoilamino)- benzoic methyl ester (Exo1),
an activator of GAP for Arf1. Intracellular injection of N-terminal peptide of Arf1 markedly suppressed the DA-induced response. In contrast,
application of those of Arf6 did not affect the response to DA. Furthermore, intracellular application of α-synuclein, a specific blocker of PLD,
significantly depressed the K+-current response to DA. In contrast, all
these reagents had no significant effect on the Na+-current response
induced by acetylcholine in the same type of cells. These results suggest
that Arf1 and subsequent PLD may regulate the K+-current response
induced by DA.
Analysis of gene expressions using RTmultiplex PCR on rat hippocampal neurons
in acute slices
5-HT1B receptor-mediated presynaptic
inhibition at the calyx of Held
Mizutani, Haruo; Tsujimoto, Tetsuhiro; Takahashi,
Tomoyuki (Grad. Sch. Med. Univ. Tokyo, Tokyo, Japan)
5-hydroxytryptamine (5-HT; serotonin) inhibits transmitter release via
activating GTP binding (G) proteins, but the target of this effect in the
nerve terminal is not determined. We addressed this question at the calyx
of Held synapse in brainstem slices of developing rats. In 5-day-old rats
bath-application of 5-HT (10 µM) attenuated the amplitude of evoked
EPSCs and facilitated paired-pulse ratio, whereas 5-HT had no effect on
the amplitude of spontaneous miniature EPSCs. The 5-HT1B receptor
agonist CP93129 mimicked the inhibitory effect of 5-HT, but the 5HT1A agonist 8-OHDPAT had no effect. 5-HT1B receptor antagonist
NAS-181 blocked the inhibitory effect of 5-HT. These results suggest
that 5-HT activate 5-HT1B receptors in the nerve terminal, thereby
inhibiting transmitter release. In whole-cell recordings from calyceal
nereve terminals, 5-HT attenuated voltage-gated Ca2+ currents, but had
no effect on voltage-gated K+ currents. Upon repetitive application 5-HT
showed tachyphylaxis with its effect on both EPSCs and presynaptic
Ca2+ currents becoming weaker in the second application. Surprisingly
10 mM BAPTA loaded into the nerve terminal abolished the
tachyphylaxis. The presynaptic inhibitory effect of 5-HT was robust at
postnatal day 5, but became weaker as animals matured. We conclude
that 5-HT1B receptors can mediate presynaptic inhibition of transmitter
release in immature calyceal terminals via inhibiting voltage-gated Ca2+
channels. Upon repetitive activation 5-HT1B receptors may be
internalized or desensitized by a Ca2+-dependent mechanism.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Neurons & synaptic functions
The rate of refilling of synaptic vesicles
with glutamate
Kaneko, Masahiro; Hori, Tetsuya; Takahashi, Tomoyuki
(Dept. Neurophys., Univ. Tokyo Grad. Sch. Med., Tokyo, Japan)
At the excitatory synapse neurotransmitter glutamate is released from
synaptic vesicles. After exocytosis vesicles are retrieved by endocytosis
and recycled for reuse, thereby maintaining synaptic transmission. During recycling vesicles are refilled with glutamate by vesicular glutamate
transporters (VGLUTs), using electrochemical gradient produced by
vacuolar-type H+-ATPase (V-ATPase). Multiple recycling mechanisms
of different speeds are thought to operate in the nerve terminal. However,
the speed of vesicle refilling with glutamate is not known. Here we manipulated vesicular glutamate directly in the nerve terminal, depleting it
by whole-cell dialysis and refilling it by the photolysis of caged
glutamate. Vesicle refilling, monitored by postsynaptic currents, was
abolished by blocking VGLUT or V-ATPase, and attenuated by increasing cytosolic Cl– concentrations. The refilling time constant ranged 2-13
s depending upon the magnitude of refilling, with 100% refilling time
constant being 18 s. This rate is faster than the "full-fusion"-type slow
recycling time (1 min), but slower than "kiss-and-run"-type fast recycling time (<1s). We conclude that transmitter glutamate can fully refill
vesicles recycled via slow, but not fast, pathways.
Glutamate NMDA receptors and MAPKs
within the amygdala participated in the
modulation effect of glucocorticoids on
Lu, Kwok-Tung1; Chao, Po-Kuan1; Yang, Yi-Ling2
(1Department of Life Science, National Taiwan Normal University,
Taipei, Taiwan; 2Institute of Biotechnology, National Chia-Yi
University, Chia-Yi, Taiwan)
The present study was aimed to elucidate the possible mechanism of glucocorticoids on the conditioned fear extinction by using fear-potentiated
startle paradigm. We found that (1) Systemic administration of dexamethasone (DEX, 1.0 mg/kg, i.p.) accelerated extinction of conditioned
fear. (2) Administration of glutamate NMDA receptor antagonists (±)HA966 (6.0 mg/kg, i.p.) and intra-amygdala infusion of MK801 (0.5 ng/
side, bilaterally) or D,L-2-amino-5- phosphonovaleric acid (AP5, 2.0 ng/
side, bilaterally) blocked the DEX facilitation effect. (3) Blockade of
corticosteroid synthesis inhibitor metyrapone (25 mg/kg. s.c.) on
extinction was removed by co-administration of NMDA receptor agonist
D-cycloserine (DCS, 5.0 mg/kg, i.p.). (4) Co-administration of DEX and
DCS in a sub-threshold dose provided a synergistic facilitation effect on
extinction (0.2 mg/kg and 5 mg/kg, respectively). However, DEX and
DCS co-administration did not alter the expression of conditioned fear.
(5) The facilitation effect of DEX was blocked by intra-amygdala
infusion of mitogen activated protein kinase (MAPKs) inhibitors
PD98059 (500 ng/side, bilaterally) or U0-126 (20 µM/side. bilaterally).
DEX significantly enhanced the phosphorylation of MAPKs which
induced by the extinction training. These results suggested that
glutamate NMDA receptors and MAPKs within the amygdala
participated in the modulation effect of glucocorticoids on extinction.
Activation of mGluRs during
preconditioning low-frequency stimulation
determines direction of synaptic plasticity
in hippocampal CA1 neurons
Fujii, Satoshi; Kaneko, Kenya; Yamazaki, Yoshihiko;
Miyazaki, Keita; Kato, Hiroshi (Dept. Neurophysiol. Yamagata
Univ. Sch. Med.,Yamagata,Japan)
In hippocampal CA1 neurons, delivery of low-frequency afferent stimuli
<LFS;80 pulses at 1 Hz> induced LTP in both the field EPSP and population spike <PS>. In the same cells, reversal of LTP in the EPSP and PS
was achieved by the same LFS given 20 min after the first LFS <second
LFS>. We investigated the effects of metabotropic glutamate receptor
<mGluR> antagonists on LTP or LTD induced by the second LFS. When
the first LFS was given in the standard solution, both the field EPSP and
PS were attenuated by the second LFS. In contrast, when the first LFS
was delivered in the presence of MCPG, a broad spectrum mGluR antagonist or 4CPG, a type 1 mGluR antagonist, the field EPSP and PS were
enhanced by the second LFS. Thus, activation of mGluRs during preconditioning LFS stimulation determines the direction of synaptic plasticity
at CA1 neurons. Then, we investigated the effects of an NMDA receptor
antagonist, AP5 on synaptic plasticity induced by the second LFS. When
the first LFS was given in the standard solution or in 4CPG but the second LFS was given in the presence of AP5, both the EPSP and PS were
enhanced after the second LFS. These results indicate that the synaptic
plasticity induced by the second LFS depends on NMDA receptor activation at CA1 synapses. Thus, it is possible that activation of mGluRs
during prior synaptic activation resulted in enhancement of NMDA receptor activation, leading to reversal of LTP.
Effects of drebrin A knock down on
glutamate receptor activities in developing
hippocampal neurons
Kato, Kenichi1,2; Shirao, Tomoaki1,2; Mizui, Toshiyuki2;
Takahashi, Hideto2; Sekino, yuko1,2,3 (1CREST, JST,
Kawaguchi, Japan; 2Dept. of Neurobiol. and Behav., Gunma Univ.
Grad. Sch. of Med., Maebashi, Japan; 3Div of Neuronal Network,
Ins of Med Sci, Univ of Tokyo, Tokyo, Japan)
Drebrin A (DA), an F-actin binding protein, is involved in spine morphogenesis. We have recently demonstrated that knock down (KD) of DA
expression attenuates synaptic clustering of PSD-95, and decrease the
number of dendritic protrusions in developing hippocampal neurons.
Furthermore, in-vivo experiments indicate that DA-KD in the rat hippocampus impairs pre-pulse inhibition. These data suggest that DA plays a
role in the regulatory mechanism of glutamate receptor activity in addition to that of spine shapes. To investigate the effect of DA-KD on
glutamate receptor activities in vitro, 12-DIV hippocampal neurons were
treated with antisense oligonucleotide specific to DA (AOD) and reverse
AOD (ROD). Then at 14 DIV, NMDA and AMPA currents in mocktreated, AOD-treated, and ROD-treated neurons were measured using
whole cell patch-clamp technique. We applied glutamate (1, 10, 30, 100
and 300 µM) in the presence of 50 µM AP5, and NMDA (1, 10, 30, 100
and 1000 µM) in the presence of 20 µM CNQX. The AMPA currents
seemed larger in AOD-treated neurons than mock-treated and RODtreated neurons. There were no clear differences in the NMDA currents
between AOD-treated and ROD-treated neurons. The data suggest that
drebrin A is involved in the regulatory mechanism of glutamate receptor
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Sensory functions
Myosin II plays a pivotal role in glutamateinduced translocation of drebrin-binding
actin filaments from dendritic spine to
parent dendrite
Regulatory roles of gonadal hormones on
the stimulatory effects of cocaine- and
amphetamine-regulated transcript (CART)
peptide in midbrain dopaminergic systems
Mizui, Toshiyuki1,2; Sekino, Yuko3,4; Shirao, Tomoaki1
Shieh, Kun-Ruey1,2; Yang, Shu-Chuan1,2,3 (1Institute of
(1Gunma Univ., Grad. Sch. Med., Maebashi, Japan; 2JSPS Research
Fellow; 3Division of Neural Network, Inst. Med. Sci. University of
Tokyo, Tokyo.; 4CREST)
Neuroscience, Tzu Chi University, Hualien, Taiwan; 2Institute of
Integrative Physiology and Clinical Sciences, Tzu Chi University;
3Department of Nursing, Tzu Chi College of Technology)
We have previously shown that glutamate induces the translocation of
drebrin and F-actin from dendritic spines to the parent dendrites, which
induces the morphological changes of dendritic spines into filopodialike protrusion. Timelapse imaging of GFP-drebrin expressing neuron
revealed that translocation occurred within 60 sec. Since myosin II B is
involved in actin-drebrin complex in the brain, contribution of actin-myosin system to the translocation was investigated in this study. In the
present study, we analyzed the effect of blebbistatin, a specific inhibitor
of myosin II ATPase, on the glutamate-induced translocation of drebrin
and F-actin in cultured hippocampal neurons. We prepared the low density cultures of rat hippocampal neurons from 18-day embryo. After pretreatment of 21 DIV neurons with 100 µM (±) blebbistatin for 60 min,
we applied 100 µM glutamate for 10 min and analyzed the location of
drebrin and F-actin immunocytochemically. Blebbistatin completely
inhibited the glutamate-induced translocation of drebrin and F-actin.
These data demonstrate that myosin II activity is required for
translocation of drebrin and F-actin from dendritic spines to parent
dendrites in hippocampal neurons.
Dense expression of cocaine- and amphetamine-regulated transcript
(CART) mRNA and peptide in the nucleus accumbens (NA) and striatum (ST) are found. NA and ST are the main projection sites of mesolimbic (ML) and nigrostriatal (NS) dopaminergic (DA) systems which are
involved in the extrapyramidal motor system and ewarding and emotional behaviors, respectively. Whether gonadal hormones, estradiol (E) and
testosterone (T), play the regulatory roles on the stimulation of CART
peptide in the MLDA and NSDA systems were examined in SpragueDawley rats in this study. DA neuronal activities were determined by
measuring the concentration of DOPAC (3,4-dihydroxyphenylacetic acid), the major metabolite of DA, in the NA and ST by HPLC-ECD. Intracerebroventricular administration of CART peptide increased the
DOPAC content of NA and ST in ovariectomized (OVX) priming E, but
not in OVX only female rats. The stimulation by CART peptide on the
DOPAC contents of NA and ST was found only in intact, castrated
(CAS) with E or T priming, but not in CAS only male rats. Finally, E and
T antagonists blocked T effects, but only E antagonist could block E effects. All these findings indicate that gonadal hormones play the regulatory roles on the stimulation of CART peptide in MLDA and NSDA
systems, and suggest that E is through intracellular genomic rather than
extracellular non-genomic action.
Nature of Neural Clustering in
Inferotemporal Cortex of Macaque Monkey
Sato, Takayuki; Uchida, Go; Tanifuji, Manabu (BSI,
RIKEN, Wako, Japan)
Sensory functions
Extracellular recordings in inferotemporal (IT) cortex suggest that there
are columnar organizations in terms of the optimal stimuli for individual
neurons (Fujita, et al., 1992). This finding is consistent with optical imaging experiments showing that a visual stimulus elicits a localized activation of the spots in IT cortex (Wang, et al., 1996, 1998). However, it is
also known that object selectivity of nearby cells is not necessarily the
same. Thus, our understanding of columnar organizations in IT cortex is
still partial.
Here, we identified active spots revealed by optical imaging, and then
recorded single unit activities and multiple unit activities (MUAs) from
these spots. To quantify the similarity of object selectivity, we calculated
correlation coefficients of responses to 100 object stimuli.
We found that only 30% of pairs of nearby single units showed
significant correlation in object selectivity. However, many pairs of
MUAs (63%) showed significant correlation in object selectivity. The
difference in number of significant pairs suggests (1) that there is a
common property among single cells, and (2) that averaging of
responses of individual cells, such as MUA, decreases cell-to-cell
variability in object selectivity and disclose common property among
cells within a spot. In fact, when all MUAs in a spot were averaged,
single unit activity and the averaged MUA showed significant
correlation in object selectivity in 63% of single cells within the spot.
However, we could not find such significant correlation between the
averaged MUA of a spot and single unit activity recorded from outside
of the spot.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Sensory functions
Changes in expression of G proteins
induced by dexamethasone and chronic
pain explain for their inhibitory effects on
development of analgesic tolerance to
morphine administration
Ahmadiani, Abolhassan2;
Fereshteh2; Kazemi, Bahram2 (1Deparment
Physiology, School of Medical Science, Tabiat Modares University,
Tehran, Islamic Republic of Iran; 2Neurosci. Res. Cent., Shaheed
Beheshti Med. Sci. Univ., Tehran, Iran.)
P2Y receptor-mediated enhancement of
inhibitory synaptic transmission in
substantia gelatinosa neurons of the rat
spinal cord
Nakatsuka, Terumasa; Fujita, Tsugumi; Koga, Akiko;
Liu, Tao; Kumamoto, Eiichi (Dept. Physiol., Facult. Med., Saga
Univ., Saga, Japan)
The inhibitory effect of pain on tolerance development to analgesic effect of opioids is reported to be mediated by stress aspect of pain and activation of HPA axis. We tried to investigate whether the chronic pain
and co-administration of dexamethasone (Dex) is able to reverse the tolerance and to evaluate expression of Gαi/o and Gβ subunits of G proteins
following chronic pain, chronic Dex, tolerance and their
combination.Tolerance was induced by chronic intraperitoneal (i.p.)
administration of morphine to male Wistar rats and analgesia was
assessed using tail flick test. Lumbar spinal tissues were assayed for
expression of G proteins using "semi-quantitative PCR" normalized to
beta-actin.Both chronic pain and chronic Dex could reduce and reverse
the tolerance. Chronic morphine did not change Gαi/o gene expression,
while chronic pain and Dex both increased its expression. Expression of
Gβ was increased following chronic morphine, but not following
chronic pain and Dex. None of these increases were observed when
morphine was co-administered with pain or Dex. It seems that the
development of tolerance to analgesic effect of morphine is partially
mediated by increased Gβ gene expression. The increase in Gαi/o genes
expression produced by chronic pain and chronic Dex can facilitate
opioid signaling pathway and compensate for morphine-induced
To date, eight subtypes of metabotropic P2Y receptors, P2Y1, P2Y2,
P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y14 receptors, have been identified in mammals. Although numerous studies have demonstrated that
ionotropic P2X receptors play a crucial role in facilitating pain transmission in the spinal cord, possible roles for P2Y receptors in nociceptive
signaling have received limited attention. In this study, we examined
whether the activation of P2Y receptors can modulate synaptic transmission in substantia gelatinosa (SG) neurons of adult rat spinal cord slices
using whole-cell patch-clamp recordings. Bath application of UTP (100
µM), an agonist for P2Y2 and P2Y4 receptors, and UDP (100 µM), an
agonist for P2Y6 receptors, affected neither excitatory (glutamatergic)
nor inhibitory (glycinergic and/or GABAergic) synaptic transmission in
all 11 SG neurons recorded. 2-Methylthio ADP (30 µM), an agonist for
P2Y1, P2Y12 and P2Y13 receptors, also did not change excitatory
transmission in all 11 SG neurons examined, but significantly increased
the frequency and amplitude of spontaneous inhibitory (glycinergic and/
or GABAergic) postsynaptic currents in 12 of 52 SG neurons recorded.
These results indicate that the activation of 2-methylthio ADP-sensitive
P2Y receptors enhances inhibitory but not excitatory synaptic
transmission, probably through a direct action on spinal inhibitory
interneurons. Thus, in contrast to P2X receptors, P2Y receptors can be
involved in an inhibitory effect on pain transmission in the spinal dorsal
Brain regions that distinguish muscle pain
from skin pain
Takahashi, Ken1; Taguchi, Toru1; Tanaka, Satoshi2;
Sadato, Norihiro2; Kakigi, Ryusuke2; Mizumura, Kazue1
(1Dept. Neural Regulation, Res. Inst. Environ. Med., Nagoya Univ,
Japan; 2Natl. Inst. for Physiol. Sci., Japan)
Recent brain imaging studies have been revealing central processing of
muscle pain. However, there is hardly a consensus on a brain region specifically responsible for muscle pain, not for skin pain. To seek out this
specific region, we used event-related functional magnetic resonance
imaging (MRI) and the 3-T MRI scanner. Twelve healthy male subjects
participated in this study. Electric stimulation with 1-ms duration was
applied to two sites: the left anterior tibial muscle and the skin just above
it. The stimuli consist of three levels in strength both for skin and muscle
stimulation, i.e. nonpainful, painful (5/10 in visual analog scale, VAS)
and more painful (7/10 in VAS). Group analysis revealed that brain regions activated by muscle stimulation included ipsilateral superior frontal gyrus, postcentral gyrus (primary somatosensory cortex, SI),
posterior cingulate; contralateral precentral gyrus, medium dorsal nucleus (thalamus), superior temporal gyrus; bilateral middle frontal gyrus,
cingulate gyrus and lenticular nuclei. These regions are similar to those
reported by other researchers and some of them are known to be activated during pain. Brain regions specifically activated by muscle stimulation, not by skin stimulation, were ipsilateral culmen, cingulate gyrus;
contralateral lenticular nucleus, pons, paracentral lobule (SI), substantia
negra, medial frontal cortex and bilateral middle temporal gyrus. These
regions may play an essential role to distinguish muscle pain from skin
pain in the brain.
Evidence from median nerve
somatosensory evoked potentials for the
activation of inhibitory cortico-coritcal
pathway from area 4 to area 1 during finger
movement in man
Weerasinghe, Vajira S; Senanayake, Nimal (Department
of Physiology, Faculty of Medicine, University of Peradeniya, Sri
Previous studies show that the spinal cord and subcortical components
of the somatosensory evoked potentials are not attenuated during movement in man. The objective of the present study was to investigate the
effect of simultaneous fractionated finger movement on cortically generated somatosensory evoked potentials. Left median nerve somatosensory evoked potentials were recorded from a scalp array of 21 electrodes
over the right scalp in 9 normal subjects aged 18-31 years. They reclined,
eyes closed as recordings were made at rest and during fractionated finger movements of the left hand. Latency and peak-to-peak amplitude of
the parietal N20 and frontal P20 waves did not show statistically significant difference between rest and movement conditions. This represents
activity in the tangentially arranged pyramidal neurons in area 3b. Amplitude of the parietal P25 wave, representing activity in radially arranged area 1 pyramidal neurons was attenuated during finger movement
by 70% (P<,0.01; Wilcoxon's test). Fractionated finger movements utilise the corticospinal pathway from the motor cortex. In animal studies
it has been shown that there is evidence for an inhibitory cortico-cortical
pathway from area 4 (motor) to area 1 (sensory) cortex but not to area
3b. We propose that activity in this pathway is responsible for our findings. This can be considered as a physiological evidence for the function
of a cortico-cortical pathway in man.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Sensory functions
The dynamics of neural network and
microcirculation: interhemispheric
interactions and neurovascular coupling
Signal amplification in the olfactory
sensory cilia
Nemoto, Masahito1; Hoshi, Yoko1; Sato, Chie1;
Terakawa, Susumu2 (1Tokyo Institute of Psychiatry, Tokyo,
Biosciences, Osaka University, Osaka, Japan.)
Japan; 2Hamamatsu University School of Medicine, Hamamatsu,
Interhemispheric neural interactions between bilateral cortical regions
are critically dependent on interhemispheric time lag of cortical activation. In somatosensory systems the time lag information is processed for
integrating bilateral stimulus inputs. Here we investigated interhemispheric interactions between bilateral somatosensory cortices by simultaneous recording of neural and hemodynamic signals and by analyzing
their dependence on stimulus time lag between conditioning stimuli
(CS) and test stimuli (TS). We measured electrophysiological signals
(local field potentials, 1<LFPs<100 Hz; multiunit spiking activity,
300<MUA<5k Hz) and optical intrinsic signals (586 nm, cerebral blood
volume, CBV; 605 nm, oxygenation) in rat somatosensory cortex evoked
by electrical pulses to the contralateral hindpaw (TS) while delivering
electrical pulses to the ipsilateral hindpaw (CS) under α-chloralose
anesthesia. Both responses to CS-TS were normalized by the responses
to TS without CS. The results showed that both electrophysiological
(LFPs and MUA) and optical (586 nm monophasic and 605 nm biphasic
activity) responses were significantly suppressed around 40-60 ms time
lag and slightly augmented at 0 ms. Average and trial-by-trial correlation
analyses revealed that CBV-related optical signals have high fidelity to
integrated MUA and LFP negative components. Activity-related
microcirculatory responses may more faithfully reflect neural
interactions through brain network than we imagined.
Molecular mechanisms underlying olfactory signal amplification were
investigated by monitoring cAMP dynamics in the intact sensory cilia.
[cAMP]i increased superlinearly with time during odorant stimuli for
over a second. This time course was remarkably different from that obtained with the rapid quench method previously applied to the in vitro
preparation, in which [cAMP]i change has been reported to be transient.
The superlinear increase of [cAMP]i was due to a gradual increase of
cAMP production rate that was consistent with the thermo-dynamical interaction model between elemental molecules, as has been revealed on
the rod photoreceptor cell. It thus seems likely that the fundamental
mechanism for molecular interactions between olfactory transduction elements is similar to that of the rod. In olfaction, however, cAMP production was extremely small (~200,000 molecules/s/cell at the maximum),
in contrast to the cGMP hydrolysis in the rod (250,000 molecules/photon). The observed numbers indicate that the olfactory receptor cell has
lower amplification at the enzymatic cascade. Seemingly, such low amplification is a disadvantage for the signal transduction, but this unique
mechanism would be essential to reduce the loss of ATP. Transduction
by a smaller number of second messenger formations would be achieved
by the fine ciliary structure that has a high surface-volume ratio. In addition, it is speculated that this low amplification at their enzymatic processes may be the reason why the olfactory receptor cell has acquired
high amplification at the final stage of transduction channels, utilizing
Ca2+ as a third messenger.
Activation of TRPV4 by hyperosmolality
Suzuki, Makoto; Mizuno, Atsuko (Department of
pharmacology, Jichi medical, Tochigi, Japan)
TRPV4 is first reported to be a "hypoosmolality-sensing" cation channel. On the following studies with knockout mice (Trpv4-/-), we have reported that response of vasopressin to hypertonicity was exaggerated but
another group has reported that it was abolished in Trpv4-/-. Although
controversial in response, both reports suggest that TRPV4 can be responsible to hypertonic stimuli. To elucidate "hyperosmolality-sensing"
in TRPV4, we designed to re-examine the response in vivo and investigate whether TRPV4 was sensitive to hyperosmolality in cultured neuronal cells. Trpv4-/- and Trpv4+/+ mice were subjected to dehydration
from 24 to 96 hrs. Then serum osmolality and water intake were measured. There was not remarkable difference in serum osmolality at any
period of dehydration but a significant decrease in serum osmolality of
Trpv4-/- at 72 hrs dehydration. Water-crave behavior and amount of water intakes after the dehydrations were not changed. Thus TRPV4 channel may respond to hyperosmolality. Neuronal cell lines with and
without TRPV4 were established from a cell line. Hyperosmoliality (500
mOsm) induced robust Ca influx in TRPV4 (+) cells by the method of
fluorescence quenching, while not in TRPV4 (-) cells. The influx was
partially blocked with genistine, a blocker of tyrosine kinase, and blunted with pBPB, a blocker of PLA2. Therefore, TRPV4 is hyperosmolality-sensng channel through several biochemical cascades.
Takeuchi, Hiroko; Kurahasi, Takashi (Dept of Frontier
Analysis of bitter taste inhibition by fatty
Yasumatsu, Keiko1; Saito, Sachiko2; Murata, Yuko3;
Ming, Ding4; Kobayakawa, Tatsu5; Margolskee, Robert
F4; Ninomiya, Yuzo1 (1Sect. Oral Neurosci., Grad. Sch. Dent.
Sci., Kyushu Univ.; 2Saito Sachiko Taste and Smell Research
Institution; 3National Res. Institute of Fisheries Sci.; 4Dept. of
Physiol. & Biophys., Mount Sinai Sch. Med.; 5National Institute of
Advanced Industrial Science and Technology)
The effect of unsaturated fatty acids on taste sensitivity was explored by
human psychophysical study, molecular biological study and mouse
neuroethological study. Inhibitory effects of fatty acids on bitter taste including QHCl and denatonium were demonstrated in perceived taste intensity test in human, the mouse behavioral experiment using a short term lick test (10 sec) and responses in the chorda tympani (CT) and the
glossopharyngeal (GL) nerve in wild type mice. In contrast, fatty acids
have no effect on bitter tasting L-amino acids, NaCl, HCl, sucrose and
MSG in all study performed. To investigate involvement of Ggustducin
in this inhibitory effect of fatty acids, nerve recording from gustducin
KO mice and in vitro G-protein activation assay using bovine taste membrane were employed. Results from gustducin KO mice showed no suppression in bitter taste responses both in the CT and the GL. In vitro Gprotein activation assay using bovine taste membrane showed that the
activation of both gustducin and transducin by denatonium was significantly inhibited by DHA and oleic acid, and that the activation of transducin by rhodopsin was not inhibited. These results suggest that fatty
acids specifically inhibit responses to bitter stimuli by suppression of activation of T2R receptors which coupled with Ggustducin and Gtransducin.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Sensory functions
Depolarization of isolated horizontal cells
acidifies their immediate surrounding by
activating V-ATPase.
Akinori1; Hara, Masayuki1; Kaneko, Akimichi3; Yamada,
Masahiro1 (1Dept. of Biol. Sci., Tokyo Metropolitan Univ., Hino,
Tokyo, Japan; 2Astellas Pharma. Co., Yodogawa, Osaka, Japan;
of Rehabilitation, Seijoh Univ., Tokai, Aichi, Japan)
Voltage-gated ionic channels of cholinergic
amacrine cells in the mouse retina
Kaneda, Makoto1; Shigematsu, Yasuhide2; Morishima,
Yosuke3; Shimoda, Yukio2 (1Dept. Physiol., Keio Univ. Sch.
Med., Tokyo, Japan; 2Medical Research Institute,Tokyo Womens'
Medical Univ., Tokyo, Japan; 3Dept. Cognitive Neuroscience, Grad.
Sch. Med., Univ. Tokyo, Tokyo, Japan)
It has been suggested that pH of the invaginating synaptic cleft of the
cone terminal is related to the membrane voltage of horizontal cells
(HCs), low in the dark and high during surround illumination (1). High
pH enhances the release of L-glutamate from the cone terminal, resulting
in the formation of the centre-surround receptive field and the colour opponency in the visual system. We measured the pH of the immediate external surface (pHo) of an HC isolated from carp or goldfish retina to
study the mechanisms producing the pH change by a ratio imaging technique, using a pH-sensitive lipophilic dye, 5-hexadecanoylaminofluorescein. When HCs were depolarized by application of 20 µM kainate or
by high extracellular K+, pHo acidified. The amount of pH acidification
was monotonically dependent on the depolarization, as much as
0.21±0.05 pH unit by 100 mM K+ (approx. 94 mV depolarization).
Acidification of the HC surface was suppressed by 0.4 µM bafilomycin
A1, a specific inhibitor of vacuolar type H+-ATPase (V-ATPase),
suggesting the existence of an outward electrogenic H+ pump enhanced
by the HC depolarization. These are consistent with the hypothesis that
proton released from the depolarized HCs can act as the inhibitory
feedback transmitters onto cone synaptic terminals. (1):Hirasawa, H.
and Kaneko, A. (2003) J.Gen.Physiol.,122:657-71.
Recent studies showed that the cholinergic amacrine cells have a unique
membrane properties but the membrane properties of the cholinergic
amacrine cells have not been characterized systematically. Here, we
studied the voltage-gated ionic channels of transgenic mouse line whose
cholinergic amacrine cells were selectively labeled with GFP electrophysiologically and immunohistochemically. Voltage-gated K currents
were inhibited by 4-aminopyridine (A current) and tetraethylammonium
(delayed rectifier). Voltage-gated Ca currents had ω-conotoxin GVIAsensitive component (N-type) and ω-AgaIVA-sensitive component (P/
Q-type). Tetrodotoxin-sensitive Na current and dihydropyridinesensitive Ca current (L-type) were not observed. The immunoreactivity
for K channels subunits (Kv. 3.1 (delayed rectifier) and Kv. 3.3 (Acurrent)) and Ca channel subunits (α1A (P/Q-type) and α1B (N-type))
was colocalized with GFP signals. Immunoreactivity for Ca channel
subunits (α1C (L-type)) did not colocalize with GFP signals.
Immunoreactivity for Na channel subunit existed in the nuclear region
but not in the cell surface of the GFP positive cells. Our findings indicate
that the signal propagation of the cholinergic amacrine cells are
mediated by the combination of the voltage-gated K channels (A-type K
current and delayed rectifier K current) and the voltage-gated Ca
channels (P/Q-type and N-type) in the mouse retina.
Structural and Functional Properties of
Homologous Electrical Synapses between
Retinal Amacrine Cells
Axonal regeneration of cat retinal ganglion
cells into the crushed optic nerve with a
Rho/ROCK inhibitor
Hidaka, Soh (Department of Physiology, Fujita Health University
Watanabe, Masami1; Matsui, Hiroe2; Terasaki, Hiroko2;
Nakamura, Hiroko2; Tokita, Yoshihito1 (1Inst. Dev. Res.,
School of Medicine, Toyoake, Aichi, Japan)
Retinal amacrine cells (ACs) regulate activities of retinal ganglion cells,
the output neurons to higher visual centers, through cellular mechanism
of lateral inhibition in the inner plexiform layer (IPL). Electrical properties of gap junction networks between ACs in the IPL were investigated
using combined techniques of intracellular recordings, Lucifer yellow
and Neurobiotin injection, dual patch-clamp recordings and high voltage
electron microscopy in isolated retinas of cyprinid fish. Six types of gapjunctionally connected ACs were classified after recordings of their
light-evoked responses to light flashes. Among them gap junction networks of three types of ACs were studied with structure-function correlation analysis. Cellular morphology of intercellular connections
between three homologous cell classes was characterized. High voltage
electron microscopy (Hitachi 1250M, NIPS, Okazaki, co-operative program 2005-HVEM02) revealed localization of gap junctions between
the dendritic tips of Neurobiotin-coupled cells. Receptive field size,
space length constant, response latency and conduction velocity were
measured. Simultaneous dual patch-clamp recordings revealed that the
lateral gap junction connections between homologous ACs expressed bidirectional electrical synapses passing Na+ spikes. Lateral inhibition regulated by ACs in the IPL appears to be associated with directional
extension of the dendrites and orientation of dendrodendritic gap junctions (J. Integr. Neurosci. 4(3): pp 313-340, 2005).
Kasugai, Japan; 2Nagoya Univ. Sch. Med. Ophthalmol., Nagoya,
We examined whether Y39983, a novel Rho/ROCK inhibitor, can make
injured RGC axons regenerate into the crushed optic nerve (OpN) of
adult cats. Methods: In vitro: retinal pieces were cultured with Y39983
for 14 days to count number of TUJ-1-positive neurites. In vivo: the left
OpN of anesthetized cats was crushed with 0.2 N tension for 60 s. On day
12, 0.5 mg of WGA-HRP was injected into the vitreous to label regenerated axons. On day 14, the cats were perfused with fixative, the OpN was
dissected, embedded in gelatin. Frozen sections were reacted for HRP
with TMB. Results: Retinal culture: To obtain the optimum concentration for axonal regeneration, we examined effect of Y39983 on neurite
outgrowth of cultured retinal pieces. The number of TUJ-1 positive processes was greatest at the concentrations of 3 and 10 µM in central to
peripheral retinal areas. Similarly, length of neurites in retinal pieces was
longest at 3 and 10 µM. Axonal regeneration in crushed OpN: We
injected Y39983 at 10 and 100 µM. An injection of 10 µM Y39983
increased regenerated axons longer than 0.5 to 2 mm from the crush site.
The second injection of 10 µM Y39983 at day 7 increased the number 2
to 3.5 fold than the number in one injection. Single or double injections
of 100 µM 39983 increased the number of regenerated axons.
Conclusion: A Rho/ROCK inhibitor, Y39983, enabled injured axons of
RGCs of adult cats to regenerate into the crushed OpN.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Motor functions
Spectral cue responses of sustaned
response neurons in the primary auditory
Sato, Yu; Chimoto, Sohei; Sakai, Masashi (Dep. Physiol.
Grad. Sch. Univ. Yamanashi, Tamaho, Japan)
Previous studies have shown that sustained-response neurons in the primary auditory cortex (A1) of awake animals have sensitivities to the
spectral-edge and fundamental frequency (F0) and that the sensitivity
originates from the specific inhibition pattern of frequency receptive
field (FRF). This study investigated cell-type correspondence between
the different experiments by estimating the excitatory and inhibitory
summation patterns for harmonic components with a given F0. The findings show that there are, at least, four types of sustained-response neurons sensitive to specific spectral features of the complex tone: energyintegrator cells integrate sound energy on the excitatory FRF; high-edgesensitive cells are sensitive to the spectral high edge on the best frequency (BF); low-edge sensitive cells detect the spectral low edge on BF; F0sensitive cells are sensitive to two F0s of harmonics corresponding to BF
and an octave below, but not to noise with a similar spectral location. The
spectral-cue sensitivity originates from specific inhibitory FRF: less
dominant inhibition for energy-integrator cells, asymmetric inhibition
for high- and low-edge-sensitive cells, and selective inhibition of nonpreferred harmonics for F0-sensitive cells. A1 operates as filters with
pass bands and reject bands, which correspond to the peak and trough in
FRF. The filter specification is well organized for decoding three acoustic features: sound energy in a given spectral region, spectral edges, and
F0 of harmonics.
Vergence eye movements elicited by nondisparity factors in 2D movies
Iijima, Atsuhiko1,3; Kiryu, Tohru2; Ukai, Kazuhiko3;
Bando, Takehiko1 (1Div. Integrative Physiol., Grad. Sch. Med. &
Dent. Sci., Niigata Univ., Niigata, Japan; 2Div. Inform. Sci., Grad.
Schi. & Tech., Niigata Univ., Niigata, Japan; 3Dept. Appl. Phys.,
Sch. Sci. & Tech., Waseda Univ., Tokyo, Japan)
We have analyzed traces of vergence eye movements while subjects
watch three-dimensional (3D) and two dimensional (2D) movies, and
found that 2D images without binocular disparity sometimes evoked
convergence similar to those found in the 3D condition. In this study, we
investigated factors that drove convergence other than binocular disparity. As previously presented, 3D/2D movies were projected on an 80
inch-screen in a dark room (10 lux) by two liquid crystalline projectors
with/without polarized filters to provide binocular disparity, field-sequentially. To monitor the gaze of the subject, we measured binocular
eye movements and head movements with a new binocular video oculography (Newopto) and a magnetic motion sensor (Polhemus). A 2D
movie representing driver's view of a go-cart (160 sec), which included
forward scenes and expanding optic flows, was shown to fifteen subjects. The motion vectors localized to their gaze points were calculated,
and the optic flow was estimated based on flame-by-flame analyses of
images. The traces of vergence eye movements consisted with the
changes in the optic flow. It is suggested that most of factors that drove
convergence in 2D movies are closely related to optic flow.
Motor functions
Longitudinal study of the involvement of
several motor cortical regions in functional
recovery after the Lesion of the
Corticospinal Tract at Cervical Spinal Cord
in Monkeys
Yukio, Nishimura1,5; Onoe, Hirotaka2,5; Perfiliev,
Sergei3; Tsukada, Hideo4,5; Isa, Tadahi1,5 (1Dev. Physiol.,
Nat. Inst. Physiol. Sci., Okazaki, Japan; 2Psychol., Tokyo Metro.
Inst. Neurosci., Tokyo, Japan; 3Physiol., Göteborg University,
Göteborg, Sweden; 4Hamamatsu Photo. K.K., Hamamatsu, Japan;
It was reported that after the lesion of the corticospinal tract (CST) at C5,
recovery of the finger dexterity was completed in 1-3 months (Sasaki et
al. 2004, Nishimura et al. 2005). In this study, to clarify the neuronal
mechanism of the functional recovery, we performed following two experiments; 1) In 3 monkeys, we performed functional imaging by
positron emission tomography (PET) during precision grip. At postoperative 1 month (during early recovery), increased activity was observed
in bilateral primary motor cortex (M1). At postoperative 3 months (after
complete recovery), in addition to bilateral M1, activities in bilateral
ventral premotor cortex (PMv) were increased. 2) In 2 monkeys we investigated the effect of reversible inactivation of areas where we found
increased activity in the PET study. Inactivation of M1 contralateral (c)
to the lesion caused deficit in control of independent finger movements
both at postoperative 1 and 3 months. Inactivation of M1 ipsilateral (i) to
the lesion caused deficit in precision grip at postoperative 1 month. Inactivation of iPMv also caused deficit in precision grip at postoperative
3 months. These results indicate that bilateral M1 and iPMv play an important role in the functional recovery.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Motor functions
Synaptic mechanisms acting on
motoneurons with reference to the basal
ganglia control of locomotion
The effect of the extinction of the initial
fixation target on ocular following
Taksakusaki, Kaoru; Saitoh, Kazuya (Asahikawa Medical
Miura, Kenichiro; Taki, Masakatsu; Tabata, Hiromitsu;
Kawano, Kenji (Grad. Sch. Med. Kyoto Univ., Kyoto, JAPAN)
College, Asahikawa, Japan)
The midbrain locomotor region (MLR) is located in the lateral part of the
mesopontine tegmentum. Either electrical or chemical stimulation of the
MLR first increases the level of postural muscle tone and then initiates
locomotion. The MLR receives GABAergic basal ganglia output from
the substantia nigra pars reticulata (SNr). The present study was designed to understand synaptic mechanisms acting on motoneurons involved in the basal ganglia control of locomotion. Intracellular recording
was made from hindlimb motoneurons (n=47) in two types of decerebrate cats (n=11). One was mesencephalic cats decerebrate at precollicular-postmammilarly level, and the other was hypothalamic cats
decerebrated at precollicular-premammilarly level. In mesencephalic
cats, short trains of stimuli applied to the MLR (3 pulses, 5 ms intervals,
30-50 µA) induced a sequence of EPSPs and IPSPs. Although
stimulation of the SNr (20-60 µA, 50-100 Hz) alone neither changed
membrane potentials nor input resistance of motoneurons, it greatly
reduced the amplitude of the MLR-induced IPSPs, resulting in an
enhancement of the EPSPs. In hypothalamic cats, fictive locomotion,
sequences of membrane oscillations with depolarizing and
hyperpolarizing phases, was induced in motoneurons. SNr stimuli
reduced the hyperpolarizing phases and finally stopped the oscillations
of both extensor and flexor motoneurons. These results suggest that
enhancement of the basal ganglia output to the brainstem may stop
locomotion by suppression of the postsynaptic inhibitory drive acting on
motoneurons and initiate locomotion by removal of the inhibition.
Initial tracking responses were larger when smooth pursuit eye movements were executed after a steady fixation and the initial fixation target
disappeared before the onset of the pursuit target motion (Miura et al.
JJP, 55 (Suppl), 2005). To study whether this phenomenon is common in
the genesis of visually-guided reflexive ocular behaviors, we observed
human ocular following response, a reflexive eye movement elicited by
the motion of a wide-field visual stimulus. At the beginning of each trial,
a stationary fixation point (placed at the center of the screen) and a stationary random-dot pattern (covering the wide visual field) were presented. The subjects fixated the fixation point and immediately (no-gap
condition) or 200 ms after the extinction of the fixation point (gap condition), the random-dot pattern moved briefly at 20 deg/s rightward or
leftward for 0.2s, and then turned off. The latency of the ocular following
responses elicited by the random-dot pattern motion was not affected by
the presence of the gap. In all 3 subjects, the change in eye position during the open-loop period of the ocular following responses was significantly larger under the gap condition than under the no-gap condition.
The effect of the gap on the ocular following responses started about 15
ms after the onset of the ocular following responses. This result suggests
that the efficacy of visuomotor transmission for ocular following responses was facilitated by the release from the fixation before the onset
of the random-dot pattern motion, as was seen in the smooth pursuit initiation.
Simple-spike activity of Purkinje cells in
cerebellar dorsal vermis during vergence
eye movements
Nitta, Takuya; Akao, Teppei; Kurkin, Sergei; Fukushima,
Kikuro (Department of Physiology, Hokkaido University School of
Medicine, Sapporo)
For pursuit of small objects moving slowly and smoothly in space close
to the observer, two independent eye movement systems are used: frontal smooth pursuit and vergence-tracking. Signals for both systems must
be synthesized for pursuit of a target moving in three dimensions. Recent
studies in our laboratory have demonstrated that among the cerebral cortical pursuit areas, three dimensional (3D) pursuit signals are generated
primarily in the frontal eye fields (e.g. Akao et al. 2005). To drive ocular
motoneurons, 3D pursuit signals must be sorted into signals for each eye
movement system and finally into oculomotor signals. Studies in our
laboratory indicate that such conversion was not detected in the cerebeller floccular region (Tsubuku et al. Soc Neurosci Abstr 2004). The
cerebellar dorsal vermis is well known as another pathway for frontal
pursuit. To examine whether vergence signals are present in this area, we
examined simple-spike discharge of vermal pursuit Purkinje (P-) cells in
2 monkeys. Of a total of 64 P-cells that were examined during both frontal pursuit and vergence-tracking, 50% discharged for both, 37.5% only
for vergence-tracking, and 12.5% only for frontal pursuit. These results
indicate that about 90% of vermal pursuit P-cells discharged for vergence-tracking and that half of them still had 3D pursuit signals. Majority (71%) of these P-cells discharged before onset of vergence eye
movements with the typical lead time of 50 ms, suggesting their involvement in the initiation of vergence eye movements.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Higher CNS functions / Autonomic nervous functions
Higher CNS functions
Facilitation of plastic function in the brain
during food intake
Oomura, Yutaka1; Aou, Shuji2; Fukunaga, Kouji3;
Moriguchi, Sigeki3; Sasaki, Kazuo4 (1Grad. Sch. Med.
Kyushu Univ. Fukuoka, Japan; 2Grad. Sch. Life. Sci. Kyushu Inst.
Tech. Kitakyushu, Japan; 3Grad. Sch. Pharm. Pharmacol. Sendai,
Japan; 4Grad. Sch. Eng. Life Inform. Toyama, Japan)
During food intake 2-3 mM glucose concentration in CSF become twice.
When 6 mM glucose is injected into the hippocampus spatial learning
and memory are facilitated. We analized this mechanism. In vitro experiments, glucose concentration in Krebs Ringer solution was changed
from 3 to 6 mM for15 min and returned to the original 3mM glucose. By
the change the amplitudes of CA1 synaptic potentials were augmented
more than 3 times and continued for more than 40 min. Furthermore presynaptic transmitter release and postsynaptic responses to NMDA applied at the apical dendrites were also significantly facilitated. Neurochemically phosphorylations of presynaptic synapsin 1-3, postsynaptic
PKCα, ERK, CaMK II of CA1 neurons were all significantly facilitated
by the glucose change.LTP of CA1 neurons produced by a tetanic
stimulation of the Schaffer collateral / commissure whichwas applied
just before returning from 6 to 3 mM glucose was significantly
facilitated, while only STPwas produced in 3 mM glucose. During LTP
meintenance only phosphorylations of MARCKS (related to plasticity)
and PKCα were significantly facilitated. We are now studying the effect
of blockers of ATP sensitive K channels on CA1 neurons.These
evidences indicate the importance of food intake for reinforcement of the
higher brain function..
Autonomic nervous functions
Attenuated defense response induced by
stimulation of amygdala in orexin neuron
ablated mice
Zhang, Wei1; Sakurai, Takesi2; Kuwaki, Tomoyuki3
(1Department of Molecular and Integrative Physiology, Chiba
University Graduate School of Medicine, Chiba , Japan;
2Department of Pharmacology, Graduate School of Comprehensive
Human Sciences, University of Tsukuba, Tsukuba 305-8577, Japan;
3Department of Molecular and Integrative Physiology,Department
of Autonomic Physiology Chiba University Graduate School of
Medicine, Chiba 260-8670, Japan)
We have previously shown that the defense response against stressor was
attenuated in prepro-orexin gene knockout mice and orexin neuron-ablated mice (Jpn J Physiol 55: S87, 2005). We have proposed that orexin
plays as a master switch to elicit multiple efferent pathways of the defense response. It is still open question, however, how information of
stressor activates the orexinergic neurons. In this experiment, we examined possible contribution of the amygdala as one of the afferent nuclei
to activate orexinergic neurons. In urethane-anesthetized mice, a
GABA-A receptor antagonist, bicuculline, was microinjected into the
amygdala, of which electrical stimulation induced simultaneous increases in blood pressure, heart rate, and respiratory frequency. Bicuculline
dose-dependently induced cardiorespiratory excitation in both orexin
neuron-ablated mice and wild-type controls. However, dose-response
curve was rightward shifted in the orexin neuron-ablated mice. We conclude that the amygdala constitutes one of the afferent pathways to the
orexinergic neurons that involved in the defense response against stressor.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Autonomic nervous functions
Penile erection during RFEM sleep
regulated by the laterodorsal tegmental
Koyama, Yoshimasa1,2; Takahasi, Kazumi2; Iwasaki,
Hiroshi3; Kawauchi, Akihiro4; Miki, Tsuneharu4;
Kayama, Yukihiko2 (1Clst. Sci. Technol. Fukushima Univ.
Fukushima, Japan; 2Dept. Physiol. Fukushima Med. Univ. Scl.
Med.; 3Dept. Urol. Minami Osaka Hospt. Osaka, Japan; 4Dept.
Urol. Kyoto Prefect. Univ. Kyoto, Japan)
The cholinergic neurons in the brainstem (laterodorsal tegmental nucleus: LDT) have a role in the regulation of REM sleep. M. Schmidt have
suggested that the cholinergic input to the preoptic area is crucial for induction of penile erection during REM sleep. In the present study, we examined the possible involvement of the cholinergic LDT neurons in the
regulation of penile erection during REM sleep. Unanesthetized, headrestrained rats were used. Single neuronal activity was recorded through
a glass pipette electrode. Penile erection is composed of two components; slow increase of the corpus spongiosum penis (CSP) pressure and
sharp peaks riding on the slow increase. Several types of the LDT neurons showed firing patterns in close relation with penile erection including 1) the cholinergic neurons which showed tonic firing increase 20-30
sec before the erection. The firing increase of this type of neurons was
well correlated with the slow increase of CSP pressure. 2) the cholinergic
neurons which showed phasic firing in synchronous with the sharp CSP
peaks. 3) the non-cholinergic neurons which decreased or stopped firing
during erection. These results suggest that the cholinergic neurons in the
LDT are involved in induction and excursion of penile erection during
REM sleep, while the non-cholinergic neurons have inhibitory influences on penile erection.
A- and C- reflexes elicited in ovarian
sympathetic nerves by single shock to a
somatic afferent nerve include spinal and
supraspinal components in anesthetized
Uchida, Sae; Kagitani, Fusako; Hotta, Harumi (Dept.
Auton. Nerv. Syst., Tokyo Metropol. Inst. Gerontol., Tokyo, Japan)
The spinal and supraspinal components of both A- and C- reflexes were
studied in the somato-ovarian sympathetic reflex discharges elicited by
a single shock either to a spinal (T9-11) afferent nerve or to a limb (tibial)
afferent nerve in urethane anesthetized rats. In central nervous system
(CNS) intact rats, a single shock to a T9-11 spinal afferent nerve produced early and late A-reflex discharges with latencies of about 51 ms
and 117 ms, respectively, and a C-reflex with a latency of about 200 ms
in a ovarian sympathetic efferent nerve. After spinalization at the third
thoracic level, stimulation of the same spinal afferent nerve produced an
A-reflex with the same latency as the early A-reflex in CNS-intact rats
and a C-reflex discharge with a latency of about 112 ms. On the other
hand, a single shock to a tibial afferent nerve evoked an A-reflex discharge with latency of about 91 ms, and a C-reflex discharge with a latency of about 228 ms in CNS-intact rats. In most cases, the A-reflex
could be divided into two subcomponents of different latencies. These
A- and C- reflex discharges elicited by stimulation of a tibial afferent
nerve were not observed after spinalization. It was concluded that ovarian sympathetic A- and C- reflex discharges evoked by stimulation of a
segmental spinal afferent nerve in CNS-intact rats are of spinal and supraspinal origin, and those evoked by tibial nerve stimulation are of supraspinal origin.
Effects of basic tastes stimuli on
autonomic nerve activity in anesthetized
Central nNOS activity in Dahl hypertensive
rats is normalized in the brainstem and
suppressed in the diencephalon
Niijima, Akira1; Torii, Kunio2; Uneyama, Hisayuki2
Tandai-Hiruma, Megumi; Hirakawa, Haruhisa;
Kemuriyama, Takehito; Nishida, Yasuhiro (The Second
(1Niigata Univ. Schoo of Med., Niigata, Japan; 2Institute of Life
Sciences, Ajinomoto Co., Inc., Kawasaki-shi, Kanagawa, Japan)
This report deals with effects of five basic taste stimuli on the activity of
sympathetic and vagal nerves innervating visceral organs in anesthetized
rats. Under urethane anesthesia, basic taste stimuli such as sweet (10%
sucrose or 5% glucose), or salty (5% NaCl), sour (0.6% acetic acid), bitter (0.2% quinine sulfate)and Umami (2.8% MSG) were applied into the
oral cavity for 10 minutes. Efferent nerve activity was recorded from
central cut end of sympthetic branches (adrenal and WAT) and vagal
branches (gastric and celiac). Results: Sensory stimulations with four basic taste stimuli (salty, sour, bitter and umami) evoked an activation of
sympathetic nerve activity to adrenal gland and WAT, however,taste stilulation with sweet taste caused a suppresseion in sympathetic efferents
as well as vagalgastric efferents. On the contrary, vagal celiac efferents
were activated by sweet taste and suppressed by other stimuli (salty,
sour, bitter. and UMami). These observtions suggest that the visceral
functions are regulated by taste stimulithrough autonomic outflows.
Dept. Physiol., Natl. Defense Medical College, Saitama, Japan)
We have demonstrated the distribution of nNOS neurons in the brainstem and diencephalon, which was upregulated in hypertensive Dahlsalt sensitive (DSS) rats compared with normotensive DSS rats. In this
study, we directly compared nNOS activity in the brainstem and diencephalon between hypertensive DSS and normotensive Sprague-Dawley
(SD) rats. The DSS and Dahl-salt resistant (DSR) rats were fed on 8%
NaCl food (DSS8% and DSR8%) or 0.4% NaCl (DSS0.4% and
DSR0.4%). SD rats were fed only on 0.4% NaCl food (SD0.4%). The
level of nNOS activity in the brainstem of SD0.4% was almost the same
as that of DSS8%, which had been significantly higher than those of
DSS0.4%, DSR0.4% and DSR8% (normotensive Dahl rat strain). Although the level of nNOS activity in the diencephalon of SD0.4% was
significantly higher than that of DSS8%, which had been almost the
same as those of the normotensive Dahl rat strain. All these results indicated that at normal blood pressure, the nNOS neuronal system in both
the brainstem and diencephalon of Dahl rat strain might be downregulated compared with SD rat strain. In hypertensive DSS rats, the nNOS
neuronal system specifically in the brainstem seems to be reversed to the
level in SD rats, although the nNOS neuronal system in the diencephalon
stays to be downregulated compared with SD rats.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Behavior & biological rhythm
Feedforward control of human
thermoregulation by skin sympathetic
nerve activity
Iwase, Satoshi1; Sawasaki, Naoki2; Michikami,
Daisaku3; Mano, Tadaaki4; Sugenoya, Junichi1; Cui,
Jian5 (1Dept. Physiol. Aichi Med. Univ. Aichi, Japan; 2Tokai
Hospital; 3Ohtsuka Pharmaceutical Co. Ltd.; 4Tokai Central
Hospital; 5Pennsylvania State University, PA, USA)
Feedforward control of human thermoregulation by skin sympathetic
nerve activitySatoshi IWASE1, Naoki SAWASAKI2, Daisaku
MICHIKAMI3, Tadaaki MANO4, Junichi SUGENOYA1, Jian CUI51.
Department of Physiology, Aichi Medical University, 2. Department of
Surgery, Tokai Hospital3. Ohtsuka Pharmaceutical Co. Ltd.4. Director,
Tokai Central Hospital5. Pennsylvania State UniversityTwo ways of
thermoregulation has been recognized, feedforward and feedback mechanisms. Feedforward mechanism employs neural afferent pathway,
whereas feedback uses convection of the blood stream from the peripheral to the core. We investigated the relation between the microneurographically recorded skin sympathetic nerve activity (SSNA) and the
tympanic temperature (Tty) measured as the core. Four exposure conditions, 1) local cold, 2) generalized cold, 3) local heat, 4) generalized
warming, were loaded to the subjects and the response of SSNA and Tty
were analyzed in time series analysis. The abilities to activate and to suppress SSNA were correlated to the changes in Tty, indicating that the individuals who are excellent in activating/suppressing SSNA are
excellent in thermoregulation. The time lag of neural activation in Tty
was within 1 min, whereas that of convectional Tty change was approx.
10 min. We concluded that skin sympathetic regulation is critical in rapid
thermoregulation in humans.
Effect of CO2 water immersion on cardiac
autonomic nerve function in humans
Sato, Maki1; Shimizu, Yuuki1; Iwase, Satoshi1;
Nishimura, Naoki1; Matsumoto, Takaaki1; Inukai, Yoko1;
Ogata, Akihiro1; Taniguchi, Yumiko1,2; Takada, Hiroki1;
Sugenoya, Junichi1 (1Dept. Physiol., Sch. Med., Aichi Med.
Univ., Aichi, Japan;
Univ., Aichi, Japan)
Food and Nut. Environ, Kinjo Gakuin
CO2 water immersion at 1000 ppm affects thermoregulation through the
increased cutaneous blood flow on the immersed skin area. However, the
effect of CO2 water immersion on cardiovascular function remains to be
clarified in humans. To examine whether CO2 bathing affects cardiac autonomic function, we analyzed heart rate variability and measured the
cardiac output by Echo (Apio XV, Toshiba, Japan) during CO2 and fresh
water immersion. Tympanic temperature (thermistor thermometry), cutaneous blood flow (laser-Doppler flowmetry) and electrocardiogram
(ECG) were monitored continuously. The subjective thermal and comfort sensations were asked every 10-min during experiments. After a rest
for 10 min, the subject immersed up to the breast level to CO2-rich water
at 1000ppm or fresh water at thermoneutral water. The results were
shown as follows: 1) HF was significantly higher in CO2 water immersion than in fresh water immersion. 2) LF/HF ratio was significantly
lower in CO2 water immersion than in fresh water immersion. 3) Heart
rate was not significantly different between CO2 water and fresh water
immersion. 4) Tympanic temperature was significantly lower during
CO2 water immersion than during fresh water immersion. 5) Cutaneous
blood flow in immersed forearm was significantly higher during CO2
water immersion than during fresh water immersion.
Behavior & biological rhythm
Early lighting condition (ELC) alters
circadian rhythm and maternal care of dam
with the subsequent disturbance of the
offspring's anxiety and memory.
Toki, Shigeru; Morinobu, Shigeru; Imanaka, Akihiro;
Yamawaki, Shigeto (Dept. Psychiat. Graduate. Sch. Biomed.
Sci. Hiroshima Univ. Hiroshima, Japan)
Although dramatic changes of lighting environments are occurring in
modern society, the influence of these changes has not been fully elucidated. It is demonstrated that, early life experiences (i.e. maternal care)
induce various changes in the function of circadian rhythm, emotionality
and memory. Here, we examined whether ELC affected circadian
rhythm and maternal care of dam, and the offspring's circadian rhythm,
anxiety and memory. Prolonged dark phase (PDP) was used to study the
effects of ELC. Dams and their litters were kept in a PDP cycle (L/
D=6:18h; postnatal days 2-14) or under a normal condition (L/
D=12:12h). Throughout this period, locomotor activity of dams was
measured and all cages were video recorded for behavioral scoring. At
age of 10-week, behavioral observations of the offsprings were undertaken. Circadian rhythm of locomotor, avoidant behavior, social interaction and object recognition memory were examined. Under PDP, the
morning offset of dams' motor activity was delayed and amounts of maternal care decreased for the first 2-7 days of lactation. In the adult offspring, circadian rhythm of locomotor was not affected. Whereas PDP
increased avoidant behavior, PDP decreased social interaction and memory. In conclusion, the PDP mothers showed impaired circadian rhythm
and maternal care, and the offsprings exhibited depressive-like behaviors. Therefore, it is conceivable that ELC may alter mother-infant interaction and subsequently change the offsprings' emotionality.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Behavior & biological rhythm
Discrimination between the tastes of
sucrose and saccharin by conditioned
preference learning in mice.
The role of GABAergic system in the
ventral pallidum on the retrieval of
conditioned taste aversion in rats
Miyamoto, Takenori1; Nagaki, Naoko1; Yasoshima,
Yasunobu2; Eda-Fujiwara, Hiroko1; Satoh, Ryohei3
Inui, Tadashi; Shimura, Tsuyoshi; Yamamoto, Takashi
(1Lab. Behav. Neurosci. Fac. Sci. Japan Women's Univ, Tokyo,
Japan; 2Dept. Mol. Gen. Inst. Biomed. Sci. Fukushima Med. Univ.
Sch. Med. Fukushima, Japan; 3Dept. Physiol. Sch. Med. Kitasato
Univ. Sagamihara, Japan)
(Dept. Behav. Physiol., Grad. Sch. Human Sciences, Osaka Univ.,
Osaka, Japan)
We examined the neural mechanism of discriminative taste preference
learning, using the procedure with some modifications of flavorpostingestive consequence learning paradigm in C57BL/6 male mice.
Wild mice were allowed to drink water for 10 min daily with the twobottle method after 16 h water- and food-deprivation. When mice were
alternately exposed to sucrose (Suc, 0.5 M) or saccharin (Sacch, 5 mM)
instead of water during 2 weeks, the amount of 0.5 M Suc consumption
markedly increased but that of 5 mM Sacch did not. Because naive mice
prefer to 0.5 M Suc much more than 5 mM Sacch, we employed 0.15 M
Suc, to which mice showed the same preference as 5 mM Sacch, instead
of 0.5 M Suc. The amount of 0.15 M Suc tended to increase with decreasing of the Sacch-consumption only when the intragastric injection
of 0.5 M Suc was done. These results suggest that mice can discriminate
between subtle difference cues of Suc from Sacch, mediating the association with intragastric sensory feedback in the brain.
It is suggested that the GABAergic system in the ventral pallidum (VP)
plays a role in taste palatability. Taste palatability shift occurs as a function of conditioned taste aversion (CTA). To elucidate the role of VP
GABAergic system on CTA, we examined the effects of microinjections
of a GABAA receptor antagonist, bicuculline, on the retrieval of CTA
memory. We measured consumption of conditioned stimulus (CS) using
one-bottle test (Experiment 1) and observed the ingestive or aversive behavior to CS using taste reactivity test (Experiment 2). Rats received 5
mM saccharin or 0.3 mM quinine hydrochloride as CS, immediately followed by an i.p. injection of 0.15 M lithium chloride (20 ml/kg). After
this conditioning, vehicle or bicuculline (12.5 - 200 ng) was bilaterally
infused into the VP immediately before re-exposure to the CS. In Experiment 1, the bicuculline microinjections significantly increased the intake of the saccharin CS, but not the QHCl CS. In Experiment 2, while
the control rats infused with vehicle showed a variety of aversive responses (e.g. gaping, chin rubbing, head shaking, forelimb flails), the
rats infused bicuculline failed to show these aversive responses. These
results indicate that the blockade of GABAA receptors in the VP attenuates aversion to saccharin CS, and this may be due to elimination of aversive responses. Thus, it is suggested that the GABAergic system in the
VP plays a critical role in the expression of CTA.
Effect of high fat diet to ICR background
clock mutant mouse adipocyte tissue
Kudo, Takashi; Kawashima, Mihoko; Tamagawa, Toru;
Shibata, Shigenobu (Dept. Pharmacol, Sch. Science and
Engineering, Waseda Univ. Tokyo, Japan)
Major components of energy homeostasis are subjected to circadian regulation that synchronizes energy intake and expenditure. Recently, relationship of circadian clock and lipid metabolism is highlighted. The
CLOCK transcription factor is a key component of the molecular circadian clock. Adipocytes play essential metabolic roles not only serving as
energy reserves but also secreting hormones and cytokines that regulate
metabolic activities. Clock mutant mice were fed with high fat diet for
13 weeks, and lipid metabolism was investigated. Both wild type and
Clock mutant mice gained body weight. But, in Clock mutant mice, increases of body weight and of adipocyte tissue were significantly attenuated. In Clock mutant mice, total cholesterol of plasma and liver, and
triglyceride of liver were significantly lowered. Again, we examined
clock controlled gene mRNA in the adipocyte by real-time RT-PCR.
Plasminogen activator inhibitor type 1 (Pai-1) which is related to cardiac
infarction was significantly down-regulated in Clock mutant mice. As a
summary, we showed that Clock mutant mice may have abnormal lipid
Different influence of moderate exercise
stress on acute phase proteins in
dependency of bright/dim light exposure
during the daytime
Kanikowska, Dominika1; Sato, Maki2; Tokura, Hiromi3;
Grzymislawski, Marian1; Wiktorowicz, Krzysztof1
(1Departament of Biology and Enviromental Sciences, Poznan
Medical University, Poznan, Poland; 2Department of Physiology,
Aichi Medical University, Japan; 3Institute of Textiles & Clothing,
The Hong Kong Polytechnic University)
The experiment aimed at knowing whether exercise stress on acute
phase proteins may be influenced differently, depending on bright (5,000
lx) or dim (50 lx) light exposure during a day.Eight healthy women volunteered as the subjects. The subject entered a bioclimatic chamber at
08:00 h on first day.The light intensity measured at eye level from 08:00
to 18:00 hours was either 50 lx in the dim light condition (first day) or
5,000 lx in the bright light condition (second day), 10 lx from 18:00 to
23:00 hours.Subjects exercised moderately for 20 min by a bicycle ergometer with 60 W intensity.Blood samples were drawn 30 minutes later
after the end of exercise on first, second and third day. Concentrations
ofα1-antichymotrypsin (ACT), transferin (Tf), α2-macroglobulin (α2M) and haptoglobulin (Hp) were analyzed by the usage of
immunoelectrophoresis. Interleukin-6 and TNFα concentrations were
analyzed by ELISA kits. There did not exist any significant differences
for acute phase proteins on first day between exercise and no exercise.
ACT was significantly higher (p<0.05) on 3rd day morning than on 2nd
day morning, suggesting that an increase of ACT due to the exercise
stress was amplified by bright light exposure during one diurnal day
before the exercise stress was applied to the subjects.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Neurochemistry
Local administrations of muscimol into Nif
(Nucleus interfacialis nidopallialis) alter
song grammar of the Bengalese finches
(Lonchura striata var. domestica).
Okumura, Tetsu1; Yamashita, Yuichi1; Okanoya,
Kazuo2; Tani, Jun1 (1Lab. Behavior & Dynamic Cognition,
RIKEN-BSI, Wako, Japan; 2Lab. Biolinguistics, RIKEN-BSI, Wako,
Songs of passerine birds (like zebra finch) are learned motor behavior
which used by males to attract females and to protect territories. Generally, bird songs are consist of several different song notes (elements), and
these notes are produced in a fixed temporal order. Among the passerine
birds, male Bengalese finches (BF) sing complex song which follows finite state syntax. The song control system of BF consists of a set of discrete nuclei including the HVC, RA and Nif. Previous lesion study
(Hosino and Okanoya 2000) showed that Nif lesioned BFs sung simpler
songs, with less phrases to phrases branching than that of prelesion birds.
This finding suggests that Nif-HVC connection plays important role in
generating song grammar in BFs. In this study, we perfused Nif with
muscimol (GABA agonist) for 30 minutes via microdialysis probes as a
perturbation on Nif-HVC system. Following a local administration of
muscimol into Nif, song grammar of BF was modified. Some of chunks
in their finite state grammar disappeared and pronounced elongation of
introductorily notes' duration was observed in first 3 hours. In addition,
we also recorded stuttering like repetitions of song notes. Nif is also
known as one of auditory relay nucleus to HVC. Some of drug effects,
therefore, are possibly caused by disruption of auditory feedback. Further detailed studies are needed to know function of Nif-HVC connection in relating to generate song grammar.
Effects of postnatal stress by cinchophen
injection on motor behavior in adult rat
Jingu, Hisaka; Koibuchi, Noriyuki (Dept. Integ. Physiol.
Gunma Univ. Grad. Sch. Med., Gunma, Japan)
To study the ontogenic change in stress susceptibility during postnatal
brain development, we have previously examined the ontogenic changes
of Fos expression in the paraventricular nucleus of the hypothalamus
(PVN) after intraperitoneal (i.p.) injection of cinchophen. We have obtained evidence that Fos was induced by cinchophen in PVN after postnatal day (P)10. Such differential responses to stress have led us
hypothesize that such stressful stimuli at various time point during postnatal development may differentially affect motor behavior in adult rat.
To test this hypothesis, we applied an open field test and a rotarod test
using rats that received i.p. injection of cinchophen on P5, P7, P10 and
P15. There were no significant differences of body weight and muscle
strength between them. In open field test, female rats that received cinchophen injection on P10 tended to stay longer in the center of the field
at P30, suggesting that they showed anxiety to a novel environment. In
rotarod test, there was no significant difference among all groups. These
results indicate that postnatal stress by cinchophen may affect in part
their motor behavior related to anxiety in adult rat.
Na+/HCO3– cotransporter 4 (NBC4)
predominantly expressed in choroid
plexus is involved in cerebrospinal fluid
Fukuda, Hidekazu1; Hirata, Taku1; Nakamura,
Nobuhiro1; Kawahara, Katsumasa2; Wakabayashi,
Shigeo3; Chang, M.H.4; Romero, M.F.4; Hirose,
Shigehisa1 (1Dept. Biol. Sci., Tokyo Inst. Tech., Kanagawa,
Japan; 2Dept. Physiol., Kitasato Univ. Sch. Med., Kanagawa,
Japan; 3Dept. Mol. Physiol., Nat. Cardiovasc. Cent. Res. Inst.,
Osaka, Japan; 4Dept. Physiol. & Biophys., Case Western Reserve
Univ., OH, USA)
Secretion of HCO3– at the apical side of the epithelial cells of the choroid
plexus is an essential step in the formation of cerebrospinal fluid (CSF).
Anion conductance with a high degree of HCO3– permeability has been
observed and suggested to be the major pathway for HCO3– transport
across the apical membrane, but the molecular entity remains unknown.
We identified the first molecular entity of apical choroid plexus HCO3–
transport, a novel variant of NBC4 (NBC4g). Electrophysiological studies and pH-recovery assay showed that NBC4g has the electrogenic,
DIDS-sensitive, and cAMP-dependent HCO3– transport activity. Furthermore, the contribution of NBC4g to choroid plexus HCO3– transport
was demonstrated by RNAi-mediated knockdown of NBC4g using primary cultured cells; treatment with siRNA of NBC4g led to a similar degree of reduction in the transport activity as that observed by treatment
with DIDS. Thus, our data strongly indicate that NBC4g is the longsought transporter responsible for the HCO3– secretion from the choroid
plexus into the ventricle thereby controlling the H+ buffering and pH of
the CSF.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Endocrine glands & hormones
Mediatory roles for forebrain AMPA/kinate
receptors in ADH secretion stimulated by
hyperosmolality or bleeding
Yamaguchi, Ken'ichi1; Yamada, Takaho2 (1Dept.
Homeostatic Regulation and Development, Niigata Univ. Grad. Sch.
Med. Dent. Sci., Niigata, Japan; 2Dept. Cellular Function, Niigata
Univ. Grad. Sch. Med. Dent. Sci., Niigata, Japan)
Endocrine glands & hormones
The anteroventral third ventricular region (AV3V), a pivotal area for autonomic functions, contains synaptic boutons with Glu and three subtypes of glutamate receptors (Glu-Rs). Although our previous data
suggest involvements of NMDA- and metabotropic Rs in ADH release
triggered by several stimuli, roles of AMPA/kinate (non NMDA)-Rs related to Na+ channels in hormone release and other functions have not
been examined as yet. This study aimed to elucidate the issue through
experiments in conscious rats with indwelling cerebral and vascular cannulae. Infusion sites in the brain were identified histologically after experiments. Topical AV3V infusion with a non NMDA-R agonist FWD
augmented plasma ADH (pADH), glucose (Glc) and osmolality (Osm),
and enhanced arterial pressure (AP) in a dose-dependent manner. All the
responses of these variables were blocked by pre-administering NBQX,
a selective non NMDA-R antagonist. When NBQX was applied to the
AV3V structures such as the median preoptic nucleus, rises of pADH in
response to systemic load of hypertonic saline or normo- or hypotensive
bleeding were inhibited remarkably. The increases of AP, Osm or Na+
provoked by the osmotic load and the responses of plasma angiotensin
II, Osm, Glc or AP caused by the bleeding were not affected significantly. These results suggest that AV3V non NMDA-Rs, as well as NMDARs, may contribute to both the hyperosmotic and hypovolemic ADH secretion.
A possible novel toxic index for dioxins/
PCBs -Is dioxin more toxic than OH-PCB?
Miyazaki, Wataru1; Iwasaki, Toshiharu1; Amano, Izuki1;
Rokutanda, Nana2; Nagaoka, Rin2; Takeshita, Akira3;
Aoki, Yasunobu4; Toyama, Chiharu4; Koibuchi,
Noriyuki1 (1Dept. Integr. Physiol., Gunma Univ. Grad. Sch. Med.,
Maebashi, Japan; 2Dept. Thorac. Visc. Org. Surg., Gunma Univ.
Grad. Sch. Med., Maebashi, Japan; 3Toranomon Hosp., Tokyo,
Japan; 4Natl. Inst. Environ. Studies, Tsukuba, Japan)
Polychrolinated biphenyls (PCBs) and dioxins (PCDD, PCDF, coplanerPCB) are the environmental chemicals that may affect the growth and
homeostasis of many organs. WHO determined toxic index of dioxins as
toxicity equivalent factor (TEF). However, we reported previously that
the most toxic dioxin (TCDD) did not affect on thyroid hormone (TH)
receptor (TR)-mediated transcription. On the other hand, a hydroxylated-PCB5005 whose TEF was almost negligible, strongly suppressed the
transcription. In this study, we performed a reporter gene assay with several PCBs and dioxins, and observed opposite tendency from TEF: many
PCBs with little TEF showed a greater effect than dioxins. Furthermore
the magnitude of suppression by PCBs and PCDF in neuroblastoma derived cell line was greater than that in fibroblast derived cell line. To analyze further the effect of these chemicals in TR-TH response element
(TRE) binding, electrophoretic mobility shift assay (EMSA) was performed. PCB congeners that suppressed TR-mediated transcription dissociated TR from TRE, indicating that PCB action is exerted through
this mechanism.These results suggest that TEF of dioxins and PCBs do
not always correctly indicate their toxicity. The suppression of TR-mediated transcription by PCBs and dioxins should be incorporated to construct a novel index of those chemicals.
Cross-talk between ACTH- and PAFinduced cortisol and aldosterone secretion
by perfused guinea pig adrenals
Shimada, Toshio; Hirose, Taeko; Matsumoto, Itsurou;
Aikawa, Tadaomi (Nagasaki Univ.Sch.Med. Nagasaki , Japan)
Platelet-activating factor (PAF) is a highly potent stimulator of the secretion of cortisol, corticosterone, and aldosterone. We previously reported
that PAF acts mainly through PAF receptor accompanied by the activation of protein kinase (PK) C. While, ACTH acts through ACTH receptor accompanied by the activation of PK A. In the present study, we
studied the cross-talk in adrenal steroidogenesis among PAF, ACTH, and
angiotensin II (ANG II). 1) The administration of 1nM PAF or 10pg/ml
ACTH significantly stimulated cortisol secretion. The rate of secretion
peaked 2.5-5 or 10-12.5min after infusion of PAF or ACTH. When concurrently applied 1nM PAF with 10pg/ml ACTH evoked cortisol secretion less than additional and peaked 5-10min. 2) Aldosterone secretion
in response to ANG II significantly stimulated at 1nM and peaked 1520min after the infusion of ANG II. The administration of 10nM PAF did
not induce significant aldosterone secretion. The concurrent administration of 1nM ANG II with 10nM PAF significantly inhibited the secretion
of aldosterone to ANG II. 3) Aldosterone secretion in response to ACTH
significantly increased at 1ng/ml and peaked 15-20min after the infusion
of ACTH. The concurrent administration of 1ng/ml ACTH with 10nM
PAF significantly evoked aldosterone secretion almost additional. The
rate of secretion peaked 0-2.5min after infusion of the mixture. These results implicate that a cross-talk between the PK A system and the PK C
system regulates the cortisol and aldosterone secretion.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Reproductive physiology
Acyl-modification of ghrelin regulates its
appetite-stimulating activity in mice
Nishi, Yoshihiro1; Tanaka, Eiichiro1; Hiejima, Hiroshi2;
kojima, masayasu2; Inokuchi, Hiroe1; Muraii,
Yoshinaka1; Higashi, Hideho1 (1Dep. Physiol., Kurume Univ.
Sch. Med., Kurume, Japan; 2Life Sci. Inst., Kurume Univ., Kurume,
Japan; 3Inst. Animal Exp., Kurume Univ., Kurume, Japan)
Ghrelin is an acylated brain-gut hormone secreted primarily from stomach. The major active form of ghrelin is a 28-amino acid peptide with an
n-octanoyl (C8) modification at Ser3 residue. There also exist other acylated forms of ghrelin, such as n-decanoyl (C10) ghrelin. The previous
study demonstrated that intravenous administration of C8-ghrelin increases appetite and food consumption in rodents. To elucidate effects of
acyl-modified ghrelins on appetite, we have therefore measured concentrations of acylated ghrelins in stomach and plasma of control and fasted
mice. In control, the C8-ghrelin concentration in stomach was higher
than the C10-ghrelin. In 48h-fasted mice, the C8-ghrelin significantly
decreased whereas the C10-ghrelin significantly increased. Consequently, the C10-ghrelin was greater than the C8-ghrelin in plasma of 48hfasted mice. Intraperitoneal (ip) injection and intracerebroventricular
(icv) injection of C8- or C10-ghrelin significantly facilitated the food
consumption 2h after the injection. Ip injection of either C8- or C10-ghrelin had a similar effect. On the other hand, 2h after icv injection of C8ghrelin, the food consumption was greater than that 2h after icv injection
of C10-ghrelin. These results suggest that the condition of energy metabolism influences the acyl-modification of ghrelin, and C10-ghrelin has a
site-dependent activity for the stimulation of appetite in mice.
A study of religious beliefs and practices
on menarche and menstruation among
middle aged women and adolescent girls of
Buddhist Hindu, Islamic and Catholic
Fernando, Delani Marie Sharaine (Department of
Physiology, Faculty of Medical Sciences, University of Sri
Jayewardenepura, Nugegoda, Sri Lanka)
Objective- A qualitative descriptive study was conducted to determine
the religious practices and beliefs related to menarche and menstruation
among middle aged women and adolescent girls in the district of Colombo within Buddhist, Hindu, Islamic and Roman Catholic religions.Method-Information was gathered from the middle aged women and the
adolescent girls by focus group discussion and by a self-administered
questionnaire respectively. Pertinent religious scripture were identified
through key informants of each religion. Results-The religious taboos
observed during menstruation included not participating in religious activities, avoidance of sex and cooking. Restricted religious activities
were observed among middle aged women of Buddhist, Hindu and Islam
religions. Buddhist adolescent girls have given up this practice. Avoidance of sexual intercourse during menstruation was observed in all four
religions. Hindu's observed unique practices such as restriction of water
during menarche and reduced household activities during menstruation.
Hindu and Islam women follow the scriptures strictly whereas the Buddhist practices seem to be influenced by the Hindu culture. Except Roman Catholics, others observe restrictions during menarche although
there are no statements in scriptures of the four religions studied.Conclusion-Unsafe practices are still continuing among women and adolescents
despite statements in religious scriptures.
Reproductive physiology
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Development, growth & aging
The aboral pore of Hydra and oral opening
of higher organisms share common
ancestral origin
Shimizu, Hiroshi; Takaku, Yasuharu; Fujisawa,
Toshitaka (National Institute of Genetics, Mishima, Japan)
Development, growth & aging
Oral opening of multicellular organisms is generally formed at the anterior end of the animal. Phylum Cnidaria is so far the only exception
where oral opening is formed at the posterior end of the animal expressing Wnt-3a homologues (1), a typical gene which is expressed specifically at the posterior end. Why this opposite oral-aboral polarity relative
to A-P polarity appeared only in this phylum remains unknown. Expression of Hox-1 homologues at the oral end has been proposed as evidence
that oral end represents anterior end even in Cnidaria (2). Here we report
that the aboral end of Hydra bears several similarities to the oral end of
higher organisms.
Analysis of migrating neurons in adult
brain using the antibodies specific to
drebrin isoforms
Classification of proteomic trajectories of
retinal proteins in mice during postnatal
Song, Mingqiao1,2; Hanamura, Kenji1; Inoue, Hiroshi1,3;
Mikuni, Masahiko2; Shirao, Tomoaki1 (1Dept of Neurobiol &
Haniu, Hisao1; Anil, Singh2; Takemori, Nobuaki2;
Komori, Naoka2; Koyama, Shozo1; Matsumoto,
Hiroyuki2 (12nd Dept. Physiol. Shinshu Univ. Sch. Med. Nagano,
Behav; 2Dept of Psychiatr & Human Behav, Gunma Univ Grad Sch
of Med, Maebashi, Japan; 3Institute of Neural Organization,
Fujioka, Japan)
Although it is known that drebrin is expressed in the rostral migratory
stream (RMS), a neurogenesis region in adult rat brain, the isoform of
drebrin is unknown. In this study, a drebrin A specific antibody (DAS2)
and another specific antibody (M2F6) recognizing drebrin E and A were
used to identify the drebrin isoform that is specifically expressed in migrating neurons. To investigate the immunocytochemical characteristics
of drebrin-positive cells, we carried out double-labeling with M2F6 and
an antibody of PSA-NCAM, GFAP, or Ki-67. In addition, we performed
double labeling of staining of RMS with DAS2 and M2F6 to identify the
drebrin isoform. We also analyzed adult rat brains whose unilateral olfactory bulb (OB) had been removed. The migrating cells in subventricular zone of RMS were strongly stained with M2F6, but not with DAS2.
This indicates that drebrin E but not A is expressed in these cells. These
packed, bi-polar cells expressed PSA-NCAM but not GFAP. Some of
them had been undergoing proliferation because their nuclei expressed
Ki-67. These findings suggest that M2F6-positive and DAS2-negative
cells are migrating neuronal precursors. Unilateral olfactory bulbectomy
significantly increased the total area of M2F6-positive and DAS2-negative cells in the ipsilateral RMS comparing with the contralateral side,
which is consistent with the previous report about the increase of migrating neuronal precursors by the bulbectomy
Japan; 2Depts. Biochem. and Molec. Biol. Univ. Oklahoma H.S.C.,
Oklahoma, U.S.A.)
Purpose: We attempt to classify the retinal proteins displayed on twodimensional (2-D) gels based on their time-dependent expression patterns, which we designate "proteomic trajectory ", along the postnatal
developmental axis. Methods: Retinas of C57/B6 mice were collected
at postnatal (P) days 1, 3, 5, 7, 9, 14, 21 and at the adult stage. After separating the proteins by 2-D gel electrophoresis, the gel images were analyzed by Progenesis workstation. Protein spots were quantified and
normalized, and the proteomic trajectory along the developmental axis
was obtained. The results were averaged from four independent experiments. The proteomic trajectories were clustered by self-organizing
mapping (SOM) using GeneCluster2. For protein identification each
spot was excised and subjected to in-gel digestion by trypsin, followed
by peptide mass fingerprinting (PMF). PMF search and confirmation
were performed by MASCOT. Results and Conclusions: We identified
ca 400 proteins. These proteins can be clustered by SOM into four major
types, each exhibiting characteristic proteomic trajectory: Juvenile-type,
showing abundant expression in the early postnatal stages and declining
along the maturation process; Transient-type, showing transient expression during the development; Adult-type, showing increased expression
in the later stages of development toward maturation; and ConstitutiveType, being expressed relatively constant during the entire developmental stages.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Nutrition, energy metabolism & body temperature
Role of Hox10 and 11 Paralogues in
Congenital Kyphoscoliotic Rats
Seki, Takayuki1; Shimokawa, Noriaki2; Iizuka, Haku1;
Koibuchi, Noriyuki2; Takagishi, Kenji1 (1Grad. Sch. Med.
Univ. Gunma, Gunma,Japan; 2Grad. Sch. Med. Univ. Gunma,
Gunma, Japan)
The genetic background and key gene for congenital scoliosis has not yet
been clarified. Ishibashi rats (IS) have congenital malformations of the
lumbar vertebrae leading to kyphoscoliosis similar to that seen in human.
This study investigated characteristics and gene expression of IS to provide insights into human congenital scoliosis.To characterize skeletal
malformations of lumbar vertebrae in IS, radiographic and staining studies were performed.Then the gene expression profile of Hox10 and 11
paralogues, whose critical roles in determination of phenotypes of lumbar and sacral vertebrae are well known, between IS and Wistar strain
rats by Real Time-PCR was studied.Significant differences on skeletal
structures between IS and Wistar were found: (1) transitional vertebrae;
(2) anterior wedged vertebra; (3) union of anterior vertebrae; (4) an additional vertebra (7th lumbar vertebra). Especially, transitional vertebra
was frequently observed.Staining studies of IS fetuses revealed the fusion of primary ossification centers in the lumbar vertebral column,
which was not observed in cervical and thoracic vertebral column.Regarding gene expression of Hox10 and 11 paralogues, the expression of
some of these paralogues had low level in lumbar/ sacral region of vertebral column compared with that of Wistar.Our results indicate that
Hox10 and 11 paralogues play critical roles in generating vertebrae of IS
phenotype. Further work is in progress to elucidate the expression profile
of Hox10 and 11 paralogues in the axial skeleton of IS.
Changes in anti-oxidant level in the blood
and the brain corresponding to wideranging fluctuation of energy metabolism
during Syrian hamster hibernation
Hashimoto, Masaaki; Osborne, Peter (Dept.Physiol.
Asahikawa Med.Univ., Asahikawa, Hokkaido, Japan)
Arousal from hibernation with a rapid increase of the energy metabolism
suggests being exposed to a strong oxidation stress whenever the animal
awakes, therefore being considered to have an innate anti-oxidation
mechanism to prevent pathological troubles. To elucidate the state of the
oxidation stress, endogenous anti-oxidants were quantified along the
time course of hibernation. Very slow flow (3.5 µL/h) brain
microdialysis enabled temperature independent sampling of the brain
extracellular fluid (ECF) during hibernation, arousal and cenothermia in
Syrian hamsters (Mesocricetus auratus). Brain tissue and dialysates were
analyzed to provide the first profile of ECF changes in levels of ascorbic
acid (AA), glutathione (GSH) and uric acid (UA) during hibernation and
the transition to cenothermia. Brain tissue content of AA and GSH were
unchanged between hibernation and cenothermia, however arousal was
associated with substantial oxidation of AA from the brain ECF and
plasma compartments. ECF-GSH increased during arousal. Brain tissue
UA content was decreased 50% during hibernation. ECF-UA levels
were unchanged in hibernation and cenothermia, however transiently
increased 100% during arousal. The results suggest that arousal from
hibernation is a suitable experimental model for examination of the
mechanisms by which non-pathological tissue integrity is maintained in
the face of the generation of free radicals during increasing metabolism,
temperature and cerebral reperfusion.
Sex difference in thermoregulation-impact
of estrogen on thermoregulationNagashima, Kei1,2,3; Konishi, Masahiro1; Kobayashi,
Akiko1; Kano, Masumi1 (1Dept. Integ. Physiol. Waseda Univ.,
Tokorozawa, Japan; 2Consol. Res. Inst. Adv. Sci. Med. Care, Tokyo,
Japan; 3Adv. Res. Cent. Human Sci., Tokorozawa, Japan)
Nutrition, energy metabolism &
body temperature
Body temperature (Tb) is different between male and female, e.g. daily
change in Tb is fluctuated with menstruation cycle in female rats. We hypothesized that estrogen plays a crucial role in the sex difference in Tb.
Methods (1) Daily change of Tb was measured after gonadectomy in
male and female rats. After the measurement, silicon tubes containing
17-beta estradiol (E2) crystalline, aimed to maintain blood estrogen constant, were subcutaneously placed in the rats. Then Tb measurement was
repeated. (2) Thermoregulation during 2-h heat exposure at 34°C or cold
exposure at 5°C was assessed in gonadectomized female rats, and the
same protocol was conducted in those with E2 tubes. Results (1)
Compared with male rats, Tb rhythm in female gonadectomized rats
became unstable, showing 2-4 h irregular oscillations. Tb rhythm
remained unchanged in male gonadectomized rats. In female
gonadectomized rats with E2 tubes, Tb rhythm returned to the normal
level. However, there was no influence of E2 on Tb rhythm in the male
rats. (2) Both in the heat and cold, gonadectomized female rats could not
maintain their Tb as those with E2 tubes. Histological analysis for the rat
brain showed that Fos-immunoreactive cells in the hypothalamus were
smaller in the rats without E2 tubes. Conclusion These results show that
estrogen is involved in the thermoregulation in female rats. Estrogen
may modulate thermal sensitivity to the environment at the level of the
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Environmental physiology
Pattern biology for an ideal cellular habitat
created by micromechanical technology
FUKUDA, JUN3; KIKUCHI, MAKOTO1 (1Department of
Medical Engineering, National Defense Medical College,
Tokorozawa, Japan; 2KURARAY Co., LTD., Kurashiki, Japan;
3Department of Physiology I, National Defense Medical College,
Tokorozawa, Japan)
Environmental physiology
[Background] The frame pattern of substance for cell seeding can act
not only on clustering of cells but also on cellular functions. However,
the cellular kinetics depending on the frame pattern of habitat is not fully
elucidated. We have established the technique for molding polymer resin
with submicron accuracy, and have constructed many kinds of microframe patterns of substances. In this research, we explored micro-frame
patterns suitable for neural network construction using PC12 cell line
and evaluated the cellular functions on each micro-frame pattern.
[Method] Micro-frame patterns were fabricated on the polymer resin of
which the optical transparency was sufficient for the microscopic observation. [Results] One micro-domain was populated by 1-3 PC12 cells
and a cellular network was formed by connecting one another through
the open windows of the micro-domain. In addition, alterations in cellular growth and network formation occurred when the micro-domain
structure was changed.[Conclusion] The results suggest that microframe patterns of substances play a critical role in cellular configuration.
Ibhazehiebo, Kingsley (Department of Physiology, University
of Benin, Nigeria)
An Epidermiologic study was carried out in the dry season on 250 commercial motor bike riders from five different parks. 69% of the bike riders were in the 31-40 and 41-50 age range while 31% were in 21-30, 5160 and 61-70 age range. Half of the population studied were normotensive.Arterial hypertension was found in 25%of the examined workers
(p<0.05), borderline hypertension was found in 26%of the workers
(p<0.05). The severity of the hypertension increased with the age of the
workers and the 31-40 age range had the highest incidence of hypertension accounting for 24(38%) of the total 63 frank cases of hypertension.
The severity of the hypertension increased linearly with their duration of
exposure to commercial motor bike riding(r=0.6, P<0.05). heart rate
showed a progressive increase with age but a drop was observed in the
51-60 age range. The characteristics of the hypertension structure and it's
interesting relation to age, number of years of commercial bike riding,
heart rate and body weight is discussed. Of particular interest is the significant number of young adult bike riders found to be hypertensive.
Winter body temperature in the blacklipped pikas, Ochotona curzoniae, in their
natural habitat
Matsumoto, Takaaki1; Sakai, Akio2; Yahata, Takehiro3;
Saito, Muneo4; Matsuzaki, Tetsuya4; Nishimura, Naoki5;
Sato, Maki5; Sugenoya, Junichi5 (1Sch. Health & Sports Sci,
Chukyo Univ., Toyota, Japan; 2Dpt. Sports Med. Sci, Grad Sch Med,
Shinshu Univ., Matsumoto, Japan; 3Dept. Nursing, Nayoro City
College, Nayoro, Japan; 4Central Inst. Exp. Animals, Kawasaki,
Japan; 5Dept. Physiol. Aichi Med. Univ. Sch. Med. Nagakute, Japan)
The pikas, Ochotona, living in cold zone or in high mountains prefer
cold and are weak to heat. They were reported to be diurnal, most active
at dawn and dusk, or active in day and night after the field observations.
We have previously studied pikas' body temperature rhythm in their natural habitat using bio-telemetry devices and showed that the pikas are essentially diurnal and may vary their activity rhythm from diurnal in the
relatively cool environment to a crepuscular (dawn and dusk) pattern in
the relatively hot environment to avoid the heat during midday. In this
study, we monitored body temperature in wild black-lipped pikas,
Ochotona cruzoniae, in their natural habitat in Qinghai, China during
mid-winter season.
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Pathophysiology
Functional roles of the spontaneous
calcium oscillations for the development of
ischemic tolerance in neuron/astrocyte coculture
Tanaka, Motoki; Kawahara, Koichi; Kosugi, Tatsuro;
Yamada, Takeshi (Laboratory of Cellular Cybernetics,
Graduate School of Information Science and Technology, Hokkaido
University, Sapporo, Japan)
Spontaneous oscillations in the intracellular concentration of calcium
(Ca2+ oscillations) contribute to the regulation of gene expression. Here
we investigated whether and how the dynamics of Ca2+ oscillations
changed after sublethal preconditioning (PC) for PC-induced ischemic
tolerance in neuron/astrocyte co-cultures. Ischemia was simulated by depriving co-cultures of both oxygen and glucose (OGD). The frequency
of spontaneous Ca2+ oscillations decreased significantly between 4 and
8 h after the end of PC in both neurons and astrocytes. The reduction in
oscillatory frequency caused by treatment with 2-APB, an inhibitor of
IP3 receptors, resulted in the development of ischemic tolerance, in a
suppression of the rise in the extracellular concentration of glutamate
during OGD, and in a down-regulation of the expression of the
glutamate transporter GLT-1. The expression of GLT-1 is known to be
up-regulated by treatment with PACAP. Treatment with PACAP6-38, an
inhibitor of PACAP receptors, decreased the oscillatory frequency and
GLT-1 protein levels, and induced ischemic tolerance. In contrast, treatment with PACAP38 increased the oscillatory frequency, and antagonized both the PC-induced down-regulation of GLT-1 expression and
ischemic tolerance. These results suggested that the sublethal PC insult
suppressed the spontaneous Ca2+ oscillations regulating various gene expressions for the development of the PC-induced ischemic tolerance.
Pathophysiological roles of ischemiainduced reverse mode operation of
glutamate transporters in astrocytes.
Brain monoamine levels in viral injection
model rat produced by poly I:C: in vivo
brain microdialysis study
kosugi, tatsuro; kawahara, koichi; yamada, takeshi;
tanaka, motoki (Laboratory of Cellular Cybernetics, Graduate
Katafuchi, Toshihiko; Take, Sachiko; Yoshimura,
Megumu (Grad. Sch. Med. Sci. Kyushu Univ., Fukuoka, Japan)
School of Information Science and Technology, Hokkaido
We have recently found that an intraperitoneal (i.p.) injection of a synthetic double-stranded RNA, polyriboinosinic: polyribocytidylic acid
(poly I:C), which mimics viral infection, induces interferon-α (IFN-α)
and serotonin (5-HT) transporter (5-HTT) in the brain. To explore the
functional significance of their expression, we determined extracellular
concentrations of 5-HT and other monoamines such as noradrenaline
(NA) and dopamine (DA) in the medial prefrontal cortex (mPFC) of
freely moving rats using in vivo microdialysis method. Following an i.p.
injection of poly I:C (3 mg/kg), NE levels in the mPFC transiently
increased but returned to the basal level within 6 hrs after the injection.
DA levels were not affected by poly I:C. On the other hand, 5-HT
concentration in the mPFC decreased to 60-70% of the basal level until
8 hrs after poly I:C, while levels of a 5-HT metabolite, 5-hydroxyindole
acetic acid, did not alter. The poly I:C-induced decrease in 5-HT was
significantly attenuated by local perfusion with a selective 5-HT
reuptake inhibitor (fluoxetine) in the mPFC. Microinjection of IFN-α
into the mPFC also decreased 5-HT levels, which was again attenuated
by perfusion with fluoxetine. It is considered that the poly I:C-induced
5-HTT, which is shown to be induced by IFN-α in astrocytes or
endothelial cells, may scavenge extracellular 5-HT into the blood or
cerebrospinal fluid, thereby decreasing 5-HT levels. We have reported
that the decrease in 5-HT in the brain is closely related to the central
mechanisms of fatigue.
During brain ischemia, the excessive influx of Na+ is caused, resulted in
the reversal of neuronal/astrocytic glutamate transporters; that is,
glutamate and Na+ are co-transported to the extracellular space. Previous
studies have revealed that this reversed uptake of glutamate occurs mainly via astrocytic GLT-1 and is the possibility cause of neuronal death.
The present study aims at elucidating whether this reverse mode operation of GLT-1 has any functional meanings for astrocytes themselves.
Analyses of the oxygen/glucose deprivation (OGD)-induced changes in
the concentration of intracellular Na+ and Ca2+ have revealed that OGD
produced Na+ overload, resulting in the reversal of Na+/Ca2+-exchanger
(NCX). The reversed NCX then caused Ca2+ overload leading to the
damage of astrocytes. When the cultures were treated with PACAP-38,
a neuron-delivered peptide, to express GLT-1, the OGD-induced reversed GLT-1 released Na+ out of the cell, and significantly reduced the
rise in intracellular Na+ and Ca2+ during OGD and the astrocytic cell
damage. In contrast, however, OGD resulted in the co-transport of Na+
and glutamate out of astrocytes via reversed GLT-1, and the marked rise
in the extracellular glutamate in neuron/astrocyte co-cultures produced
excitotoxic neuronal death. These results suggested that ischemia-induced reverse mode operation of GLT-1 was toxic to neurons but beneficial to astrocytes by maintaining their Na+ gradient across cell
J. Physiol. Sci., Vol. 56, Suppl., 2006
ORAL: Miscellaneous—modeling & simulation, methodology, history, etc.
Decrease of connexin 43 expression in
ventricular myocytes and prolongation of
QRS duration on electrocardiogram in type
II diabetes mellitus model OLETF rats
Sunagawa, Masanori; Bae, Maeng; Hanashiro,
Kazuhiko; Nakamura, Mariko; Kosugi, Tadayoshi (1st
Dept. Physiol, Unit Physiol, Sch. Med, Univ. the Ryukyus, Okinawa,
Diabetes mellitus (DM) frequently accompanies with contractile dysfunction and arrhythmia. L-type Ca2+ channel current (ICa(L)), transient
outward current (Ito), delayed rectifier outward K+ current (IK(delay)) and
Na/Ca exchanger current (INCX) in ventricular myocytes were compared
between control (LETO) and a type II DM model (OLETF) rats using a
patch-clamp technique at 50 weeks of age to clarify electrophysiologic
changes in diabetic heart. Blood pressure (BP) was measured at caudal
artery by noninvasive tail-cuff method. After rats were anesthetized by
sodium pentobarbital, electrocardiogram (ECG) was recorded by apexbase lead, and then hearts were excised and perfused with collagenase
solution to isolate myocytes. Fibrosis of ventricles was histologically
evaluated using Azan stain and connexin 43 protein (Cx43) expression
was quantitated by western blot. Systolic and diastolic BPs were significantly elevated in OLETF rats. PQ interval and QRS duration were significantly prolonged and the cell sizes of myocytes were enlarged
remarkably in OLETF rats. Current densities of (ICa(L), Ito, IK(delay) and
INCX were not changed in OLETF rats as compared with those in LETO
rats. Although fibrosis was not seen in OLETF rat ventricles, Cx43 expression significantly decreased. We thought that the QRS duration was
prolonged due to the delay in conduction of excitation in OLETF rat ventricles, which might be related with the decrease in Cx43 expression.
Evidence of microglial activation in the
brain in acute stress
Sugama, Shuei (Nippon Medical School, Department of
Physiology, Tokyo, Japan)
Microglial cell has been demonstrated to be involved in various diseases,
such as Alzheimer and Parkinson diseases, HIV encephalitis and multiple sclerosis. In spite of the facts that stress plays crucial roles in the progression of clinical diseases, the involvement of stress on the microglial
activity remains to be elucidated. Based on finding that stress induced
the elevation of proinflammatory cytokines, we hypothesized; (1) physical/emotional stress may have some effect on the microglial activation,
(2) IL-18, a proinflammatory cytokine and demonstrated to be increased
in stress from the adrenal gland, may participate in the microglial activation. We employed restraint combined with water immersion stress for 2
hours as acute stress. Immediately after release from stress, rats were
sacrificed for experiments. Immunohistochemistry with OX-42 revealed
that acute stress provoked morphological microglial activation in the
thalamus, hypothalamus, hippocampus and central grey. Semi-quantitative real time PCR and immunohistochemistry showed that stress significantly induced IL-6 and iNOS from microglia. In addition,
intraperitoneal IL-18 administration (5 µg/rat) caused robust microglial
activation in the brain in a similar fashion observed in stress.
Furthermore, in-vitro studies using microglia cell line (MG6-1)
demonstrated that IL-18 administration (up to 500 ng/ml) significantly
induced iNOS, IL-6, and IL-18 in a dose dependent manner. Thus, the
present study suggests that stress may stimulate microglial cells to
produce several pro-inflammatory cytokines and iNOS at least through
stress-induced circulating IL-18.
Role of Spikar in the maintenance of
dendritic spines
Yamazaki, Hiroyuki; Mizui, Toshiyuki; Takahashi,
Hideto; Shirao, Tomoaki (Dept. of Neurobiol. and Behav.,
Gunma Univ. Grad. Sch. of Med., Maebashi, Japan)
Miscellaneous—modeling &
simulation, methodology, history,
Dendritic spines are multiple functional units that receive most excitatory inputs in central nervous system. Modification of dendritic spine number is associated with several neurological diseases and synaptic
plasticity. Spikar is a novel molecule which was isolated as a drebrinbinding protein using yeast two hybrid screening. In rat primary cultured
hippocampal neurons, GFP-Spikar was localized primarily in nucleus
and dendritic spines, and lesser amounts in soma, dendritic shafts, and
axons. In this study, we investigated the role of Spikar in cultured hippocampal neurons during development. Hippocampal neurons were
transfected with a Spikar-shRNA expression vector or an empty vector
as a control at several developmental stages. The Spikar-shRNA expression vector caused 60-90% knock down (KD) of endogenous Spikar. In
early stage of development, Spikar KD did not affect the density of dendritic protrusions. In contrast, at a stage of synapse formation, Spikar KD
reduced spine density without changing filopodia density. In more mature stage when majority of dendritic protrusions are dendritic spines,
Spikar KD reduced spine density as well. These results suggest that
Spikar plays a role in the maintenance of dendritic spines without affecting the filopodia formation.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Poster Presentations
Cellular & molecular physiology
FK506-induced Ca2+ release from
microsomal vesicles of rat pancreatic
acinar cells is biphasic
Ozawa, Terutaka (Dept. Physiol. Tohoku Univ. Grad. Sch. Med.,
Sendai, Japan)
Cellular & molecular physiology
Enhancement of Ca2+-regulated exocytosis
by indomethacin-induced arachidonic acid
accumulation in guinea pig antral mucous
Fujiwara, Shoko1; Shimamoto, Chikao2; Kato, Masumi1;
Nakanishi, Yoshihiko2; Nakahari, Takashi1 (1Dept. of
Physiol., Osaka Medical College, Takatsuki 569-8686, Japan;
of Internal Medicine (Div II), Osaka Medical College,
Takatuki 569-8686, Japan)
Ca2+-regulated exocytosis is enhanced by the PGE2/cAMP pathway in
antral mucous cells of guinea pigs. The inhibition of the PGE2/cAMP
pathway by a PKA inhibitor (H-89) or aspirin (ASA) decreased the frequency of ACh-stimulated exocytotic events by 60%. Indomethacin
(IDM), however, decreased the ACh-stimulated exocytotic events only
by 30%. Moreover, IDM increased the ACh-stimulated exocytotic
events by 50% in H-89-treated or ASA-treated cells. IDM inhibits the
synthesis of PGG/H and 15R-HPETE, while ASA inhibits only PGG/H
synthesis. Thus, IDM accumulates arachidonic acid (AA). AACOCF3 or
ACA (PLA2 inhibitors), which inhibits AA synthesis, decreased the
ACh-stimulated exocytotic events by 60%. IDM, however, did not increase the frequency in AACOCF3-treated cells. AA increased the frequency of ACh-stimulated exocytotic events in AACOCF3- or ASAtreated cells, similar to IDM in ASA-treated cells. Moreover, in the presence of AA, IDM did not further increase the ACh-stimulated exocytotic
events in ASA-treated cells. The PGE2 release from antral mucosa indicates that inhibition of PLA2 by ACA decreases AA accumulation in unstimulated and ACh-stimulated antral mucosa. The dose-response study
of AA and IDM demonstrated that the concentration of intracellular AA
accumulated by IDM is less than 100 nM. In conclusion, IDM modulates
ACh-stimulated exocytosis via AA accumulation in antral mucous cells.
The effect of the immunosuppressant drug FK506 on microsomal Ca2+
release was investigated in rat pancreatic acinar cells. When FK506 (0.1200 µM) was added to the microsomal vesicles at a steady state of ATPdependent 45Ca2+ uptake, FK506 caused a dose-dependent and a
biphasic release of 45Ca2+. Almost 10% of total 45Ca2+ uptake was
released at concentrations of FK506 up to 10 µM (Km = 0.47 µM), and
60% of total 45Ca2+ uptake was released at concentrations of FK506 over
10 µM (Km = 55 µM). Preincubation of the vesicles with cyclic ADPribose (cADPR: 0.5 µM), which is known to modulate the ryanodine
receptor, increased the FK506 (< 10 µM)-induced 45Ca2+ release (Vmax
value of the release: 8.1% without cADPR vs. 14.4% with cADPR).
Preincubation with 200 µg/ml of heparin, an inhibitor of inositol 1,4,5trisphosphate (IP3) receptor, resulted in significant inhibition of the
FK506 (30 µM)-induced 45Ca2+ release. Subsequent addition of IP3 (5
µM) after FK506 (100 µM)-induced 45Ca2+ release did not cause any
release of 45Ca2+. These results indicate that there are two different types
of FK506-induced Ca2+ release mechanisms in the endoplasmic
reticulum of rat pancreatic acinar cells: a high-affinity mechanism of
Ca2+ release, which is activation of the ryanodine receptor, and a lowaffinity mechanism of Ca2+ release, which is activation of the IP3
Properties of store-operated Ca2+ entry in
rat chromaffin cells
Warashina, Akira1; Inoue, Masumi2 (1Div. Cell Physiol.,
Niigata Univ. Grad. Sch. Med. & Dent. Sci., Niigata, Japan; 2Dept.
Cell System. Physiol., Sch. Med., Univ. Occupational &
Environmental Health)
The application of thapsigargin (TG) or cyclopiazonic acid (CPA) to
Fura-2-loaded chromaffin cells in the perfused rat adrenal medulla abolished a transient [Ca2+]c rise induced by muscarine in Ca2+-free medium
due to the depletion of Ca2+ stores. Either TG or CPA induced a sustained
increase of [Ca2+]c in Ca2+-containing medium, indicating that store-operated Ca2+ entry (SOCE) mechanism exists in this cell type. The TGinduced [Ca2+]c increase was inhibited completely with 2 mM Ni2+ but
only by 18% with 100 µM D600, whereas maintained [Ca2+]c increases
during prolonged stimulations with muscarine (100 µM) and high-K+
(40 mM) were inhibited by 100% and 51% with Ni2+, by 53% and 80%
with D600, respectively. In cells to which muscarine and Ni2+ had been
co-applied, Ca2+ stores remained depleted to induce a sustained SOCE
after the two agents were washed out. In isolated chromaffin cells, CPA
induced a much smaller extent of elevation in [Ca2+]c, compared with
that induced in cells being in the adrenal medulla, which possibly
suggests that the mechanism involved in SOCE may be fragile and was
impaired in dissociation. TG or CPA applied alone to the adrenal
medulla did not elicit a detectable amount of catecholamine secretion
despite the elevation of [Ca2+]c, nor promoted secretory responses to a
significant extent when applied during stimulation with high-K+. These
results suggest that SOCE in rat chromaffin cells may not produce a
sufficient increase in [Ca2+]c near the secretory vesicles to trigger
J. Physiol. Sci., Vol. 56, Suppl., 2006
Cellular & molecular physiology
Expression and characterization of
Calcium-sensitive myosin II
zhang, ying1; Feng, Xiaohua1; Kawamichi, Hozumi1;
Nakamura, Akio1; Yoshiyama, Shinji1; Farkas, Laszlo2;
Nyitray, Laszlo2; Kohama, Kazuhiro1 (1Dept. Mol. Cell.
Pharmacol., Gunma Univ. Grad. Sch. Med., Maebashi, Japan;
2Dept. Biochem., Eotvos Univ., Budapest, Hungary)
Purpose: Myosin II is one of the typical motor proteins and is classified
as non-regulated, phosphorylatable and Ca-binding myosins. Physarum
myosin II belongs to Ca-binding one. Myosin II regulated by Ca-binding
has not yet expressed as a recombinant protein. Here, we report the expression of heavy mero-myosin of physarum myosin II together with
preliminary characterizations. Method: We used baculovirus expression
system. Sf9 cells were infected with the virus constructs. Result: When
baculovirus of heavy chain(HC) fragments was infected together with
those of phosphorylated light chain (PLC) and Ca-binding light chain
(CaLC), Sf9 cells produced soluble HMM, which were recovered in the
supernatant together with PLC and CaLC. The HMM showed Mg-ATPase activity of 0.21 (s–1head–1), and actin-activated ATPase activity
with Vmax=1.27 (s–1head–1), and Km=1.8µM. The movement of actin
filaments on the HMM-coated glass surface was sensitive to Ca2+. We
will show the effect of Ca2+ on the movement of HMM associated with
various kinds of light chains.
CaMKI-induced Phosphorylation
Regulates Drp-1 Dynamics and
Mitochondrial Morphology in Hippocampal
han, xiaojian; Matsushita, masayuki; Lu, Yunfei;
Tomizawa, kazuhito; Matsui, Hideki
(Dep.Phsiol.Grad.Sch.Med.& Dent., Univ.Okayama,
Mitochondrial morphology is regulated by balance of fission and fusion
events. Certain dynamin family members such as dynamin-related protein 1 (Drp-1) are involved in the regulation of mitochondrial fission.
Drp-1 specifically controls mitochondrial outer membrane fission. However, very little is known about the mechanism that initiates mitochondrial fission by Drp-1. In the present study, we detected Drp-1 was
phosphorylated by CaMKI in vitro. In primary cultured hippocampal
neurons, high K+ stimulation induced phosphorylated Drp-1 increment
and Drp-1 transition from cytoplasm to mitochondria and mitochondrial
fragmentation. The effect of high K+ was inhibited by KN93 (CaMK inhibitor). In vitro experiment, we found phosporylation promoted the
Drp-1 complexes formation. Although overexpression of GFP-hFis1-C
did not alter mitochondrial morphology, it inhibited high K+ induced mitochondrial fission in neurons. These results suggest that Drp-1 dynamics and mitochondrial morphology may be regulated by CaMKI-induced
Drp-1 phosphorylation.
Analysis of IP3 dynamics during the
intracellular Ca2+ oscillations in
mammalian eggs
Shirakawa, Hideki1,3; Sato, Moritoshi2; Umezawa,
Yoshio2; Miyazaki, Shunichi3 (1Dept. Appl. Phys. Chem., Univ.
Electro-Comm., Tokyo, Japan; 2Grad. Sch. Chem., Univ. Tokyo,
Tokyo, Japan; 3Dept. Physiol., Tokyo Women's Med. Univ. Sch. Med.,
Tokyo, Japan)
At fertilization of mammalian eggs, the repetitive Ca2+ releases from intracellular stores through inositol 1,4,5-trisphosphate (IP3) receptors are
thought to be induced by the soluble factor originated from the sperm.
Although phospholipase C zeta (PLCζ) is the possible candidate of such
'sperm factor', there are no evidence to show the elevation of intracellular
IP3 concentration at fertilization. We measured the changes in IP3
concentration during Ca2+ oscillations in mouse eggs, using a novel
FRET-based IP3 probe, fretino. Eggs expressing fretino showed a
transient decrease in FRET signal in response to the microinjection of
IP3, but not to inositol 1,3,4,5-tetrakisphosphate (IP4). The signal
decayed exponentially with a time constant of 100-180 s. When Ca2+
oscillations were induced in the eggs by insemination, the signal of
fretino showed no detectable changes to indicate the increase in IP3
concentration. On the other hand, eggs expressing a large amount of
PLCζ showed significant decrease in the FRET signal from fretino.
Furthermore, the FRET signal oscillated with Ca2+ in such eggs,
suggesting the enhancement of PLCζ activity by cytoplasmic Ca2+. The
magnitudes of Ca2+-induced IP3 production in the fertilized eggs and in
the eggs expressing PLCζ or PLCδ1, were also compared.
Na+ entry via store-operated Ca2+ channels
in mouse submandibular acinar cells.
Fukushi, Yasue; Ohsaga, Atsushi; Maruyama, Yoshio
(Dept. Physiol. Facult. Med. Univ. Tohoku, Sendai, Japan)
We reported previously that two store-operated Ca2+ channels (SOCs)
could be separated by using Zn2+ in the submadibular acinar cells. In
short, after depleting the Ca2+ stores with thapsigargin, SOC signals during readmission of external Ca2+ were detected. The signal showed two
phases; the initial large transient and subsequent sustained phase. External Zn2+ inhibited the former but not the latter. External Ni2+ or excess
of outside K+ markedly reduced both. Based on this observation, we
studied Na+ entry through SOC. Loading benzofrane isophthalate
(SBFI)-AM for 2 h at 37oC to the cells, internal [Na+]i was monitored
with digital imaging methods. Prior elimination of external Na+ (replaced with an impermeable cation, NMDG+) induced a substantial increase in Na+ entry by Na+ readmission, and it was strengthened by a
simultaneous elimination of external Ca2+. When Ca2+ stores were actively depleted with thapsigargin under Ca2+- and Na+-free condition, the
largest Na+ signals were counted by the Na+ readmission. In contrast to
the pattern of Ca2+ entry, that of Na+ was monophasic and inhibited by
external Zn2+, Ni2+ and Ca2+ as well. The finding that external Ca2+ reduced Na+ signals suggests that Ca2+ store depletion induces Na+ entry
through SOCs in a competitive manner with Ca2+. Collectively, after depletion of Ca2+ stores, Na+ may enter into the cells through the divalent
cation-sensitive Ca2+-entry pathway in mouse submandibular acinar
J. Physiol. Sci., Vol. 56, Suppl., 2006
Cellular & molecular physiology
Q268X binds with wild-type HNF4α or its
repressor SHP and accumulates in the
nucleolus in cultured cells
Enhancement of the priming step of
exocytotic events caused by Cl–-free
Ogata, Makiko1; Awaji, Takeo2; Iwasaki, Naoko1;
Iwamoto, Yasuhiko1; Miyazaki, Shunichi2 (1Diabetes
Kato, Masumi1; Fujiwara, Shouko1; Shimamoto,
Chikao2; Nakahari, Takashi1 (1Dept. of Physiol., Osaka
Center, Tokyo Women's Medical University School of Medicine,
Tokyo, Japan; 2Department of Physiology, Tokyo Women's Medical
University School of Medicine)
Medecal College, Takatsuki, Japan; 2Dept. of Internal Medicine (Di
II), Osaka Medical College, Takatsuki, Japan)
Although the HNF4 α protein is known to function as a dimer and
Q268X heterozygotes carrying mutations in its allele cause MODY1, it
is still unclear whether Q268X and wild type HNF4 α can dimerize, and
what causes such a distinct phenotype. We visualized the practical and
mutual interactions of HNF4 α and Q268X HNF4 α using Fluorescence
Resonance Energy Transfer (FRET). A transition in cellular localization
was seen in Q268X-HNF4 α complexes from the nucleoplasm to the
nucleolus, where wild type HNF4α is normally localized in COS7 and
CHO cells. Furthermore, FRET microscopy showed that Q268X-HNF4
α bound to wild type HNF4α and accumulated in the nucleolus. SHP,
which is a repressor of HNF4 α, also bound to Q268X and translocated
to the nucleolus. The cellular localization of a deletion mutant of HNF4
α showed that the site contributing to nucleolar accumulation is P333 to
I338. Some proteins displayed an altered cellular function after
localization to the nucleolus. According to these results, transfer to the
nucleolus of the heterodimer Q268X-HNF4 α must affect the function
of HNF4 α. Consequently, this study showed that Q268X-HNF4 α
dimerizes with wild type HNF4 α and also binds with the repressor and
changes its localization to the nucleolus. These effects, together with
transcription function, may lead to the distinct phenotype of MODY.
The final steps of exocytosis are consisted of three steps, docking, priming, and fusion, in which the priming step is maintained by ATP. We examined the effects of Cl–-free solution on the priming step.The isolated
antral mucous cells were obtained by a collagenase treatment, and observed using video-microscopy. In Cl–-free solution, Cl– was replaced
with NO3–.Acetylcholine (ACh 1 µM) increases the frequency of
exocytosis ; an initial phase followed by a sustained phase. The Cl–-free
solution enhanced the initial peak frequency of ACh-evoked exocytosis
approximately 4 fold respectively.To examine effects of Cl–-free
solution on the priming step, intracellular ATP was depleted by anoxia
(aerated with N2 100%) or dinitrophenol (DNP 100 µM). Depletion of
ATP eliminated the initial phase of ACh-evoked exocytotic events. After
ATP depletion, Cl–-free solution did not evoke any initial
phase.However, when cells were first perfused with Cl–-free solution,
and then ATP was depleted, ACh induced an initial phase. Moreover,
cells were first stimulated with ACh, which depelets the primed
granules, and after a short interval (9min), cells were stimulated with
ACh again. The initial phase was induced by the second ACh stimulation
during perfusion with Cl–-free solution, while it was not during
perfusion with the control solution.Based on the observation, Cl–-free
solution increases number of the primed granules, which enhances AChevoked exocytotic events in antral mucous cells.
Effects of a time-varying magnetic field on
intracellular organelles of bovine adrenal
chromaffin cells in culture.
Change in expression and distribution of
tight junction-related proteins in primary
cultured parotid acinar cells
Ikehara, Toshitaka1; Sasaki, Hiromi2; Minami, Yuki2;
Hosokawa, Keiko1; yamaguchi, Hisao3; Kitamura,
Mitsuo1; Shono, Masayuki1; Kawazoe, Kazuyoshi2;
Yoshizaki, Kazuo1; Kinouchi, Yohsuke4; Miyamoto,
Hiroshi1 (1Univ.Tokushima Grad. Sch., Tokushima, Japan;
Qi, Bing; Yoshigaki, Junko; Michikawa, Hiromi;
Katsumata, Osamu; Dohke, Yoko; Furuyama,
Shunsuke; Sugiya, Hiroshi (Dept. Physiol., Nihon Univ. Sch.
Univ. Hospital, Tokushima, Japan; 3Tokushima Bunri
Univ., Tokushima, Japan; 4Univ. Tokushima, Tokushima, Japan)
We tested the effects of exposure to a switched 1.5 Tesla magnetic field
on transient increase in intracellular Ca2+ stimulated by neurotransmitters in bovine adrenal chromaffin cells. [Ca2+]i was increased transiently
by addition of acetylcholine (ACh) in Ca2+-free medium and the AChinduced increase was inhibited significantly by 2 hr-exposure to the
magnetic field. The exposure caused not only to decrease the peak value
but also to slow the decay phase of [Ca2+]i after peak. Delay of the decay
phase was also caused by addition of KCN in the presence of ACh. The
intracellular ATP content and oxygen consumption were influenced by
the exposure in glucose-free medium. Measurement of mitochondrial
membrane potential by using fluorescent probe, JC-1 (Molecular Probe),
showed depolarization of the membrane in both cells exposed to antimycin and the magnetic field. The cellular content of F-actin stained with
fluorescent probe (Alexa fluor 488 phalloidin) was also decreased by the
exposure. These effects of magnetic field would be related to the eddy
Dent. at Matsudo,Japan)
Tight junction is the essential structure for salivary epithelial cells to
keep polarity and to secrete fluid unidirectionally. Previously, we established a system for primary culture of parotid acinar cells. Acinar cells
isolated from the rat parotid glands formed large colonies and attached
to the basement of dishes at 24 h after the dispersion. After 2 days, most
cells spread as a monolayer whereas a part of cells formed hemispherical
lumps. Analysis with electron microscopy suggests that cells in the
lumps retained original tight junctions and lumens. On the other hand,
cells in monolayer also formed tight junctions. Immunofluorescence microscopy showed claudin-1 was observed in the lumps, while claudin-4
was detected at the tight junctions in monolayer. Most cells in the monolayer that had claudin-4-positive tight junctions retained secretory granules containing amylase. Therefore, the claudin-4-positive cells were
probably derived from acinar cells, but not from ductal cells. Immunoblotting analysis showed that claudin-4 was expressed at 24 h and its expression increased time-dependently during the culture, although it was
not detected just after the dispersion. These results suggest that the expression of claudins changed from isotype 1 to 4 while the morphology
of the acinar cells changed to monolayer. Claudins possibly have a correlation with the formation and maintenance of culture configuration in
parotid acinar cells.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Cellular & molecular physiology
Effects of ELF magnetic fields on
differentiation of cultured osteoblast-like
Cyclic GMP modulates ACh-stimulated
exocytosis in guinea pig antral mucous
Yamaguchi, Hisao1; Hosokawa, keiko2; Ikehara,
Toshitaka2; Shichijo, Hiroe3; Kitamura, Mitsuo2;
Yoshizaki, Kazuo2; Kinouchi, Yohsuke3; Miyamoto,
Hiroshi2; Aizawa, Katuo4 (1Dept. Envion. Physiol., Fac. Human
Saad, Adel H1; Nakahari, Takasi2; Fujiwara, Shoko2;
Shimamoto, Chikao3; Marunaka, Yoshinori1 (1Mol. Cell
Life Sci., Tokushima Bunri Univ., Tokushima, Japan; 2Dept. Physiol.,
Inst, Health Biosci., Univ. Tokushima, Tokushima, Japan; 3Dept.
Electric & Electronic Engr., Fac. Engr. Univ. Tokushima, Tokushima,
Japan; 4Fac. Sci. & Engr., Waseda Univ. Tokyo, Japan)
In this study, the effects of extremely low frequency (ELF) region on the
osteoblast-like cells (MC3T3-E1) for the differentiation was examined.
Sinusoidal (60Hz) magnetic fields were about 3 mT. Collagen protein
contents of cultures were measured microscopically by using ImSpector
system. Using insulin-like growth factor I (IGF-I), the difference between the effect by exposure was examined with these during effects for
the collagen content of these cells. From these results, the effects of exposure and IGF-I treatment caused significant increase on collagen synthesis of osteoblasts. It is supposed that the effects of magnetic fields go
through the intercellular signaling pathway. Therefore, we experiment
by the use of some inhibitors which block the intercellular signal transduction and examine which route the exposure passes in order to influence on differentiation of osteoblasts. As a results, it is suggested that
ELF magnetic fields stimulate collagen synthesis because of activation
of p38 MAPK and induce the cell differentiation. These results indicate
that the mechanisms of differentiation related to IGF in the osteoblasts
were altered by the magnetic fields of extremely low frequency.
Physiol., Grad. Sch. Med. Sci., Kyoto Pref. Univ. Med., Kyoto,
Japan; 2Physiol., Osaka Medical College, Takatsuki, Japan;
3Internal Medicine, Osaka Medical College, Takatsuki, Japan)
In guinea pig antral mucous cells, acetylcholine (ACh) induces a biphasic increase in the Ca2+-regulated exocytosis: an initial transient phase
followed by a sustained one. We studied the effects of cGMP on AChstimulated exocytosis in guinea pig antral mucous cells using video microscopy. Cyclic GMP enhanced the frequency of ACh-stimulated exocytotic events, while cGMP alone induced no exocytotic events under
the ACh-unstimulated condition. Cyclic GMP did not affect either Ca2+
mobilization or cAMP accumulation. cGMP shifted the Ca2+ dose-response curve upward with no shift to the lower-concentration, indicating
that cGMP increases responsibility of the Ca2+-regulated exocytosis, but
not the Ca2+ sensitivity. When cGMP was added after ATP depletion by
dinitrophenol (DNP) or anoxia (N2 bubbling), ACh evoked only a sustained phase in the exocytosis without any initial transient phase. In contrast, when cells were pretreated with cGMP before ATP depletion, ACh
evoked the biphasic exocytotic events. These observations indicate that
cGMP modulates ATP dependent priming of Ca2+-regulated exocytotic
events. In conclusion, cGMP increases the number of primed granules
via acceleration of the ATP-dependent priming step, which enhances the
Ca2+-regulated exocytotic events stimulated by ACh.
Rapid recruitment of Na,K-ATPase to the
cell surface
Murata, Miyahiko1; Imura, Akihiro2 (1Integrative Brain Sci.,
Vesicle disruption and plasma membrane
bleb formation caused by illumination with
blue light in acridine orange-loaded cells
Grad. Sch. Med. Kyoto Univ., Kyoto, Japan; 2HMRO, Grad. Sch.
Med. Kyoto Univ., Kyoto, Japan)
Hiruma, Hiromi; Katakura, Takashi; Simada, Jun'ici;
Kawakami, Tadashi (Dept. of Physiol. Kitasato Univ. Sch. of
Na,K-ATPase is a membrane-bound protein that maintains intracellular
ionic concentrations, i.e., low Na+ and high K+, using the energy from
hydrolysis of ATP. We have now observed a rapid recruitment of Na,KATPase to the cell surface in responce to extracellular low calcium concentration in the parathyroid cell that releases PTH in responce to low
calcium. Using isotope-labelled ouabain that is a specific ligand for
Na,K-ATPase, we observed that the recruitment occurs in a few minutes.
Biotinylation of the cell-surface proteins revealed that a considerable
amount of Na,K-ATPase is present in intracellular region and bound to a
specific protein. In analysis of the protein-deficient mice, the molecular
association is required for a novel mechanism of rapid recruitment of
Na,K-ATPase. This rapid recruitment is essential for PTH release that is
the first step of calcium regulation of the whole body. This indicates the
importance of NA,K-ATPase in the calcium homeostasis.
Med., Sagamihara, Japan)
Acridine orange (AO), a weakly basic fluorescent dye, is permeable to
plasma and vesicle membranes and preferentially remains in intracellular acidic regions. Using fluorescence microscopy, we observed dynamic
changes in AO-loaded cultured mouse cells during illumination with
blue light. Immediately after the start of illumination, the successive disruption of vesicle membrane was observed as a flash of fluorescence,
and shortly after that, blebs were formed on the plasma membrane regardless of the occurrence of vesicle disruption. Vesicle disruption was
almost completely inhibited when cells were treated with the vacuolar
H+-ATPase inhibitor bafilomycin A1 followed by staining with AO, but
not when bafilomycin A1 was treated after AO staining. Thus, the filling
of AO in the vesicle, which is driven by vacuolar H+-ATPase, is initially
required for vesicle disruption. In contrast, bafilomycin A1 did not prevent plasma membrane blebbing, indicating that the blebs are formed independently of the vesicle disruption. Both the vesicle disruption and the
formation of plasma membrane blebs were partially inhibited by removal of oxygen from the cell environment and by singlet oxygen scavengers, sodium azide, ascorbic acid, and L-histidine, but not by the
hydroxyl radical scavenger dimethyl thiourea. Thus, both phenomena
are likely caused at least in part by the generation of singlet oxygen.
These photosensitive features of plasma and vesicle membranes may be
based on the use of the photodynamic effect, such as cancer therapy.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Cellular & molecular physiology
Live cell tracking of GLUT4 molecule in
3T3L1 adipocyte using Qdot nano-crystals
Fujita, Hideaki; Watanabe, Tomonobu; Nedachi, Taku;
Higuchi, Hideo; Kanzaki, Makoto (TUBERO, Tohoku Univ.,
Tokyo, Japan)
Electrophysiological analysis of
electrogenic Na+-HCO3– cotransporter
activity in bovine parotid acinar cells
Yamaguchi, Souichirou; Ishikawa, Toru (Dept. Biomed. Sci.
Grad. Sch. Vet. Med. Hokkaido. Univ., Sapporo, Japan)
Insulin stimulates glucose transport into adipocyte and muscle cells by
inducing the translocation of the insulin responsive glucose transporter
GLUT4 from intracellular storage compartments to the plasma membrane. GLUT4 translocation is a complicated process involving budding
and fission at the storage compartment, trafficking to the plasma membrane, and fusion at the plasma membrane. Here, we have established a
new method to visualize the movement of single GLUT4 molecule in
living cells. 3T3L1 adipocyte expressing exofacial-myc-GLUT4-eCFP
was labeled with Qdot-conjugated Myc antibody in the presence of insulin. Qdot-GLUT4 complex was then endocytosed by washing out of insulin. Observation was performed under video-rate confocal microscope
equipped with high sensitivity EMCCD camera so that movement of
GLUT4 molecule can be tracked for ~10sec. Movement of GLUT4 molecules was obtained before or after the 2nd insulin stimulation for 5, 15,
and 30 min. Analysis of the diffusion of GLUT4 molecules showed that
movements can be classified in either 1) free diffusion, 2) confined diffusion, or 3) transport and all these movement existed both at basal state
and after insulin stimulation. Overall movement tended to be in confined
diffusion at the basal state, but fraction of transported GLUT4 increased
by insulin stimulation. Diffusion coefficient of GLUT4 was higher after
insulin stimulation than basal state. Together, insulin increases the mobility of GLUT4 and enhances its translocation to the plasma membrane.
Electrogenic Na+-HCO3– cotransporter (NBCe) plays an important role
in mediating HCO3– efflux and influx across the basolateral membrane
in various HCO3–-transporting epithelia, including kidney proximal tubules and pancreatic ducts. Its activity has been generally assessed by
monitoring intracellular pH, Na+ concentration, or membrane potential,
but electrophysiological properties of the cotransporter at the native state
still remain largely unknown. Using the whole-cell patch clamp technique, we have recently, for the first time, identified and characterized
membrane currents attributable to the activity of a NBCe expressed in
acutely dissociated acinar cells (BPA cells) from bovine parotid that secretes large volumes of a HCO3–-rich fluid. Under voltage-clamp conditions, the currents were dependent upon extracellular Na+ and HCO3–
and DIDS-sensitive. Further analysis of the currents indicated that the
stoichiometry of the native cotransporter is most likely to be 2 HCO3– :
1 Na+. We could also demonstrate that BPA cells express transcripts of
NBCe1-B (bNBCe1-B) and that recombinant bNBCe1-B currents in
HEK293 cells shares common electrophysiological and pharmacological properties with those of the native currents. This study represents an
initial attempt to provide electrophysiological characterization of a
NBCe expressed in a native HCO3–-secreting exocrine gland.
Selective collection of catecholaminergic
(CA) neurons in the brain and its
application to functional analyses using
tyrosine hydoroxylase (TH) - green
fluorescent protein (GFP) transgenic mice
Ishii, Yoshiyuki1; Kobayashi, Kazuto3; Itoi, Keiichi1,2
(1Grad. Sch. Info. Sci. Tohoku Univ., Sendai, Japan; 2Grad. Sch.
Med. Tohoku Univ., Sendai, Japan; 3Fukushima Med. Univ.,
Fukushima, Japan)
CA neurons are involved in a wide spectrum of physiological functions
in the brain including sensory, motor, emotional, autonomic and endocrine regulation. Most CA neurons are localized in the brainstem and hypothalamic regions and typically make clusters of cells, among which
the noradrenergic (A1, A2, A6) and dopaminergic (A9, A10, A12) neurons predominate. In order to explore functional roles of these neurons,
we sought to collect CA neurons selectively using TH-GFP transgenic
mice in which GFP expression was driven under TH-promoter. Fetal
(E14.5, E16.5, or E18.5) brain was extracted, and neurons were dispersed after treating with trypsin, then GFP-positive cells were sorted
out by flow-cytometry (FACS). RNA was extracted from the GFP-positive (TH) neurons, reverse-transcribed, and analyzed by PCR.
Concentration-sensitive Na+ channel (NaC)
is involved in the regulation of proliferation
in rat C6 glioma cells
Hagiwara, Teruki; Yamaguchi, Hiroyuki; Tanaka,
Hokuto; Takeuchi, Takashi; Morimoto, Yoshiyuki;
Yoshida, Shigeru (Dept. Life Sci., Sch. Sci. & Engineer., Kinki
Univ., Higashi-Osaka, Japan)
The concentration-sensitive Na+ channel (NaC; c:concentration) works
as a Na+ sensor and it opens when [Na+]o changes. The present study was
carried out to clarify the function of the NaC as one of the regulating factors in cell growth, using rat C6 glioma cells, since they have NaC in
quantity. The image analysis of Na+ dynamics, using a Na+ indicator
(SBFI) and ARGUS-50 (Hamamatsu Photonics), revealed an elevation
of [Na+]i when [Na+]o was raised from normal (140 mM) to 190 mM.
This increase was augmented when Na+ efflux was suppressed by inhibitors of the Na+ pump (ouabain) or of the Na+/K+/Cl– cotransporter (bumetanide). Osmolarity alteration was not important because addition of
mannitol to the external solution did not introduce any changes in [Na+]i.
The expression of immediate early gene egr-1, measured by the realtime PCR method, was reduced when [Na+]o was raised or when [Na+]i
was elevated by a Na+ ionophore, monensin (Cell Biol Int 29:261-268,
2005). These procedures suppressed the rate of cell proliferation. When
the expression of NaC was selectively inhibited by RNA interference
(RNAi) techniques, both [Na+]i and the growth rate of C6 cells were less
affected by [Na+]o changes, indicating that NaC was involved in cell
growth (by controlling gene expression through introduction of Na+ into
the cell). It is concluded that Na+ ions enter C6 glioma cells mainly
through NaC, and Na+ ions regulate cell growth by controlling expression of proliferation-related genes.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Cellular & molecular physiology
Voltage- and pH-dependence of proton flux
through the plasmalemmal vacuolar-type
H+-ATPase in osteoclasts
Volume-sensitive chloride channel
involved in necrotic neuronal death by
Sakai, Hiromu1; Kawawaki, Junko2; Moriura, Yoshie1;
Mori, Hiroyuki1; Morihata, Hirokazu1; Kuno, Miyuki1
Inst. Physiol. Sci., Okazaki, Japan)
(1Dept. Physiol., Osaka City Univ. Grad. Sch. Med., Osaka, Japan;
2Central Lab., Osaka City Univ. Grad. Sch. Med., Osaka, Japan)
The vacuolar-type H+-ATPase (V-ATPase) is an electrogenic H+ pump
that is distributed in living organisms. By carrying out uphill H+ transport, V-ATPases acidify lysozomes and energize intracellular membranes. In osteoclasts, the V-ATPases are enriched in the plasma
membrane faced to the bone surface (ruffled membrane) and serve as a
major acid-secretion pathway required for bone resorption. In this study,
we attempted to identify the pump currents of osteoclasts electrophysiologically and investigated their dependence on the membrane potential
and/or H+ gradient, which may change widely under different functional
states. Outward H+ currents were increased by intracellular dialysis with
ATP up to 10 mM in dose-dependent manner. The V-ATPase current was
evaluated by blockers for the V-ATPase, bafilomycin A1 and N,N'-dicyclohexylcarbodiimide, in the presence of 5 mM ATP. The V-ATPase currents were decreased by hyperpolarization, but were still outward at -80
mV under pHo/pHi of 7.3/5.5. The current amplitude was decreased by
either intracellular alkalization or extracellular acidification, but did not
show current reversal. Significant outward H+ currents were seen at 0
mV even under pHo/pHi of 5.5/7.3. The data showed that the V-ATPasemedaited currents depends on both voltage- and pH gradients across the
plasma membrane. The pump, however, could secrete H+ upon exposure
to strong acids as far as energy is supplied sufficiently.
Inoue, Hana; Okada, Yasunobu (Dept. Cell Physiol., Natl.
Excitotoxicity is associated with stroke, brain trauma and neurodegenerative disorders. Focal swellings along dendrites called varicosities are a
hallmark of acute excitotoxic neuronal injury. We previously reported
that cultured mouse cortical neurons express the volume-sensitive outwardly rectifying (VSOR) chloride channel, which is involved in volume regulation after osmotic swelling. Here we studied a role of the
VSOR chloride channel in excitotoxic neuronal injury in cultured mouse
cortical neurons. The blockade of the VSOR chloride channel activity by
NPPB (40 µM), phloretin (100 µM) or IAA-94 (1 mM) during
excitotoxic stimulation inhibited varicosity formation and necrotic
neuronal death. On the other hand, a GABAA receptor/chloride channel
blocker, bicuculline (10 µM) or picrotoxin (100 µM), failed to inhibit
neuronal necrosis induced by excitotoxicity. On-cell patch-clamp
studies revealed robust VSOR chloride channel activity on varicosities
during exposure to NMDA. These results suggest that the VSOR
chloride channel is involved in aggravation of excitotoxicity by serving
as the pathway for chloride influx, which induces varicosity formation
and cell swelling leading to necrotic cell death.
Effects of K+ and Cl– on Na+-dependent
Mg2+ efflux from rat ventricular myocytes
Inhibition of a glial K channel by various
tricyclic antidepressants
Tashiro, Michiko; Tursun, Pulat; Konishi, Masato (Dept.
Su, Suwen; Inanobe, atsushi; Lossin, Christoph; Hibino,
Hiroshi; Kurachi, Yoshihisa (Grad.Sch.Med. Univ. Osaka,
physiol., Tokyo Medical Univ., Tokyo, Japan)
We measured intracellular free Mg2+ concentration ([Mg2+]i) with fluorescent Mg 2+ indicator furaptra (mag-fura-2) in Ca2+-free condition (0.1
mM EGTA) at 25°C. After the cells loaded with Mg2+ in 24 mM-Mg2+
solution for 3 h, reduction of [Mg2+]o to 1 mM caused a decrease in
[Mg2+]i in the presence of extracellular Na+ (Na+-dependent Mg2+
efflux). To study the effects of K+ on Na+-dependent Mg2+ efflux, the
initial rate of decrease in [Mg2+]i (initial δ[Mg2+]i) /δt) was compared at
high extracellular [K+] (75 mM) and K+-free (replaced by N-methyl-Dglucamine) conditions. [Na+]o was kept constant at 70 mM, and
membrane potential was set at -13 mV with amphotericin-B-perforated
patch clamp technique. With the K+-based pipette solution, the initial
δ[Mg2+]i/δt values obtained in the presence of 75 mM K+ and 0 mM K+
in the perfusate were not significantly different (79.0±6.0% and
65.6±5.0%, respectively, of the control values measured at 140 mM
[Na+]o without any modification of extracellular and intracellular K+ and
Cl–). Intracellular perfusion with K+-free (Cs+-substituted) solution from
the patch pipette in combination with removal of extracellular K+ did not
significantly change the initial δ[Mg2+]i) /δt (77.7±8.2% of the control).
Finally, the initial δ[Mg2+]i/δt was unchanged by extracellular and
intracellular perfusion with K+-free and Cl–-free solutions (71.6±5.1% of
the control). These results suggest that K+ and Cl– are not involved in the
Na+-dependent Mg2+ efflux.
Around 70% of brain tissue is composed of glial cell, which regulates the
homeostasis of various neurotransmitters, ions and water in the brain.
However, little studies have been performed on the effects of CNS-acting drugs on glial function. We have examined the effect of various tricyclic antidepressant agents: amitriptyline, imipramine, nortriptyline,
and desipramine, on a K+channel responsible for the glial K+-buffering
action. The glial K+ -buffering channels are composed either of homomeric assembly of Kir4.1 or of heteromeric assembly of Kir4.1 and
Kir5.1. In this study, Kir4.1 homomeric channels were exogeneously expressed in tsA201 cells and whole-cell currents were recorded using a
patch-clamp technique. Application of each of the various tricyclic antidepressants immediately and reversibly caused a reduction of inward
and outward currents through this channel. The inhibition was stronger
as the membrane was more depolarized. Development of the current
blockage was well fitted with a single exponential function. These results indicate that the block of Kir4.1 channels by these antidepressants
was clearly in a voltage- and time-dependent fashion. Thus, various tricyclic antidepressants may act as inhibitors at the glial Kir4.1 channels.
We conclude that the inhibition of the glial Kir4.1 channels by these
drugs underlies the therapeutic effects and some of the side effects, particularly seizures in overdose.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Cellular & molecular physiology
Inhibition of hypertonicity-induced cation
channel sensitizes HeLa cells to shrinkageinduced apoptosis
Gene deletion and silencing refutes the
long held hypothesis that maxi-anion
channel is a plasmalemmal VDAC
Shimizu, Takahiro1; Wehner, Frank2; Okada, Yasunobu1
Sabirov, Ravshan1,3; Sheiko, Tatiana2; Liu, Hongtao1;
Deng, Defeng2; Okada, Yasunobu1; William, Craigen2
Inst.Physiol. Sci., Okazaki, Japan;
Cell shrinkage is a hallmark of apoptosis. We previously demonstrated
that the apoptotic volume decrease, which represents an early-phase
event of apoptosis, is induced by K+ and Cl– efflux. On the other hand, it
is known that osmotic cell shrinkage directly leads to apoptotic death in
cells that lack the ability of volume regulation, called regulatory volume
increase (RVI). In HeLa cells that can exhibit RVI, however, strong hypertonic stimulation failed to induce cell death. Since we have recently
showed that the hypertonicity-induced non-selective cation channel
(HICC) plays an important role in the RVI process in HeLa cells, we
used flufenamate, a HICC blocker, to induce persistent cell shrinkage.
Hypertonicity-induced cell death and activation of caspase-3 was enhanced by flufenamate in a concentration-dependent manner. The concentration dependency was in good accord with that for HICC current
inhibition. These results suggest that HeLa cells are sensitized by inhibition of HICC to shrinkage-induced apoptosis.
We have recently demonstrated that maxi-anion channels constitute a
major pathway for the regulated release of ATP. It is widely held that a
voltage-dependent anion channel (VDAC) located in the plasmalemma
that normally functions in the mitochondrial outer membrane is the most
likely candidate protein of this channel. This hypothesis was based on
the similarity of shared biophysical properties, such as the large unitary
conductance and bell-shaped voltage dependency of the maxi-anion
channel and mitochondrial VDAC. In the present study, we deleted each
of the three genes encoding the VDAC isoforms individually and collectively. We have demonstrated that maxi-anion channel (around 400 pS)
activity in VDAC-deficient mouse fibroblasts was unaltered. The channel activity was similar in VDAC1/VDAC3 double-deficient cells and in
double-deficient cells with VDAC2 protein depleted by RNA interference. VDAC deletion slightly down-regulated, but never abolished, the
swelling-induced ATP release. The lack of correlation between VDAC
protein expression and maxi-anion channel activity strongly argues
against the long held hypothesis of plasmalemmal VDAC being the
maxi-anion channel. Details of the biophysical profile, such as the different potassium-to-chloride and glutamate-to-chloride selectivity and a
different pattern of the voltage-dependent gating provide independent
support for our conclusion.
Iptakalim hydrochrolide inhibits ATPsensitive potassium channel activity of rat
pancreatic B-cells
Suga, Sechiko1; Wakui, Makoto1; Nakano, Kyouko2;
Wu, Jie3 (1Hirosaki Univ. Sch. Med. Hirosaki, Japan; 2Hirosaki
Univ. Sch. Health Sci. Hirosaki, Japan; 3Barrow Neurological
Institute, Phoenix, AZ, USA)
Iptakalim hydrochloride (IPT) is a novel ATP-sensitive potassium channel (K(ATP)) opener which has a different chemical structure from any
other known K(ATP) opener, and produces vasodilution. In this study,
we examined the effect of IPT on rat pancreatic beta-cell functions. In
the perifusion experiment for islets, an application of IPT increased insulin secretion, tested with 5.5 mM glucose in the extracellular solution.
Examined in isolated beta-cells loaded with fura-2, IPT elevated intracellular calcium concentration, which was restored by diazoxide. Under
the patch-clamp whole-cell configuration, IPT induced depolarization in
isolated beta-cells followed by action potential firing. The depolarization was associated with a decrease in membrane conductance resulting
from a decrease in K(ATP) activity. Further, IPT applied into the bath solution inhibited K(ATP), recorded in the cell-attached mode. IPT applied
to the intracellular surface of the membrane also inhibited K(ATP), recorded in the inside-out mode. These results indicate that IPT acts on
pancreatic beta-cells as a K(ATP) blocker, which in turn causes electrical
excitation of the cell and insulin secretion.
(1Dept.Cell.Physiol.Natl.Inst.Physiol.Sci.,Okazaki,Japan; 2Dept.
Mol. Human Genet., Baylor Coll. Med., Houston, USA; 3Dept.
Biophys., Natl. Univ., Tashkent, Uzbekistan)
Functional characterization of Cl-/HCO3exchanger by using Cl- indicator dye
Hayashi, Hisayoshi; Suzuki, Yuichi (Lab. of Physiol., Food
and Nutri. Sci., Univ. of Shizuoka)
Chloride ions subserve many physiological functions, including regulation of cell volume, intracellular pH, fluid secretion, and stabilization of
the resting membrane potential. Cl– is absorbed from the gastrointestinal
tract is mediated by Cl–/HCO3 – exchanger. Recent studies have suggested that a major Cl–/HCO3 – exchanger is SLC26A3. Since multiple isoforms of the Cl–/HCO3– exchanger are co-expressed in an intact colonic
cell, complicating the functional analysis of an individual isoform, we
generated an N-terminal hemagglutinin epitope-tagged human
SLC26A3 construct and expressed transiently in CHO cells by using inducible gene expression systems. Using this system, we have previously
characterized SLC26A3 by measuring of its activity with fluorescent
pH-sensitive indicators, BCECF. To assess the validity of pH measurements, we measured the Cl–/HCO3– exchange activity by using chloridesensitive dye, MQAE. We first measured Cl–/HCO3– exchange activity
by MQAE and then measured its activity by using pH sensitive dye in
the same cells. Cl–/HCO3– exchange rate measured by using MQAE was
10-20-fold greater than the rate measured by using BCECF. In addition,
a carbonic anhydrase inhibitor acetazolamide partially inhibited Cl–/
HCO3– exchange activity. These results suggest that even in the presence
of a carbonic anhydrase, its reaction rate is not enough for intracellular
pH measurements to assess the Cl–/HCO3– exchange activity in CHO
J. Physiol. Sci., Vol. 56, Suppl., 2006
Cellular & molecular physiology
Src kinase-dependent osmoregulation of
Na+ reabsorption in renal A6 cells
Niisato, Naomi; Marunaka, Yoshinori (Dept. Mol. Cell
Hypoxia activates maxi-anion channels
and thereby induces ATP release from
Physiol. Kyoto Pref. Univ. Med., Kyoto, Japan)
Liu, Hongato1; Ravshan, Sabirov2; Okada, Yasunobu1
We have previously reported that hypotonic shock stimulates Na+ reabsorption by activating a PTK-dependent pathway and that hypotonic
shock causes a decrease in cytosolic Cl- concentration ([Cl-]c) through
regulatory volume decrease (RVD) in renal epithelial A6 cells. In this report, we found that hypotonic shock increased tyrosine phosphorylation
of src kinase at pY416 (an active site of src kinase) in a manner dependent on the RVD-induced decrease in [Cl-]c. We further found that a decreasse in [Cl-]c caused a significant increase in tyrosine
phosphorylation of src kinase at pY416 under an isotonic condition without any effect on tyrosine phosphorylation state of src kinase at pY527
(an inactive site of src kinase). Furthermore, pretreatment with PP2 (a
specific inhibitor of src kinase) abolished the hypotonic shock-induced
stimulation of Na+ rebasorption and alpha-subunit of epithelial Na+
channel (ENaC) mRNA expression. Taken together these results, it is
suggested that hypotonic shock stimulates Na+ rebasorption through induction of alpha-ENaC gene expression by activating src kinase through
the RVD-dependent decrease in [Cl-]c. Supported by Grants-in-Aids
from JSPS (17590191 and 17390057).
(1Dept Cell Physiol., Natl. Inst. Physiol. Sci., Okazaki, Japan; 2Dept.
Biophys., Natl. Univ., Tashkent, Uzbekistan))
Regulation of maxi-anion channel by
calcium, magnesium and ATP
Toychiev, Abduqodir1; Sabirov, Ravshan1,2; Okada,
Yasunobu1 (1Department of Cell Physiology, National Institute
for Physiological Sciences, Okazaki, Japan; 2Department of
Biophysics, National University of Uzbekistan)
The maxi-anion channel is widely expressed in animal cells. We have recently demonstrated that this channel fulfils a general physiological
function as an ATP-conductive gate for cell-to-cell purinergic signaling.
However, the regulatory mechanisms of the maxi-anion channel remain
poorly understood. We studied the activation mechanism of the maxi-anion channel in inside-out patches excised from mammary C127 cells.
The channels activated upon excision in Ca-free solution in the presence
of 1 mM Mg2+ and absence of ATP. Increasing both Ca2+ and Mg2+ ion
concentration led to a dramatic increase in the rate of channel activation.
Half-maximal activation occurred at the concentration of 0.0012 mM for
Ca2+ ions and 2.8 mM for Mg2+ ions. MgATP added to bath (intracellular) solution greatly suppressed the channel activation with half-maximal inhibition at 0.037 mM. A non-hydrolysable analogue of ATP,
AMP-PNP, did not suppress the channel activation suggesting that ATP
hydrolysis (presumably, the channel phosphorylation) is necessary for
the channel inactivation. When all Mg2+ ions were washed out, the free
ATP still suppressed the channel, indicating that binding of free ATP can
also close the pre-activated maxi-anion channel. Thus, the regulatory
control mechanism of the maxi-anion channel involves divalent cationdependent steps and possibly phosphorylation.
Recent studies have shown that permeability of some chloride channels
to organic anions, such as glutamate and ATP, is involved in cell-to-cell
communication mediated by released organic anions. Previous our studies demonstrated that a maxi-anion channel serves as a conductive pathway for ATP release in a mouse mammary cell line (Sabirov et al., 2001),
rabbit kidney macula densa cells (Bell et al., 2003) and rat cardiomyocytes (Dutta et al, 2004). In the present study, the possible relation between expression of maxi-anion channel and ATP release was tested in
mouse astrocytes in primary culture. In response to hypoxia stress, astrocytes exhibited both activation of maxi-anion channel and massive release of ATP. Hypoxia-induced ATP release was inhibited by blockers of
maxi-anion channel, but not by those of other candidate pathways for
ATP release, such as gap junction hemi-channel, CFTR channel, exocytosis and volume-sensitive outwardly rectifying (VSOR) anion channel.
Using a biosensor technique based on ATP responses of P2X2 receptors
expressed in HEK293 cells, the local ATP concentration on a single astrocyte surface was found to increase to about 5 µM during hypoxia.
Therefore, it is concluded that the maxi-anion channel serves as a major
pathway for ATP release from astrocytes under hypoxia.
Restoration of volume-sensitive chloride
current in cisplatin-resistant human
epidermoid cancer KB cells decreases
their cisplatin resistance
Lee, Elbert L.; Shimizu, Takahiro; Takahashi, Nobuyuki;
Okada, Yasunobu (Dept. Cell Physiol., Natl. Inst. Physiol. Sci.,
Okazaki, Japan)
The platinum-based drug cisplatin is a widely used anticancer drug
which acts by causing the induction of apoptosis. Some types of cancer
have intrinsic or acquired resistance to cisplatin, however. A model of
cisplatin resistance is provided by the cisplatin-resistant KB/CP4 human
epidermoid cancer cell line. It was found previously in our laboratory
that activity of the volume-sensitive, outwardly rectifying chloride channel (VSOR-ClC) is virtually absent in KB/CP4 cells. We hypothesized
that the lack of VSOR-ClC current may contribute to cisplatin resistance
in these cells. An attempt was made to restore the current in KB/CP4
cells so that the effect of its expression on cisplatin resistance could be
tested. Treatment of KB/CP4 cells with trichostatin A (TSA), a histone
deacetylase inhibitor, caused VSOR-ClC current to be partially restored.
A cell viability assay showed that in response to cisplatin, viability of
cells treated with TSA for 48 h decreased significantly compared to control cells. Moreover, a caspase-3 activity assay showed that TSA-treated
cells underwent significantly increased apoptosis induced by cisplatin.
These effects were blocked by simultaneous treatment of the cells with
a VSOR-ClC blocker. From these results, we conclude that restoration of
VSOR-ClC functional expression by TSA treatment leads to a decrease
in cisplatin resistance and an increase in cisplatin-induced apoptosis in
KB/CP4 cells.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Cellular & molecular physiology
Electrophysiological properties of acidactivated anion channels in HeLa cells
wang, haiyan1,2; Shimizu, Takahiro1; Okada, Yasunobu1
Molecular structure responsible for
nuclear translocation of phospholipase Czeta
(1Department of Cell Physiology, National Institute for
Physiological Sciences, and Department of Physiological Sciences,
School of Life Science, Okazaki, Japan; 2Department of Cardiology,
TangDu Hospital, the Fourth Military Medical University, XI'AN
Shaanxi Province, China)
Kuroda, Keiji1; Ito, Masahiko1; Shikano, Tomohide1;
Awaji, Takeo1; Takeuchi, Hiroyuki2; Kinoshita,
Katsuyuki2; Miyazaki, Shunichi1 (1Dept. Pysiol., Tokyo
It has recently been reported that extracellular acid pH activates anion
channels in several cell types. In the present study, we also found functional expression of proton-activated Cl – channel in human epithelial
HeLa cells. Whole-cell currents were rapidly activated by extracellular
acidic solution (pH < 5.0) in a reversible manner. This current exhibited
prominent outwardly rectification under symmetrical Cl – conditions,
time-dependent activation at positive potentials, and low-field anion permeability sequence of I – > Br – > Cl – > aspartate –. A Cl – channel blocker, 4, 4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) or
phloretin, could inhibit the current in a concentration-dependent manner
with the IC50 value of 0.12 or 18.6 µM, respectively. Anion selectivity
and sensitivity to Cl – channel blockers of this proton-activated current
are similar to those of the volume-sensitive outwardly rectifying
(VSOR) Cl – channel current in HeLa cells. However, the other
properties are distinct from those of the VSOR Cl – channel which is
inhibited by acid and exhibits modest outward rectification and
inactivation kinetics at positive potentials.
Phospholipase C-zeta (PLCζ) is a strong candidate of the mammalian
sperm factor that induces IP3-mediated Ca2+ oscillations and subsequent
embryonic development. PLCζ consists of 4 EF-hand domains (EF1-4)
in the N terminus, X and Y catalytic domains, and C2 domain in the C
terminus. PLCζ expressed by injection of cRNA into mouse eggs causes
fertilization-like Ca2+ oscillations, and then it is accumulated into the
formed pronucleus as the sperm factor is. The nuclear translocation
ability (NTA) was investigated by expressing PLCζ mutants tagged with
a fluorescent protein by RNA injection into eggs or 1-cell embryos.
Point mutation analysis revealed a lysine-rich nuclear localization signal
(NLS) sequence between Lys374 and Lys381 in the X-Y linker region.
Truncation of EF1 resulted in the loss of NTA, and point mutation
revealed a responsible sequence in the N terminus of EF1. However,
even if EF1 was present, NTA was lost when EF2-4 or C2 domain was
deleted. Both NTA and Ca2+ oscillation-inducing ability are lost in these
truncation or deletion mutants. Similar results were obtained in cultured
COS cells after transfection with cDNA of mutants. It is predicted from
the 3-D structure of PLCδ1 that PLCζ is folded at the hinge region in the
X-Y linker and that EF-hand domains and C2 domain make extensive
contact. Besides NLS, highly coordinated overall structure of PLCζ is
responsible for NTA as well as Ca2+ oscillation-inducing activity.
The importance of Fyn tyrosine kinase in
Ca2+-sensitization of vascular smooth
muscle contraction induced by a
sphingosylphosphorylcholine and Rhokinase pathway.
Kawamichi, Hozumi; Miao, Junying; Kishi, Hiroko;
Kajiya, Katsuko; Guo, Fengling; Xu, Dan; Kobayashi,
Sei (Dept. Mol. Physiol., Yamaguchi Univ., Sch. Med.)
Whereas the Ca2+-dependent contraction of vascular smooth muscle
(VSM) which regulates physiological vascular tone, the Rho-kinase
(ROK)-mediated Ca2+-sensitization of VSM contraction contributes to
abnormal VSM contraction such as vasospasm. We previously found
that sphingosylphosphorylcholine (SPC) is an upstream messenger for
the ROK-mediated Ca2+ sensitization and that inhibitors of Src family
tyrosine kinase (Src-TK) blocked the SPC-induced contraction and activation of ROK. In the present study, we attempted to determine the enzyme molecule in a family of Src-TK which contributes to the Ca2+sensitization mediated by a SPC/ROK pathway. In order to accomplish
this purpose, we performed knockdown of the target molecule by using
siRNA which was transfected into the human coronary artery smooth
muscle cells (CASMCs) with the efficiency of about 100%. The siRNAmediated knockdown of Fyn inhibited the SPC-induced contraction of
CASMC, whereas non-silencing control siRNA lacked any effect. These
results provide the first direct evidence that Fyn mediates the Ca2+-sensitization of VSM contraction induced by a SPC/ROK pathway. In addition, Fyn constructs (wild, constitutively active, and dominant negative
types) were transfected to CASMCs with high efficiency (> 50%), although CASMCs were well-differentiated contractile cells. In poster
presentation, the effects of transient overexpression of Fyn constructs on
the contraction of CASMCs will be also discussed.
Women's Med. Univ. Sch. Med; 2Tokyo, Japan; 2Dept. Obs. Gyn. ,
Juntendo Univ. Sch. Med.)
PACAP/VIP receptors in the guinea pig
Naruse, Satoru1; Fujiki, Kotoyo2,3; Wei, Muxin2,3; Zhang,
Sumin2,3; Ozaki, Tsuyosi3; Ishiguro, Hiroshi2; Kondo,
Takaharu2 (1Nagoya Univ. Grad. Sch. Med., Nagoya, Japan;
Univ. Grad. Sch. Med. Nut., Nagoya, Japan; 3Natl. Inst.
Physiol. Sci., Okazaki, Japan)
PACAP and VIP are closely related neuropeptides and act primarily as
inhibitory transmitters on most of the gastrointestinal and vascular
smooth muscle cells. However, their actions are opposite in the guinea
pig gallbladder. In this study we have tried to identify PACAP/VIP receptor subtypes expressed in the guinea pig gallbladder. Total RNA was
extracted from the guinea pig gallbladder. RT-PCR was conducted using
the primers with high sequence homology among human, mouse, and rat
PAC1, VPAC1, VPAC2 receptors. PAC1, VPAC1 and VPAC2 receptor
mRNAs were abundantly expressed in the gallbladder. Sequence analysis of guinea pig PAC1 receptor revealed a high homology (91% in nucleotide sequence and 99% in amino acid) to human PAC1 receptor.
There was an isoform of PAC1 receptor that contained an additional 84
nucleotides encoding 28 amino acids in the third intracellular loop. The
amino acid sequence was identical to that of the hop variant reported in
rats and humans. The nucleotide and amino acid sequences of guinea pig
VPAC1 and VPAC2 receptors also had high homologies to the respective human (90% and 95%), rat (91% and 93%), and mouse (93% and
91%) sequences. The guinea pig gallbladder express PAC1, hop variant,
VPAC1 and VPAC2 receptor mRNAs. The expression of the hop variant
of PAC1 receptor may be related to the contractile response observed in
the gallbladder.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Cellular & molecular physiology
Signal mechanisms of regulatory volume
increase (RVI) in HeLa cells and of RVI
inhibition under apoptotic stimulation.
Functional analysis of signal molecules of
abnormal vascular contraction in lipid raft
Takahashi, Nobuyuki; Muthangi, Subramanian; Okada,
Yasunobu (Dep. of Cell Physiology, Nat. Inst. Physiol. Sci., Nat.
Kajiya, Katsuko; Kishi, Hiroko; Kawamichi, Hozumi;
Miwa, Saori; Kobayashi, Sei (Dept. Mol. Physiol., Yamaguchi
Inst. Natural Sci.)
Univ. Sch. Med., Ube, Japan)
Signal mechanisms of regulatory volume increase (RVI) in HeLa cells
and of RVI inhibition under apoptotic stimulationTakahashi, Nobuyuki;
Subramanian, Muthangi; Okada, Yasunobu (Dept. Cell Physiol., Natl.
Inst. Physiol. Sci., Okazaki, Japan)Most cells show cell volume recovery, called regulatory volume increase (RVI), after osmotic shrinkage.
However, under apoptotic conditions, cell volume persistently decreases
without exhibiting RVI. In human epithelial HeLa cells exposed to hypertonic solution, RVI was significantly inhibited by an Akt blocker.
Moreover, exogenous expression of the dominant negative form of Akt
inhibited RVI under hypertonic conditions. Akt was phosphorylated by
hypertonicity, and this phosphorylation was inhibited by apoptotic stimulation by staurosporine, H2O2, or TNF-α. Either of these apoptotic
stimuli suppressed RVI and then induced apoptotic cell death. Apoptosis
signal-regulating kinase 1 (ASK1) was found to be activated by either
apoptosis inducer. Overexpression of the kinase dead mutant of ASK1
restored both shrinkage-induced Akt phosphorylation and RVI under
apoptotic conditions. Thus, it is concluded that Akt activation induced
by hypertonicity is involved in the RVI mechanism in HeLa cells and
that shrinkage-induced Akt activation is inhibited by ASK1 activated by
various apoptotic stimuli thereby leading to persistent cell shrinkage in
apoptotic cells.
Hypercholesterolemia is a major risk factor of cardiovascular events. A
Rho-kinase-mediated Ca2+ sensitization of vascular smooth muscle
(VSM) plays a critical role in abnormal vascular contraction such as vasospasm. We found that sphingosylphosphorylcholine (SPC) sequentially activated Fyn and Rho-kinase to induce the Ca2+ sensitization. We
observed the strong link between the SPC-induced contraction and the
tissue and cellular cholesterol in VSM, suggesting the involvement of
the cholesterol-enriched membrane microdomains, membrane lipid
rafts. In membrane-permeabilized VSM, SPC induced contraction in the
absence of cytosolic GTP which is required for the activation of G-proteins and thus of GPCRs. Taken together with the localization of Fyn in
the membrane lipid rafts, these findings suggest the importance of cholesterol and are compatible with the interaction of SPC with the other
membrane components than GPCRs and/or the direct interaction between SPC and lipid membrane, which may in turn affects the function
of membrane proteins. Therefore, we examined the interaction of SPC
with raft model membranes. The surface plasmon resonance measurement (BIACORE system) revealed that SPC highly associates with the
model membrane microdomains, lipid rafts and that cholesterol in the
model membrane enhances the incorporation of SPC into the membrane.
We propose that cholesterol and its enriched membrane lipid rafts may
play a role in Ca2+ sensitization mediated by a SPC-Fyn-Rho kinase
Effect of CXCL12 stimulation on matrix
metalloproteinases-1 (MMP-1) expression
of NK cells
Inoue, Hiroshi1; Domae, Naochika2; Nishikawa, Yasuo1
Serum-dependence of AMPA receptormediated proliferation in glioma cells
Yoshida, Yukari1; Tsuzuki, Keisuke1; Ishiuchi, Shogo2;
Ozawa, Seiji1 (1Dept. Neurophysiol., Gunma Univ. Grad. Sch.
(1Dep. of Physiology. Osaka Dental Univ., Osaka,Japan; 2Dep. of
Internal Medicine Osaka Dental Univ., Osaka,Japan)
Med., Maebashi, Gunma, Japan; 2Dept. Neurosurg., Gunma Univ.
Grad. Sch. Med., Maebashi, Gunma, Japan)
NK (Natural killer) cells have the ability to migrate and eliminate tumor
cells. We evaluated the role of matrix metalloproteinases-1 (MMP-1) on
regulating chemokine-dependent invasion of NK cells into type I collagen. We revealed that CXCL12 promoted the invasion program on
freshly isolated human NK cells in a MAP kinase dependent manner, because CXCL12 enhanced NK cells invasion was significantly inhibited
by p38MAP inhibitor SB203580 and MEK 1/2 inhibitor U0126. Next we
examind whether CXCL12 especifically enhanced the production of
MMP-1 from NK cells. This production wes significantly inhibited by
SB203580 and U0126. Immunofluorescence confocal microscopic studies suggested that MMP-1 was co-localized with alpha2 integrin on the
NK cell surface stimulated by CXCL12. The co-localization of MMP-1
and alpha2 integrin was significantly inhibited by SB203580 and U0126.
Immunoprecipitation assy showd that productional MMP-1 associated
with alpha2 beta1 integrin on NK cells stimulated by CXCL12. This association was significantly inhibited by SB203580 and U0126. These results suggested that MMP-1 associated with the cell surface was
involved in NK cell invasion into type I collagen, and MMP-1 associated
with alpha2 integrin on the cell surface may be a critical step in facilitating pericellular matrix degradation during cell invasion.
Glutamate may cause Ca2+ entry through Ca2+-permeable glutamate receptors, which in turn stimulates the anti-apoptotic signaling cascade in
glioma cells. Here, we found that a human glioma cell line, U-87 MG,
expressed GluR1, GluR2 and GluR3 subunits of α-amino-3-hydroxy-5methyl-4-isoxazolepropionate
(AMPARs). Approximately 20% of GluR2 was expressed in the
unedited form, which is known to assemble Ca2+-permeable AMPARs.
Ca2+ entry through the activation of these receptors by AMPA was
detected clearly in approximately 20% of U-87 MG glioma cells. Cell
proliferation assays revealed that the application of AMPA or glutamate
facilitated cell proliferation by activating AMPARs in low-serum
medium containing 0.5% fetal calf serum (FCS). Unexpectedly, cell
proliferation by the activation of AMPARs was not detected in serumrich medium containing 10% FCS. Overexpression of the unedited form
of GluR2 (GluR2Q) by adenoviral-mediated gene transfer markedly
increased the Ca2+ entry into U-87 MG cells. This treatment in the
presence of glutamate facilitated proliferation and migration of U-87
MG cells in the low-serum condition, whereas it had again no effect in
the serum-rich condition. It is therefore likely that cell proliferation and
migration of U-87 MG cells are under the regulation of growth factors
contained in the serum as well as Ca2+ entry through AMPARs, and that
the latter regulation becomes evident only when serum factors are
deprived of culture medium.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Cellular & molecular physiology
deprivation induces persistent cell
shrinkage and apoptotic cell death
Nukui, Miho; Shimizu, Takahiro; Okada, Yasunobu (Dept.
Cell Physiol., Physiol. Sci., Okazaki, Japan)
Although cell shrinkage is one of the phenotypical features of apoptosis,
it has been controversial whether it is a prerequisite to apoptosis induction. In this study, we examined whether a persistent decrease in cell volume could per se initiate apoptotic cell death without any apoptotic
stimulus. When HeLa cells were incubated in isotonic Na+-free solution,
the mean cell volume immediately began to decrease and reached 84%
of the original value within 30 min. After persistent shrinkage, activation
of caspase-3 and reduction of cell viability were observed. Application
of a blocker of the Na+-K+-2Cl– cotransporter (NKCC), bumetanide (100
µM) or furosemide (1 mM), or that of the Na+/Ca2+ exchanger (NCX),
KB-R7943 (25 µM), inhibited Na+ deprivation-induced shrinkage and
attenuated apoptotic cell death. These results suggest that shrinkage of
HeLa cells exposed to Na+-free solution is induced by efflux of Na+, K+
and Cl– mediated by operation of NKCC and reversed operation of NCX,
and that an isotonic volume decrease per se triggers the apoptotic
Wakui, Makoto1; Suga, Sechiko1; Mizukami, Hiroki3;
Yagihashi, Soroku3; Takeo, Teruko2 (1Hirosaki Univ. Sch.
Med. Hirosaki, Japan; 2Hirosaki Univ. Sch. Health Sci. Hirosaki,
Japan; 3Hirosaki Univ. Sch. Med. Hirosaki, Japan)
Sphingosine-1-phosphate (S1P) receptor is known to show a variety of
actions including endothelial permeability regulation. Multiple S1P receptors including S1P-1 are expressed on pancreatic islets, and S1P, a
S1P-1 receptor agonist, was shown to potentiate insulin secretion. However, a precise mechanism of the receptor action is not clear at moment.
Using Cre-LoxP system, we made mice specifically lacking S1P-1 receptor gene in pancreatic islet B-cells. The blood glucose levels in fasting state are the same in knockout and control mice. After intraperitoneal
glucose challenge, knockout mice were significantly less able to normalize blood glucose levels than were the control mice. In the perifusion experiment for isolated islets, glucose stimulation increased insulin
secretion in the control islets, whereas it failed in islets from knockout
mice. In isolated pancreatic B-cells from both control and knockout
mice, glucose stimulation caused depolarization followed by action potential firing. The membrane capacitance measurement revealed that calcium pulse stimulation could not increase the exocytosis of insulin
granules. These results indicate that S1P-1 receptors are essential for Bcell insulin secretion in the exocytotic process at least distal to calcium
Gq/PLC-coupled receptor-induced
transient reduction in plasma membrane
Fujii, Yusuke; Omatsu-Kanbe, Mariko; Matsuura,
Hiroshi (Shiga Univ. Med. Sci. Otsu, Shiga, Japan)
Phosphatidylinositol-4,5-bisphosphate (PIP2) is well known to be a
source of the important second messengers inositol-triphosphate (IP3)
and diacylglycerol (DAG). To investigate the intracellular localization of
PIP2, it has been commonly utilized pleckstrin homology (PH) domain
fused with green fluorescent protein (PH-GFP) as a marker. However
this method is not suitable for cells difficult to be transfected. In the
present study, we established the assay method of receptor-induced transient dissociation of PIP2 from the plasma membrane in rat brown adipocytes using anti-PIP2 antibody. Cells incubated with or without stimuli
for 0-120 sec were fixed immediately, blocked with BSA and incubated
with anti-PIP2 antibody. After washing, the cells were incubated with Alexa Fluor 546-labeled IgG and fluorescent signals were observed using
confocal laser scanning microscope. In control cells, PIP2 displayed
staining which outlined the cells periphery. When the cells were stimulated with 1µM noradrenaline (NA), plasma membrane PIP2 was rapidly
decreased within 2.5 sec. Dissociation of PIP2 from the plasma
membrane was transient and relocalized to the plasma membrane within
2 min. Stimulation of the cells with 50µM ATP showed similar response
to NA. 5µM wortmannin inhibited relocalization of PIP2 to the plasma
membrane in NA- or ATP-stimulated cells, indicating that
phosphatidylinositol-4-kinase (PI4K) plays an important role in PIP2
The role of sphingosine-1-phosphate-1
receptors in pancratic B-cell functions
Prostaglandin E2 release from antral
mucosa of guinea pig: Differrent role COX1 and COX-2
Nakanishi, Yoshihiko1; Shimamoto, Chikao1; Kato,
Masumi2; Fujiwara, Shoko2; Nakahari, Takashi2 (1Dept. of
Internal Medicine, Osaka Medical College, Takatsuki 569-8686,
Japan; 2Dept. of Physiol., Osaka Medical College, Takatuki 5698686, Japan)
Contributions of COX-1 and COX-2 in basal and ACh-stimulated prostaglandin E2 (PGE2) release were studied in antral mucosa of guinea pig.
PGE2 was released from antral mucosa spontaneously. Acetylcholine
(ACh), which increases [Ca2+] i, increased the PGE2 release from antral
mucosa in a dose dependent manner. COX-1 or COX-2, which generates
PGE2 from arachidonic acid (AA), was regulated by [Ca2+]i. Basal and
ACh-stimulated PGE2 release were increased by the addition of AA, and
was inhibited by a PLA2 inhibitor and COX inhibitors. SC560 (100 nM,
a selective inhibitor of COX-1) decreased ACh-stimulated PGE2 release
without any decrease in basal PGE2 release, while ionomycin increased
PGE2 release. NS398 (20 µM, a selective inhibitor of COX-2) decreased
basal PGE2 release without any decrease in ACh-stimulated PGE2
release. Moreover, in isolated antral epithelial cells, SC560 inhibited
ACh-stimulated-PGE2 releases, however, NS398 did not. Thus, in antral
mucosa, basal PGE2 release is maintained via COX-2 of interstitial cells
and ACh-stimulated PGE2 release is maintained via COX-1 of antral
epithelial cells. These observations suggest that PGE2 released via COX2 in the interstitial cells maintains an integrity of the resting antral
mucosa and that released via COX-1 in antral epithelial cells maintains
an autocrine mechanism, which enhances Ca2+-regulated exocytosis in
ACh-stimulated antral mucosa, such as during meals.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Cellular & molecular physiology
Germ cell apoptosis in rat testis is induced
by oxidative stress via oral administration
of di(2-ethylhexyl)phthalate, and is
significantly prevented by treatment of
antioxidant vitamins or rare sugars
Effects of various local anesthetics on
axonal transport in cultured mouse dorsal
root ganglion neurons
Arai, Tamie1; Hiruma, Hiromi2; Katakura, Takashi2;
Hoka, Sumio1; Kawakami, Tadashi2 (1Dept. Anesthsiol.,
Yamaguchi, Fuminori1; Suna, Shigeru2; Tokuda,
Masaaki1; Jitsunari, Fumihiko2 (1Facult. Med. Kagawa Univ.,
Kitasato Univ. Sch. of Med., Sagamihara, Japan; 2Dept. of Physiol.
Kitasato Univ. Sch. of Med., Sagamihara, Japan)
Kagawa Japan; 2Facult. Med. Kagawa Univ., Kagawa Japan)
We previously reported that intrathecal administration of various local
anesthetics resulted in the dorsal root axonal degeneration near entry into
the spinal cord in rats. The level of neurotoxicity of local anesthetics in
the dorsal root depended on the kind of anesthetics and this was comparable to the level of side effects occurred in the clinical use. In the present
study, we assessed the effects of various local anesthetics on axonal
transport in cultured mouse dorsal root ganglion neurons. Lidocaine,
bupivacaine, and ropivacaine (concentrations: 1-50 mM) all decreased
axonal transport, but in different degree. The order of potency was
lidocaine<bupivacaine<ropivacaine. At a concentration of 50 mM,
lidocaine and bupivacaine but not ropivacaine caused the rupture of plasma membrane in some dorsal root ganglion neurons. Considering with
the pharmacological potency, lidocaine:bupivacaine:ropivacaine =
1:4:4, the neurotoxocity of ropivacaine is likely much less than that of
lidocaine. These in vitro experiments may be useful for determining the
neurotoxicity of local anesthetics.
Phthalate esters have been used as plasticizers of synthetic polymers. Recent studies revealed that they induce atrophy of the testis, although its
pathogenesis remains unknown. Testicular atrophy with aspermatogenesis was induced by feeding with 2% DEHP-containing diet. The biochemical and immunohistochemical analysis revealed that DEHP
increased the reactive oxygen species generation, with concomitant decrease of glutathione and ascorbic acid, and selectively induced apoptosis of spermatocytes, thereby causing atrophy. Oxidative stress was
selectively induced in germ cells, but not in Sertoli cells, treated with
mono(2-ethylhexyl)phthalate (MEHP), a hydrolysed metabolite of DEHP. Furthermore, MEHP selectively induced the release of cytochrome
c from mitochondria of the testis. These results indicate that oxidative
stress elicited by MEHP principally injured mitochondrial function, and
induced apoptosis of spermatocytes and atrophy of the testis. Using the
2% DEHP-dose, the effect of simultaneous administration of vitamins C
and E was examined. The vitamin supplementation significantly prevented the testicular injury. Results suggest that antioxidant vitamins can
protect the testes from DEHP-toxicity. Some of rare sugars (i.e. D-psicose and D-allose) are also effective in prevention of the testicular injury.
Microarray analysis has been applied to elucidate the genes involved in
the DEHP-toxicity and the protection mechanism.
Lysophosphatidic acid (LPA)-induced cell
migration inhibition is independent on
ROCK-mediated reduction in PI3-kinase
(PI3K) products
Sugimoto, Naotoshi; Takuwa, Noriko; Takuwa, Yoh
(Grad. Sch. Med. Kanazawa Univ., Kanazawa, Japan)
PI3Ks produce 3'-phosphoinositides (3'-PIs) including PI(3,4,5)P3 and
PI(3,4)P2, whereas PTEN dephosphorylates 3'-PIs to decrease the contents of PI(3,4,5)P3 and PI(3,4)P2. Elevation of PI(3,4,5)P3 and PI(3,4)P2
contents induces activation of PDK1 and Akt, resulting in cell migration
and cell survival. Very recently, it has been shown that Rho-ROCK stimulates PTEN, resulting in inhibition of Akt and cell migration. These observations led us to investigate the effects of Rho-stimulating GPCR
agonist LPA on Akt activation and cell migration. In CHO cells that endogenously express LPA1 receptor, IGF-I stimulated Akt phosphorylation (P-Akt) and chemotaxis in a PI3K inhibitor-sensitive manner. In
PTX-treaed cells, LPA inhibited IGF-I-induced P-Akt and chemotaxis.
Y-27632, a ROCK inhibitor, prevented LPA-inhibition of IGF-I-induced
P-Akt, indicating the ROCK mediates inhibition of P-Akt. However, Y27632 failed to abrogate LPA-inhibition of IGF-I-induced chemotaxis.
Thus, there was a discrepancy between LPA-induced inhibition of the
cellular 3'-PIs amount, which is reflected by the extent of P-Akt, and inhibition of cell migration. These results suggest that LPA-induced inhibition of cell migration is not dependent on ROCK-mediated stimulation
of PTEN and, thereby, reductions of cellular 3'-PIs contents
Hippocalcin-mediated regulation of Mixed
Lineage Kinase (MLK) 3 activity
Kobayashi, Masaaki; Takamatsu, Ken (Dept. Physiol. Toho
Univ. Sch. Med. Tokyo, Japan)
Hippocalcin is a member of the neuronal calcium sensors (NCS) family
predominantly expressed in the hippocampal pyramidal cells. We have
found that hippocalcin binds to C-terminal region of Mixed Lineage Kinase (MLK) 3. Here we analyzed activity of MLK3 in hippocalcin deficient mice. Immunoblot analysis using substrate specific anti-phosphoantibodies revealed that MKK3, one of the substrate of MLK3, in hippocalcin deficient mice was higher phosphorylated than that in wild
type. Kinase activity of immunoprecipitated MLK3 was examined using
bacterially expressed MKK4 as a substrate. The resting MLK3 activity
in hippocalcin deficient mice was higher than that in wild type. By adding recombinant hippocalcin (100ng) to the assay condition, the activity
of MLK3, which was immunoprecipitated from wild type mice, was inhibited. These results indicate that the binding of hippocalcin to C-terminal domain of MLK3 directly inhibits MLK3 kinase activity.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Cellular & molecular physiology
Sphingosine-1-phosphate accelerates
ischemia-induced angiogenesis in the
mouse limb
Oyama, Osamu1,2; Takuwa, Noriko1; Sugimoto,
Naotoshi1; Koizumi, Junji2; Takuwa, Yoh1 (1Grad. Sch. Med.
Kanazawa Univ., Kanazawa, Japan; 2Grad. Sch. Med. Kanazawa
Univ., Kanazawa, Japan)
S1P has a critical role in vascular maturation during mammalian development. However, little is known about the role of S1P in ischemia-induced angiogenesis in adults. We investigated the effect of both exo- and
endogenous S1P on angiogenesis in ischemic skeletal muscle in adult
mice. Unilateral hindlimb ischemic model is a well-established in vivo
angiogenesis assay system. We monitored post-ischemic angiogenesis
by blood flow recovery with laser doppler imager and capillary density
with anti-CD31 immunohistochemistry after surgery. First, we injected
S1P into ischemic muscle everyday after surgery in C57BL6/J mice.
Limb blood flow was 2.5 fold elevated in S1P(10–8M)-injected mice at
day 7. Capillary density was 1.5-fold increased in S1P group at day 10.
These effects were comparable to bFGF administration. Trying to create
long acting S1P, we prepared the slow-release microsphere containing
S1P with polylactide-co-glycotide and intramuscularly administrated it
just once immediately after surgery. Mice received microsphere containing S1P increased blood flow in ischemic limb. We also examined the effect of endogenous S1P overproduction by generating sphingosine
kinase 1 transgenic (SphK1-Tg) mice, and blood flow was slightly increased compared with littermate wild type mice. S1P1 receptor selective
agonist SEW2871 also accelerated blood flow recovery. We showed S1P
accelerates ischemia-induced angiogenesis, most likely via S1P1 receptor in adult mice. S1P and SEW2871 is a potential therapeutic for ischemic diseases.
Yang, Ding-I; Yang, Jir-Jei (Institute of Neuroscience, Tzu Chi
University, Hualien, Taiwan)
1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) is commonly used for adjuvant chemotherapy to treat malignant glioma including glioblastoma
multiforme (GBM). BCNU kills tumor cells via multiple actions including carbamoylation and alkylation. Herein we test the effects of NO donors on alkylating cytotoxicity to rat C6 glioma cells. The alkylating
agents tested included methyl methanesulfonate (MMS), N-methyl-Nnitrosourea (MNU), and N-ethyl-N-nitrosourea (ENU). The synergistic
effects of three NO donors, namely S-nitrosoglutathione (GSNO), diethylamine NONOate (DEA/NO), and sodium nitroprusside (SNP) on alkylating agents were determined by colony-formation assay. We found that
inclusion of NO donors substantially reduced the extents of colony formation following exposure of glioma cells to all three alkylating agents.
Among the three NO donors, GSNO appeared to be the most potent one.
GSNO also exerted similar synergistic actions reducing the extents of
colony formation when co-administrated with 1,2-bis(methylsulfonyl)1-(2-chloroethyl)-hydrazine, another alkylating agent that mimics the
chloroethylating action of BCNU. O6-Methylguanine methyl-DNA
transferase (MGMT) is a DNA repair enzyme that removes the cytotoxic
O6-alkylguanine adducts induced by alkylating agents. Western analysis
indicated that expression of MGMT was reduced in the presence of GSNO, suggesting the possibility that GSNO enhanced alkylating cytotoxicity via, at least in part, decreasing cellular MGMT contents.
Granule localization of neurotrophin-3 in
mouse alveolar macrophages
Hikawa, Shiori1; Hiruma, Hiromi2; Masuda, Noriyuki1;
Kawakami, Tadashi2 (1Dep. of Med., Kitasato Univ. Sch. of
Med.; 2Dep. of Physiolo., Kitasato Univ. Sch. of Med.)
Neurotrophins are growth factors that exert multiple actions on neuronal
and nonneuronal cells. We have previously shown that neurotrophins,
NT-3 and NT-4/5, are expressed in mouse alveolar macrophages. In the
present study, we further attempted to clarify the intracellular localization of NT-3 and its co-localization with interleukin IL-1, fibroblast
growth factor FGF, and histamine in alveolar macrophages. Immunocytochemical staining with antibodies against NT-3, lysosomal membrane
protein LAMP-2, IL-1, FGF, and histamine was performed on alveolar
and peritoneal macrophages obtained from the adult mouse. Approximately 80% of alveolar macrophages were immunoreactive for NT-3. In
contrast, peritoneal macrophages were rarely immunoreactive for NT-3
(<5%). In these NT-3-immunoreactive macrophages, NT-3 was localized in intracellular granules. The granule localization of NT-3 was confirmed, since the NT-3-containing granules were immunoreactive for
LAMP-2, which labels intracellular granule membrane. Almost all alveolar and peritoneal macrophages were immunoreactive for IL-1, FGF,
and histamine. IL-1 and FGF were diffusely distributed in the cytoplasm.
Histamine was localized in granules. In some granules, histamine was
co-localized with NT-3. Since a large number of alveolar macrophages
contain NT-3 in their granules and NT-3 is co-localized with histamine
in some granules, they may be co-released in an exocytotic manner from
alveolar macrophages and may play synergic roles in pulmonary pathophysiology.
Nitric oxide donors enhance alkylating
cytotoxicity in rat C6 glioma cells
Microglia expressing NG2 chondroitin
sulfate proteoglycan in normal and
pathologic brains as multipotent neural
Tanaka, Junya; Sakamoto, Aiko; Matsumoto, Hiroaki;
Imai, Yoshinori (Sch. Med., Ehime Univ., Ehime, Japan)
Rat primary microglia (MG) acquired a multipotent property to give rise
to neuroectodermal cells through two-step culture in 10 and 70% serumsupplemented media for 5 d (Yokoyama et al., Glia 2004; 45, 96-104).
Such multipotent MG called promicroglioblasts (ProMGB) formed cell
aggregates, which generated cells with neuroectodermal phenotypes
shortly after transfer into serum-free medium. As revealed by immunohistochemistry, there were a few MG expressing NG2-chondroitin sulfate proteoglycan (NG2) in the neonatal rat brain. Primary culture from
the neonatal brain contained NG2+ MG, which appeared being the
source of NG2+-ProMGB aggregates. The aggregates were MG-markers+/GFAP+/NCAM+/S-100b- and possessed an alkaline phosphatase
activity. Marked accumulation of NG2+ MG was observed in the close
vicinity of stab wounds made in mature rat brain. The NG2+ MG in the
wounds separated with trypsin-EDTA formed NG2+ aggregates in 70%
serum-supplemented medium and then turned into cells with neuroectodermal phenotypes in serum-free medium. Although it is quite difficult
to separate viable neurons from mature brains, cells from the stab
wounds generated process bearing b-tubulin III+ cells easily. These data
suggest that NG2+ MG in normal developing or pathologic brains are involved in genesis or regeneration of the brain.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Cellular & molecular physiology
Microglial cells generate osteoclast-like
multinucleated giant cells and cells with
neuroectodermal phenotypes, depending
on culture conditions.
Reaction of electron-transferring
flavoprotein with D-lactate dehydrogenase
and enoyl-CoA reductase
Ii, Chisato; Takahashi, Hisaaki; Matsumoto, Hiroaki;
Imai, Yoshinori; Tanaka, Junya (Sch. Med., Ehime Univ.,
Mol. Physiol., Grad. Sch. Med. Sci., Kumamoto Univ., Kumamoto,
Ehime Japan)
The anaerobic bacterium Megasphaera elsdenii uses lactate as the carbon source and produces lower fatty acids in the rumen of cattle and
sheep. Electron-transferring flavoprotein (ETF) is a key enzyme in the
intracellular redox system of M. elsdenii. ETF receives electrons from
flavoprotein D-lactate dehydrogenase (D-LDH), which oxidizes D-lactate to pyruvate. The received electrons are then transferred to flavoprotein enoyl-CoA reductase (ECR), which reduces enoyl-CoA to acylCoA. The acyl-CoAs are eventually changed to fatty acids by CoA elimination. ETF also receives electrons from NADH, which is reduced by
many other redox reactions. ETF contains two FAD molecules as the cofactor. The functions of the two FAD molecules are presently unlcear. In
this study, we found the followings by spectrophotometric experiments
using purified flavoproteins. Here the two FAD molecules in ETF are
designated FAD-1 and FAD-2. (1) NADH reduces both FAD-1 and
FAD-2. (2) D-LDH interacts with only FAD-1. (3) ECR interacts with
both FAD-1 and FAD-2. (4) Electron transfer between FAD-1 and FAD2 can occur without NADH/NAD+, D-LDH, and ECR.
Although microglial cells are currently considered mesodermal cells, it
has not been completely determined whether they are hematopoietic or
mesenchymal origins. To obtain some insights in this issue, we induced
dedifferentiation of microglial cells in culture by incubating in 70% serum-supplemented medium for 2 d. Microglial cells were separated from
primary mixed glial culture that was started from neonatal rat forebrains
or from ischemic brain lesions of rats whose right middle cerebral artery
was transiently occluded. Cells cultured in 70% serum-medium gradually exhibited amoeboid shape and often formed cell aggregates, while increasing expression of Id genes and getting highly proliferative. Such
cell aggregates differentiated into cells with neuroectodermal phenotypes after they were transferred into serum-free medium on poly-Llysine-coated substrate. By contrast, cells that had been cultured in 70%
serum-medium gradually fused resulting in formation of multinuclear
giant cells, after they were transferred into 10% fetal calf serum-supplemented medium containing M-CSF and RANKL. As revealed by RTPCR, such giant cells elevated expressions of mRNAs encoding DCstamp, TRAP and Cathepsin K that are specific markers of osteoclasts.
Taken that osteoclasts are derived from hematopoietic stem cells (HSCs)
and HSCs are known to generate neuroectodermal cells, microglial cells
may be of hematopoietic origin.
Sato, Kyosuke; Nishina, Yasuzo; Shiga, Kiyoshi (Dept.
Multipotent amoeboid microglia appear in
brain lesions may come from blood:
immunohistochemical comparison of
microglial reaction in lesions with and
without breakdown of blood brain barrier
Isolation of a receptor cDNA on
macrophages for allogeneic MHC (H-2Kd)
Sakamoto, Aiko; Matsumoto, Hiroaki; Imai, Yoshinori;
Tanaka, Junya (Sch. Med. Ehime Univ., Ehime, Japan)
[Background] Previously, we found that allograft-induced macrophages
(AIM; H-2DbKb) were the major effector cells responsible for allograft
(e.g., BALB/c skin and Meth A tumor; H-2DdKd) rejection. In the last
meeting, we reported isolation of a cDNA, which encoded a novel receptor on AIM for allogeneic MHC (H-2Dd), by using anti-AIM monoclonal antibody (mAb; R15) and H-2Dd tetramer. We named this
receptor "macrophage MHC receptor (MMR)". In the present study, we
obtained a cDNA encoding a novel receptor on AIM for allogeneic MHC
We found that amoeboid-shaped microglia expressing NG2 proteoglycan accumulated in stab wounds in the brain. Some of the NG2-positive
microglia expressing nestin and GFAP turned into cells with neuroectodermal phenotypes in vitro. To elucidate whether such amoeboid NG2positive microglia are blood-borne or the activated form of resident microglia, we compared the nature of cells expressing microglia markers
using three kinds of brain pathology models using Wistar rats; stabwound, middle cerebral artery occlusion (MCAO), and facial nerve axotomy models. The former two models accompanies breakdown of
blood brain barrier (BBB), while the axotomy model does not. A huge
number of NG2-positive amoeboid shaped cells expressing Iba1, a
marker of microglia/macrophages, accumulated in the stab wounds and
the core lesions of MCAO. The majority of the amoeboid cells were proliferating as revealed by Ki67-immunostaining. In contrast, microglial
cells in the axotomied facial nerve nucleus enlarged somata but still kept
ramified shapes, and none of them were Ki67-negative. Most of resident
microglial cells died within 2 days after the stab-lesioning or MCAO,
while none of microglia died in the facial nerve nucleus. These observations suggest that multipotent NG2-positive microglia in the brain lesions are blood-borne and distinct from resident ramified microglial
Yamaji, Junko; Yoshida, Ryotaro; Takahashi, Takeshi;
Takeda, Sayako; Inui, Takaki; Mori, Yoshiaki; Kubota,
Takahiro (Dept. Physiol., Osaka Medical College, Osaka, Japan)
[Method] cDNA fragments were isolated by the T7 phage expression
cloning method using R12 mAb and H-2Kd tetramer. Full length of the
cDNA was obtained by the RACE method. mRNA expression was
estimated by RT-PCR. cDNAs fused to GFP cDNA were transfected to
HEK293T cells; and the binding of H-2 molecules to the transfectants
was explored under a confocal microscope. The dissociation constant
(Kd) of AIM toward H-2 molecules was assessed by flow cytometry.
[Results] We isolated the full length (ξχ5 2.4kb) of cDNA, which
encoded a receptor on AIM for allogeneic MHC (H-2Kd), and named the
receptor 'MMR2'. The MMR2 mRNA was expressed exclusively in AIM
but not in other cells infiltrating into allograft. HEK293 cells transfected
with MMR2 cDNA reacted with H-2Kd, but not with other H-2,
molecules. The H-2Kd binding was completely suppressed by R12 or
anti-H-2Kd mAb. The Kd value of AIM toward H-2Kd was 2.8×10–9 M.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Cellular & molecular physiology
Expression of the tight junction protein
claudins in salivary gland cells
Michikawa, Hiromi; Fujita-Yoshigaki, Junko; Sugiya,
Hiroshi (Nihon Univ. Sch. Dent. at Matsudo, Chiba, Japan)
Tight junction (TJ) is an important adhesion system in epithelial cells,
and also plays a role for regulation of paracellular flux across epithelial
sheets. Claudins, a component of TJ, comprise a large family consisting
of more than 20 members in mammals, and contribute to regulation of
barrier function. We have compared expression of claudins in culture
and intact duct cells of the rat submandibular gland with that in MDCK
cells, a typical epithelial cell line. SMIE cells derived from rat submandibular duct were kindly provided by Dr. B. J. Baum (NIDCR). Duct
cells were isolated from rat submandibular gland. TJ proteins were detected by western blotting. Immunofluorecence strain of TJ proteins was
observed by a confocal laser scanning microscope. SMIE cells formed
TJ as well as MDCK cells, which was confirmed by immunofluorecence
strain of the TJ proteins occludin and ZO-1. Claudin-3 protein was detected in both cells. Claudin-1 and claudin-4 proteins were detected in
MDCK cells, but not in SMIE cells, which was confirmed by immunofluorecence strain of claudins. However, in rat submandibular duct cells,
claudin-4 was detected by western blotting and immunofluorecence
strain. In SMIE cells, the transepithelial electrical resistance was lower
and the flux of FITC-dextran was higher than in MDCK cells, indicating
that SMIE cells are more permeable than MDCK cells. These results
suggest that claudin-4 expression contributes to barrier function of intact
salivary gland duct cells.
Era, Seiichi1; Hayashi, Tomoya2; Takami, Shuichiro3;
Matsuyama, Yukie1; Negawa, Tsuneo1; Shimizu,
Katsuji3 (1Gifu Univ. Grad. Sch. Med., Gifu, Japan; 2Meiji Univ.
Oriental Med., Kyoto, Japan; 3Gifu Univ. Grad. Sch. Med., Gifu,
In order to examine the redox state of cerebrospinal fluid (CSF) albumin
together with serum albumin from orthopedic patients, we have analyzed
the percentage of reduced and oxidized albumin fractions in CSF and serum by HPLC. Forty-two patients had no detectable evidences of systemic diseases such as hepatic and renal dysfunctions, and they were
divided into two groups by their age (young group, average age = 31.3
years (n = 10); elder group, 64.8 years (n = 32)). Informed consent was
obtained in all cases. Albumin is the mixture of reduced and oxidized albumins. Oxidized albumin is composed of two type of albumin, i.e.,
mixed disulfide with cysteine (tentatively called HNA-1) and oxidation
product higher than mixed disulfide with reactive oxygen species (called
HNA-2). In the elder patients, mean values for the fraction of HMA,
HNA-1 and HNA-2 were 93.1, 5.8 and 1.1% for CSF, and 69.5, 28.6 and
1.9% for serum, respectively. In the young patients, those values were
93.0, 6.7 and 0.3% for CSF, and 76.4, 22.0 and 1.6% for serum, respectively. CSF is believed to maintain the homeostasis of brain functions,
especially defense against oxidative stress. From our HPLC results, in
both groups, significant difference between the values for CSF and serum indicates the function of blood-brain barrier is maintained. Significant high value for reduced albumin level in CSF in both groups
indicates the redox state of CSF is kept highly reduced status.
Proteome analysis of livers from LEC rats
using proteins separated by twodimensional electrophoresis
Suzuki, Takahiro1; Ohishi, Masamichi3; Maeda,
Tadakazu3; Hayashi, Masanobu4; Kawahara,
Katsumasa5; Furudate, Sen-ichi2 (1Dept. Cellular and
Molecular Physiol., Kitasato Univ. Graduate Sch. Med Sci.,
Kanagawa, Japan; 2Dept.Laboratory Animal Sci., Kitasato Univ.
Sch. Med., Kanagawa, Japan; 3Dept.Biomolecular Dynamics.,
Kitasato Univ. Sch.Sci., Kanagawa, Japan; 4Dept. Veterinary
Radiology., Rakuno Gakuen Univ., Hokkaido, Japan; 5Dept.
Physiol., Kitasato Univ. Sch. Med., Kanagawa, Japan)
Long-Evans Cinnamon (LEC) rats have been used as an animal model
for human Wilson's disease. LEC rats developed hepatic abnormalities
due to accumulation of copper in the liver. The aim of this study was to
search for liver proteins related to Wilson's disease. The livers from LEC
and WKAH/Hkm rats (12 and 16 weeks of age) were homogenized and
performed using two-dimensional electrophoresis (2-DE). Their 2-DE
spots were analyzed by an image analysis software for comparison of
protein quantity. Although no significant difference was observed between the two strain rats of 12 weeks, 17 spots were different between
the two of 16 weeks. One spot of 36.5 kDa was positive in the WKAH
rat, but was negative in the LEC rat. Four spots were denser by 1.5 times
in the LEC than in the WKAH, Two of them were denser by around 7
times. On the other hand, 8 of 12 spots were much thinner by 2 times in
the LEC than in WKAH. These results suggest that two-dimensional
electrophoresis may be useful for the study of Wilson's disease.
Highly reduced status of lumber
cerebrospinal fluid monitored by the redox
state of the sulfhydryl in albumin
In vivo stable transduction of protein into
the rat brainstem using the
Hemagglutinating virus of Japan-envelope
Owada-Makabe, Kyoko; Tsubota, Yuji; Yukawa,
Kazunori; Maeda, Masanobu (Wakayama Med. Univ. Sch. of
Med., Wakayama 641-8509, Japan)
We have demonstrated for the first time that the Hemagglutinating virus
of Japan-envelope (HVJ-E) vector allows the efficient transduction of
protein into the rat brainstem using the technique of microinjection in vivo. Successful transduction of an exogenous protein, β-galactosidase (βgal), was performed via the direct injection of only 100 nl HVJ-E vector
solution into the specific restricted brain area (nucleus tractus solitarius,
NTS). To examine whether the β-gal activity was maintained in the rat
brainstem, samples were collected 3, 6 and 24 hours after transduction
and a colorimetric assay was utilized to detect and quantify β-gal
activity. A constant and high transduction level of β-gal activity was
maintained in the rat brainstem, that was not significantly reduced within
24 hours following transduction compaired with β-gal without HVJ-E
vector. This kind of targeted delivery system using the HVJ-E vector
should have wide applications of various therapeutic proteins to the
central nervous system in vivo.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Transport across cell membrane
The role of apical Na+-H+ exchanger in
HCO3- secretion in mouse pancreatic duct
Yamamoto, Akiko1; Ishiguro, Hiroshi1; Ko, Shigeru2;
Kondo, Takaharu1; Naruse, Satoru2 (1Grad. Sch. Med. Univ.
Nagoya, Nagoya, Japan; 2Grad. Sch. Med. Univ. Nagoya, Nagoya,
Transport across cell membrane
In the present study, to investigate the role of apical Na+-H+ exchangers
(NHE) in HCO3– secretion from pancreatic duct cells, we examined the
activity of apical Na+-H+ exchange in interlobular pancreatic duct segments isolated from normal mice and δF508 mice where function of
CFTR (cystic fibrosis transmembrane conductance regulator) is
microdissection.The ducts were superfused with HCO3–-free Hepesbuffered
separately.Intracellular pH (pHi) was measured by microfluorometry in
ducts loaded with pH-sensitive fluoroprobe BCECF.The duct cells were
acid-loaded with 20 mM NH4+,which was followed by a Na+-free
solution in both the bath and lumen.The rate of pHi recovery after readdition of Na+ to the luminal solution was calculated as a measure of
the activity of apical Na+-H+ exchange.The rate of pHi recovery (dpH/
dt) was 0.12 ± 0.01 pH unit/min (mean ± SD, n = 8) in wild type ducts,
which was completely inhibited by 100 µM HOE642,an inhibitor of
NHE. Forskolin reduced the apical NHE activity to 0.05 ± 0.01 pH unit/
min (n = 9, p < 0.01).The apical NHE activity in cystic fibrosis ducts was
0.20 ± 0.01 pH unit/min (n = 6),which was significantly (p < 0.01) higher
than that in wild type ducts and was accelerated to 0.66 ± 0.11 pH unit/
min (n = 6, p < 0.01) by application of forskolin. In mice pancreatic duct
cells,the activity of apical NHE was suppressed by functional CFTR and
it was stimulated by cAMP in the absence of functional CFTR.
Expressions of Na/K-ATPase αsubunit
isoforms in rat salivary glands
Characterization of Cl–-dependent
bicarbonate secretion in mouse ileum
Kurihara, Kinji1; Nakanishi, Nobuo2; Tomomura, Akito2;
Tonosaki, Keiichi1 (1Div. of Physiol., Dept. of Human Dev. &
Uchiyama, Hisakazu; Hayashi, Hisayoshi; Suzuki,
Yuichi (Lab. Physiol., Food and Nutritional Sci., Univ. Shizuoka,
Fost., Sch. of Dent., Meikai Univ. Sakado, Saitama, Japan; 2Div. of
Biochem, Dept. of Oral Biol. & Tissue Eng., Sch. of Dent., Meikai
Univ. Sakado, Saitama, Japan)
There are three isoforms in Na/K-ATPase α subunit, α1, α2 and α3. It is
known that α1 is expressed in wide range of cells/tissues and that α2 and
α3 localize abundantly in neuronal tissues. We examined the expression
of Na/K-ATPase α isoforms in salivary glands by RT-PCR. Alpha1 was
expressed in three major salivary glands. Alpha2 was expressed in
submandibular gland (SMG) and sublingual gland (SLG) and faintly
parotid gland (PG). Alpha3 was expressed in SLG alone. The DNA
sequence of α2 and α3 PCR products from salivary glands were
identical with the corresponding portion of brain. We examined mRNA
levels of these α isoforms by comparing the quantity of the PCR
products. Alpha1 levels in 3 salivary glands were same as that in brain.
Alpha2 levels in SMG, SLG and PG were determined to be 1/32, 1/16
and 1/256 of that in brain, respectively. Alpha3 level in SLG was at 1/8.
Since α3 is abundant in nerve tissues, we examined whether α3
expression is induced when pheochromocytoma PC12 cells are
differentiated into neuron-like cells by nerve growth factor (NGF). PC12
cells expressed α1 and α3, however α3 was not changed by the NGFtreatment of the cells. The α isoforms of HSY cells, a cell line of human
parotid gland adenocarcinoma, were also examined. HSY cells
expressed α1 alone. The expression pattern of Na/K-ATPase α isoforms
in cells/tissues seemed to be rather stable and it was not easily altered
with differentiation factors or carcinogenesis.
Bicarbonate secretion in mouse ileum has at least two components, one
being Cl–-dependent, and the other being Cl–-independent and activated
by cAMP. We have previously demonstrated and characterized Cl–/
HCO3– exchange activity in the mouse ileal villous cell by microfluorometric intracellular pH (pHi) measurements. The purpose of this study is
to determine if the Cl–/HCO3– exchanger that we characterized is responsible for the ileal Cl–-dependent HCO3– secretion. To this end isolated
mouse ileum was mounted in Ussing chamber and the alkaline secretion
rates (JOH(sm)) were determined by continuously titrating the luminal
buffer-free solution to pH 7.2 with 1mM H2SO4 using a pH stat device.
The serosal side was always bathed with HCO3–/CO2-buffered solution.
The Cl–-dependent JOH(sm) was inhibited by 30µM niflumic acid (by
25%) and by 100µM acetazolamide by (40%) both being added to the
mucosal side, whereas cAMP-induced JOH(sm) was not inhibited by these
compounds. Glibenclamide (100µM) added to the mucosal side had no
effect on either component. Br–, I–, NO3–, and SO42– added to the
mucosal side induced JOH(sm) of 80%, 50%, 50%, and 65%, respectively,
of the JOH(sm) induced by Cl–. These inhibitor sensitivity and ion
selectivity profiles of Cl–-dependent bicarbonate secretion generally
agreed with those of the Cl–/HCO3– exchanger we have previously
shown in the enterocytes by pHi measurement, suggesting that the Cl–/
HCO3– exchanger is responsible for Cl–-dependent bicarbonate
secretion. The molecular identity of the exchanger remains to be
J. Physiol. Sci., Vol. 56, Suppl., 2006
Heart & circulation
Activation of neuron-specific
cotransporter KCC2 by brtain-type creatine
Rapid electrical stimulation of H9c2 cells
induces apoptosis via both the death
receptor and mitochondrial pathways.
Inoue, Koichi1; Yamada, Junko2; Ueno, Shinya1;
Fukuda, Atsuo1,2 (1Dept. Physiol., Hamamatsu Univ. Sch. Med.,
Isomoto, Shojiro; Uchino, Tomoko; Ono, Katsushige
Hamamatsu, Japan; 2Dept. Biol. Info. Proc., Grad. Sch. Electric Sci.
& Tec., Shizuoka Univ. Hamamatsu, Japan)
GABA, a major inhibitory neurotransmitter in the adult CNS, is excitatory at early developmental stages due to the elevated intracellular Cl–
concentration ([Cl–]i). This functional switch is primarily attributable to
the K+-Cl– cotransporter KCC2, the expression of which is developmentally regulated in neurons. Previously we reported that KCC2 interacts
with brain-type creatine kinase (CKB). To elucidate the functional significance of this interaction, HEK293 cells were transfected with KCC2
and glycine receptor α2 subunit, and gramicidin-perforated patch-clamp
recordings were performed to measure the glycine reversal potential
(Egly), giving an estimate of [Cl–]i. KCC2-expressing cells displayed the
expected changes in Egly following alterations in the extracellular K+
concentration ([K+]o) or administration of an inhibitor of KCCs,
suggesting that the KCC2 function was being properly assessed. When
added into KCC2-expressing cells, dominant-negative CKB induced a
depolarizing shift in Egly and reduced the hyperpolarizing shift in Egly
seen in response to a lowering of [K+]o) compared to wild-type CKB.
Moreover, 2,4-dinitrofluorobenzene (DNFB), an inhibitor of CKs,
shifted Egly in the depolarizing direction. In primary cortical neurons
expressing CKB, the GABA reversal potential was also shifted in the
depolarizing direction by DNFB. Our findings suggest that in the cellular
microenvironment, CKB activates the KCC2 function.
(Dep. Cardiovasc. Sci. Oita Univ. Sch. Med, Oita, Japan)
Background: Recent studies have revealed that tachyarrhythmias cause
electrical and structural remodeling of cardiomyocytes, and that apoptosis contributes to myocardial remodeling in certain arrhythmias. We suspected that tachyarrhythmias lead to arrhythmogenic substrates through
the induction of apoptosis, and then may tend to initiate and perpetuate
themselves. In this study, we tested the hypothesis that rapid electrical
stimulation of cardiomyocytes in culture causes apoptosis. Methods
and Results: The cultured H9c2 cardiac cells were subjected to rapid
electrical stimulation for 72 hours at a pacing frequency of 5 Hz. To determine the degree of apoptosis, the percentage of hypodiploid cells, mitochondrial transmembrane potential (∆Ψm), and activities of caspases3, -8, and -9 were measured quantitatively. Compared with the cells in
the absence of stimulation, the cells subjected to rapid stimulation
showed apoptosis in terms of the appearance of hypodiploid cells
(4.7±0.5% vs 31.0±8.5%), loss of ∆Ψm (5.3±0.5% vs 19.5±2.7%), and
activation of all caspases tested (caspase-3, 0.050±0.015 vs
0.117±0.030; caspase-8, 0.038±0.004 vs 0.056±0.002; caspase-9,
0.029±0.003 vs 0.046±0.010) with statistically significant differences.
Conclusions: Rapid electrical stimulation of H9c2 cells induced
apoptosis via both the death receptor and mitochondrial pathways. The
present in vitro model may be useful to elucidate mechanisms of
tachycardia-induced myocardial remodeling via apoptosis, and to lead to
therapies for preventing the arrhythmogenic substrate development.
Molecular basis of pacemaker activity in
the rabbit sinoatrial node
Honjo, Haruo1; Tellez, James O.2; Dobrzynski, Halina3;
Yamamoto, Mitsuru1; Billeter, Rudorf2; Boyett, Mark R.3;
Kamiya, Kaichiro1; Kodama, Itsuo1 (1Res. Inst. Environ.
Med., Nagoya Univ., Nagoya, Japan; 2Univ. Leeds, Leeds, UK;
Manchester, Manchester, UK)
Heart & circulation
The ionic mechanisms underlying pacemaker activity have been extensively studied in the sinoatrial (SA) node, but little is known about the
molecular basis of ionic currents. The aim of this study was to evaluate
mRNA expression for ion channels, connexins and Ca2+-handling proteins using quantitative PCR and to visualize the distribution of these
transcripts using in situ hybridization in and around the rabbit SA node.
Quantitative PCR showed that there were significant differences in the
abundance of 60% of the 30 mRNAs studied in different tissues (SA
node center, SA node periphery and atrial muscle). Grouping analysis of
the PCR data identified two significantly different clusters: mRNAs
tended to increase (Cx45, Nav1.1, Cav1.3, HCN1, HCN4, Kv4.2, ERG,
KvLQT1, Kir2.2 and Kir3.1) and those tended to decrease (Cx43, Cx40,
RYR2, SERCA2a, Nav1.5, Cav1.2, Kv1.4, Kv4.3, KChIP2, Kv1.5,
minK, Kir2.1 and Kir6.2) from the atrial muscle to the center of the SA
node. The mRNA expression profiles of the center and the periphery of
the SA node were similar but there were significant differences for some
transcripts (e.g. Cav1.3, HCN1 and HCN4). In situ hybridization confirmed these regional differences in the mRNA expression pattern. This
study shows a complex variation in the expression of ion channel mRNAs from the atrial muscle to the SA node, which may be important in
the functional organization of the SA node as a physiological and dependable pacemaker of the heart.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Heart & circulation
Properties of
handling in
sarcoplasmic reticulum of saponin-treated
sarcolipin transgenic mouse myocardium
Morimoto, Satoshi1; Kawai, Makoto2; Kusakari,
Yoichiro1; O-Uchi, Jin1; Komukai, Kimiaki2; Hongo,
Kenichi2; Takeda, Toshihiro3; Asahi, Michio4; Otsu,
Kinya3; Kurihara, Satoshi1 (1Jikei Univ. Sch. Med. Tokyo,
Japan; 2Jikei Univ. Sch. Med. Tokyo, Japan; 3Osaka Univ. Grad.
Sch. Med. Osaka,Japan; 4Osaka Univ. Grad. Sch. Med.
We investigated whether the alteration of Ca2+ uptake rate in sarcoplasmic reticulum (SR) would influence the Ca2+ content and the leakage of
Ca2+ in SR. In this study, we used saponin-treated thin trabeculae obtained from the hearts which overexpresses sarcolipin (SLN). We reported that SLN reduced the activity of SR Ca2+-ATPase. The advantage of
saponin-treated preparation is that integrated structure around SR and
Ca2+ handling functions are well preserved. Thus, the functions of SR to
release and uptake Ca2+ can be measured under more physiological conditions compared to isolated vesicular SR. Ca2+ content was measured
by releasing all Ca2+ from SR by caffeine (50 mM) after loading Ca2+
(pCa 6.2) into SR in the presence of ATP (4 mM) for different periods.
Leaked Ca2+ was estimated by measuring the remaining Ca2+ in SR after
washing for various durations, following Ca2+ loading. Ca2+ uptake rate
was slower in SLN transgenic (SLN-TG) than that in non-transgenic
(SLN-NTG) myocardium. The maximal Ca2+ content and leakage of
Ca2+ in SLN-TG and SLN-NTG were almost identical. The results suggest that the modulation of Ca2+ uptake rate could not affect maximal
Ca2+ content and Ca2+ leakage measured at steady state. The modulation
of Ca2+ uptake rate influences Ca2+ handling in each heartbeat but does
not alter the maximal Ca2+ content and leakage at steady state.
Abnormal cardiac function is associated
with a mutation of the SERCA2a gene
Bai, Yunzhe; Minamisawa, Susumu; Mouri, Maya;
Ishikawa, Yoshihiro (Yokohama City University School of
Background: Recently, mutations of the phospholamban gene, a peculiar inhibitor of sarcoplasmic reticulum calcium ATPase (SERCA2a),
have been found in patients with familial cardiomyopathy. This result
suggests that mutations of SERCA2a gene also cause cardiomyopathy.
In our previous study, we found one patient with hypertrophic cardiomyopathy who harbored a missense mutation in the SERCA2a gene. The
mutation changed the amino acid sequence from valine (540) to alanine
in the cytoplasmic region close to the ATP binding site. Objective: We
examined whether the V540A mutation of SERCA2a could cause the abnormal cardiac function or not. Methods: Mouse SERCA2a cDNA was
cloned and the V540A mutation was inserted into it. Transgenic mice
overexpressing the V540A mutation of SERCA2a in the heart was generated. Cardiac phenotypes were observed in SERCA2a transgenic mice
(TG) using in vivo echocardiography and hemodynamic analyses. Results: By Western blot analysis, we found that the expression of
SERCA2a protein was increased by 1.7-fold in the ventricles of TG
when compared with those of non-transgenic mice. The weight of the
left ventricle was slightly, but significantly increased in TG. The maximal first derivate of left ventricular pressure in TG was significantly
lower by 16%, when compared with non-transgenic mice. Conclusion:
We concluded that the mutation of the SERCA2a gene resulted in the abnormal cardiac function in mice. Our results imply that the V540A mutation of the SERCA2a gene is a causal mutation in human
Phosphorylation status of contractile
proteins and functional characteristics in
myocardium of dilated cardiomyopathy of
Syrian hamster (TO-2 strain)
Saeki, Yasutake1 (1Dept. Physiol., Tsurumi Univ. Sch. Dent.
Med., Yokohama, Japan; 2Dept. Phys., Facult. Sci., Tokyo Univ. of
Science, Tokyo, Japan)
To understand the pathophysiology of hereditary cardiomyopathy, we
measured the phosphorylation status of contractile proteins, ventricular
light chain 2 (VLC2), troponin I (TnI), troponin T (TnT) and myosinbinding protein C (MyBP-C), and the Ca2+-dependence of tension and
ATPase activity in skinned trabeculae obtained from normal control
(F1B) and cardiomyopathic hamsters (TO-2). To change the phosphorylation status of contractile proteins, isolated trabeculae were exposed to
Tyrode's solution containing 30 mM BDM for 30 minutes before skinning. In the BDM-untreated preparations, all the contractile protein
phosphorylation levels were higher in F1B than in TO-2, while the Ca2+
sensitivities of tension and ATPase activity were substantially lower in
F1B than in TO-2. The BDM treatment did not alter the contractile protein phosphorylation levels as well as the Ca2+ sensitivities of tension
and ATPase activity in TO-2 preparations. However, the BDM treatment
decreased the contractile protein phosphorylation levels as well as increased the Ca2+ sensitivities of tension and ATPase activity in F1B
preparations to the levels similar to those in TO-2 preparations. These results suggest that the increase in Ca2+ sensitivities of tension and ATPase
activity in TO-2 hamster hearts results from the decreased basal level of
TnI and TnT phosphorylation, since the dephosphorylation of VLC2 and
MyBP-C has been reported to decrease Ca2+ sensitivities.
Developmental changes in nitric oxide
production during hypoxia and
reoxygenation in the rat striatum
Tsuchimochi, Hirotsugu; Nakamura, Shoji (Dept. Neurosci.
Yamaguchi Univ. Sch. Med. Ube, Yamaguchi, Japan)
In the brain nitric oxide (NO) is implicated in blood flow regulation, neurotransmitter release, learning and memory, neuronal plasticity, and neuronal degeneration and survival. To understand the role of NO in
physiological as well as pathological conditions, it is essential to observe
the dynamics of NO production itself. Using an NO-selective electrode
system that allows the real-time in vivo measurement of NO, we examined changes in NO production during hypoxia and reoxygenation in the
striatum of rats from late prenatal to adult stages. Under urethane anesthesia, an NO-selective electrode and laser Doppler flow probe was inserted into the striatum for measuring changes in NO concentration and
tissue blood flow, respectively. In adult rats, spontaneous rhythmic production of NO ranging from about 0.2 - 1.5 Hz was observed, which was
not related directly to cardiac or respiratory cycles. The rhythmicity in
NO production was reduced after systemic administration of the NMDA
channel blocker ketamine. In adult rats, brain hypoxia abolished the
rhythmicity, but caused a large amount of NO production in the striatum.
This overproduction of NO during hypoxia was also observed in the fetal
and neonatal rat brain, although the basal rhythmic NO production was
not detected. The immature rat brain may have less capacity to produce
NO than the matured rat brain. The appearance of the rhythmic NO production in the brain may indicate the maturity of the brain NO-producing
system. Further study is needed to clarify the origin of this rhythmic NO
production in the rat brain.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Heart & circulation
Cytochalasin D as the uncoupler of E-C
coupling for the optical monitoring of
action potentials in the isolated rat atrium
entry through the T-type Ca2+
channels causes apoptosis via the
mitochondrial pathway
Sakai, Tetsuro (Dept. Physiol., Univ. Ryukyus Sch. Med.,
Uchino, Tomoko; Isomoto, Shojiro; Ono, Katsushige
Okinawa, Japan.)
(Dep. of Cardiovasc. Sci., Oita Univ. Sch. Med., Japan)
Multiple-site optical recording methods, using a fast voltage-sensitive
merocyanine-rhodanine dye (NK2761) and a multi-element (16 X 16)
photodiode array, were employed to simultaneously monitor action potentials from many sites in the isolated rat atrium preparation. Cytochalasin D was used to reduce contraction-related optical artefacts by
suppressing muscle contraction without affecting electrical action potential. The suppression effect of this chemical (40µM) on the contractionrelated optical artefacts is time-dependent for the first 30 min. and
become steady state within 40 - 60 min. after the application to the bath.
The suppression of the artefact of this chemical is generally stronger than
that of 2,3-butanedione monoxime (BDM: 20 mM). Using this chemical,
we successively evoked tachycardia-like excitation (TE) in the isolated
rat right atrium preparation and mapped optically the excitation spread
pattern during TE.
Background: It is postulated that T-type Ca2+ channels play important
roles not only in physiological condition of various organs and tissues,
but also during the progression of various diseases. In this study, we tested the hypothesis that Ca2+ entry through the T-type Ca2+ channels causes apoptosis using recombinant CaV3.2 (α1H) T-type Ca2+ channels
expressed in HEK293 (HEK-α1H) cells. Methods and Results:
Cultured HEK293 cells and HEK-α1H cells were incubated for 12 hours
in serum-free medium containing different concentrations of Ca2+ (1.87.2 mM). To determine the degree of apoptosis, mitochondrial
transmembrane potential (δΨm) and activities of caspase-3, -8, -9 were
measured quantitatively. Intracellular free Ca2+ measured by flow
cytometry using Fluo-3 was elevated depending on Ca2+ concentration
in the medium ([Ca2+]o) in HEK-α1H cells but not in HEK293 cells. In
HEK-α1H cells, apoptosis in terms of loss of δΨm and activation of
caspase-3 and -9 was observed at [Ca2+]o of 5.4 mM or more, while
caspase-8 was not activated. In contrast, apoptosis was not induced at
any [Ca2+]o in HEK293 cells. Conclusion: We demonstrated that Ca2+
entry through the T-type Ca2+ channels causes apoptosis via the
mitochondrial pathway. Thus, T-type Ca2+ channels may be therapeutic
targets for certain pathophysiological conditions related to apoptosis.
RhoB-geranylgeranylation is involved in
Na+/Ca2+ exchanger mRNA increase by
lisophosphatidylcholine in H9c2 cells.
Maeda, Sachiko; Matsuoka, Isao; Kimura, Junko (Dept.
Pharmacol., Sch. Med., Fukushima Med. Univ., Fukushima, Japan)
Cardiac Na+/Ca2+ exchanger1 (NCX1) expression levels change under
various pathophysiological conditions, e.g. heart failure. However, its
mechanism is unknown. We previously showed that fluvastatin (Flv), an
HMG-CoA reductase (HMGR) inhibitor, decreased NCX1 mRNA and
protein expression via inhibiting a small G-protein, RhoB in H9c2 cardiomyoblasts. Flv-induced down-regulation of NCX1 mRNA was reversed by mevalonate, farnesyl pyrophosphate (FPP) or geranylgeranyl
pyrophosphate (GGPP), suggesting an involvement of isoprenylation of
RhoB. Conversely, we also found that lisophosphatidylcholine (LPC) increased NCX1 mRNA and protein by activating RhoB. RhoB requires
isoprenylation for its activation by either GGPP or FPP. Here, we investigated which isoprenoid is involved in NCX1 increasing effect of LPC.
Treatment of H9c2 cells with Flv for 24 hours decreased NCX1 mRNA
to about 60% of control. Addition of GGPP or FPP restored NCX1 mRNA, which had been decreased by Flv, to a control level within 24 hours.
No significant difference was observed between GGPP and FPP. When
LPC was applied with Flv, NCX1 mRNA remained decreased. However,
when LPC and GGPP were applied simultaneously, NCX1 mRNA was
increased to a level significantly higher than the control. Unlike GGPP,
FPP did not induce this increase. These results suggest that an isoprenoid
involved in the effect of LPC increasing NCX1 mRNA is GGPP but not
A human ventricular action potential model
for the analysis of arrhythmias elicited by
"intracellular Calcium handling
Hirano, Yuji (Dep't of Cardiovasc. Dis., MRI, Tokyo Medical and
Dental University)
Dysfunctions in the gating of ryanodine-receptors (RyR) and intracellular Ca2+ handling play critical roles to evoke lethal arrhythmias not only
in inherited arrhythmgenic syndromes such as CPVT, but also in more
common cases including heart failure. To develop a human ventricular
action model which allows analyses of the effects of altered RyR properties on arrhythmogenesis, we incorporated a calcium release system
with 4-state Markovian RyR gating (Stern, J.Gen.Physiol.1999) into a
simplified human action potential model (TenTusscher, Am.J.Physiol.2004). Introduction of Ca concentration-dependence on RyR opening
rate was a first step to model Ca-induced Ca-release. To obtain reasonable RyR gating in the control, it was necessary to add a new Ca compartment (cleft), where Ca concentration increased and decayed much
faster than cytosolic Ca transient. Under these settings, augmented sensitivity of RyR on cleft Ca easily evoked spontaneous oscillatory Ca releases during the decay phase of Ca transient. Induction of DADs
leading to triggered activity, however, could not be reproduced straightforwardly in spite of further inclusion of SR Ca-load dependence of RyR
gating. Generation of triggered action potential after repolarization required additional manipulation of sarcolemmal current systems including a reduction of IK1, a situation found in patients with terminal heart
failure. Our model provides basis to elucidate mechanisms of Ca-mediated arrhythmias, and further to find out RyR-targeted anti-arrhythmic
J. Physiol. Sci., Vol. 56, Suppl., 2006
Heart & circulation
The relationship betweeen action potential
duration and the rate of Ca2+
replenishment to the junctional
sarcoplasmic reticulum in heart muscle of
hypertensive rat
Tanaka, Midori; Tameyasu, Tsukasa
(Dept.Physiol,St.Marianna Univ.Sch.Med.,Kawasaki,Japan)
To characterize Ca2+handling by the junctional sarcoplasmic reticulum
(JSR) in heart muscle of hypertensive rat(SHR), we examined the time
course of short-term mechanical restitution(STMR) after varying magnitude of twitch contraction with the papillary muscle of adult(20 weeksold)SHR and age-matched control(WKY) and action potential duration
at various stimulus frequencies. The slope of the STMR in WKY was independent of the magnitude of the preceding twitch and similar to that of
SHR unless the preceding twitch was not so large as the rested state
twitch. The slope decreased after the rested state twitch in SHR. The
functions G(t) and H(t), representing the time courses of the JSR Ca2+ replenishment and release, respectively, were derived graphically from a
family of the mechanical restitution curves for the both strains. The action potential duration as measured at its half width was significantly
greater in SHR than WKY(p <0.005). Both a time to peak tension and a
half relaxation time were significantly longer in SHR than WKY(p
<0.0001). The result suggests slower Ca2+ replenishment to the JSR in
SHR than WKY irrespective of the longer action potential in the former
than the latter.
Effects of redox potential on acetaldehydeinduced activation of the type 2 ryanodine
Oba, Toshiharu1; Maeno, Yoshitaka2; Murayama,
Takashi3 (1Dept. Reg. Cell Physiol., Nagoya City Univ. Grad. Sch.
Med. Sci., Nagoya, Japan; 2Dept. Forensic Med. Sci., Nagoya City
Univ. Grad. Sch. Med. Sci., Nagoya, Japan; 3Dept. Pharmacol.,
Juntendo Univ. Sch. Med., Tokyo Japan)
Acetaldehyde (AcA), an oxidized product of alcohol, may contribute to
alcohol-induced cardiac muscle dysfunction. Recently, we have demonstrated that even at 1-3 µM, AcA activates the ryanodine receptor type 2
(RyR2) channel and promotes Ca2+ release from the cardiac muscle SR
(Oba et al., JJP 55:S90, 2005). AcA produces ROS and oxidizes GSH to
GSSG to lead to oxidative stress in tissues. We studied whether
cytoplasmic redox potential (RP) affects AcA-induced activation of the
RyR2 channel and mediates cardiac dysfunction. In experiments under
RP control using glutathione buffers, fixation of cytoplasmic (cis) RP at
the oxidative state activated the channel incorporated into bilayers,
whereas definition of luminal (trans) potential did not. Exposure of the
RyR2 to AcA without defining RP stimulated markedly the channel in a
dose-dependent manner (1-100 µM). When RP in cis/trans sides of the
channel was fixed at -220/-180 mV, AcA at concentrations less than 3
µM caused no longer the channel activation, but higher concentrations
increased 2 fold the open probability. In condition under cis/trans
potential at -250/-180mV, AcA failed to activate the RyR2 channel.
These results indicate that the activation of RyR2 channels by AcA
exposure is elicited even at reductive states and suggest that cytoplasmic
RP may have a protective effect against pathophysiological changes by
AcA in cardiac myocytes.
A Simulation Study of Ca2+ Regulation of
Cardiac Mitochondria
Saito, Ryuta1,2; Sarai, Nobuaki1,3; Matsuoka, Satoshi1,4;
Noma, Akinori1,4 (1Kyoto Univ. Group in Leading Project for
Two different subtypes of α1-adrenoceptor
modulate L-type Ca2+ current via different
intracellular signal transduction pathways
in rat ventricular myocytes
Biosimulation, Kyoto, Japan; 2Mitsubishi Pharma Co., Yokohama,
Japan; 3Dept. Nano-Medicine Merger Education Unit, Grad. Sch.
Med., Kyoto Univ., Kyoto, Japan; 4Dep. Physiol. And Biophys.,
Grad. Sch. Med., Kyoto Univ, Kyoto, Japan)
O-Uchi, Jin1; Komukai, Kimiaki2; Kusakari, Yoichiro1;
Morimoto, Satoshi1; Kawai, Makoto2; Hongo, Kenichi2;
Sasaki, Hiroyuki3; Kurihara, satoshi1 (1Jikei Univ., Tokyo,
To clarify complex interactions between cardiac excitation-contraction
(E-C) coupling and energy metabolism, a cardiac mitochondria model is
developed, which consists of oxidative phosphorylation, TCA cycle,
pyruvate metabolism, fatty acid β oxidation, malate shuttle and Ca2+
dynamics. Using this model, we studied regulation of mitochondrial
function by Ca2+, and investigated contribution of each functional
element of oxidative phosphorylation to mitochondrial oxygen
consumption (mVO2), mitochondrial membrane potential and NADH.
Calculation of the membrane potential was improved by integrating
charge flux across the membrane via proton pumps and transporters. The
model simulation revealed that the activation of F1F0-ATPase, adenine
nucleotide translocator enhanced mVO2 by about 60%, This result is
supported by experimental data demonstrating a large increase in mVO2
of state 3 mitochondria by Ca2+ (Territo et al. Am J Physiol. 278:C42335, 2000). The expanded computer model, which combines the
mitochondria model with an E-C coupling model of cardiomyocyte
(Kyoto model), suggested that Ca2+-dependent activation of both
oxidative phosphorylation and Ca2+-dependent dehydrogenases plays
pivotal roles in regulating cardiac mitochondrial function by stabilizing
metabolite concentrations during an increase in workload induced by
changing beating frequency.
Purpose: We have recently reported that the effects of α1-adrenoceptor
stimulation (α1ARS) on L-type Ca2+ current (ICa) can be classified in two
opposite effects (negative and positive effects) and the positive effect is
protein kinase C (PKC)-dependent. We postulate that these two effects
simultaneously occur through different subtypes of α1-adrenoceptor and
different intracellular signal transduction pathways. In this study, we
investigated the effects of α1AARS and α1BARS on ICa. Methods:
Perforated patch-clamp was used for recording of ICa from isolated adult
rat ventricular myocytes. Holding potential was set at -40 mV and
depolarization pulse to 0 mV was applied every 10 sec. Results:
Biphasic change in ICa (a transient decrease followed by a sustained
increase) was induced by a non-selective α1-adrenoceptor agonist,
phenylephrine (Phe). However, a selective α1A agonist, A61603 showed
only positive effect on ICa, and the application of Phe with a selective α1A
antagonist, WB4101, caused only sustained negative effect. After
pertussis toxin treatment, a transient decrease in ICa induced by Phe
disappeared and only sustained increase was observed. Conclusion
α1AARS showed positive effect on ICa, which was PKC-dependent. On
the other hand, α1BARS showed negative effect mediated through Gi/o
protein. Thus, α1AARS and α1BARS produce opposite effects on ICa via
different intracellular signal transduction pathways.
Japan; 2Jikei Univ., Tokyo, Japan; 3Jikei Univ., Tokyo, Japan)
J. Physiol. Sci., Vol. 56, Suppl., 2006
Heart & circulation
Formation of functional multiheteromeric
channels by KCNQ1, KCNE1 and KCNE2
Toyoda, Futoshi1; Ueyama, Hisao2; Ding, Wei-Guang1;
Zankov, Dimitar1; Matsuura, Hiroshi1 (1Dept. Physiol. Shiga
Univ. Med. Sci., Otsu, Japan; 2Dept. Biochem. Shiga Univ. Med. Sci.,
Otsu, Japan)
The KCNE proteins (KCNE1-5) are single transmembrane peptides that
function as ancillary β-subunits of voltage-gated K+ channels.
Functional coupling of KCNE1 (E1) with KCNQ1 (Q1) to form cardiac
IKs channel is well documented. However, Q1 is also found to have an
affinity for all other members of KCNEs. The present study tested the
possibility for the formation of functional channels consisting of Q1, E1
and KCNE2 (E2). We constructed a plasmid encoding a fusion protein
in which the N terminus of Q1 was fused to the C terminus of either E1
or E2, and characterized the function of heteromeric channels by
measuring whole-cell currents from COS7 cells transiently transfected
with the plasmids. It was comfirmed that E1-Q1 fusion channels
produced voltage- and time-dependent currents, similar to those
obtained by coexpression of E1 together with Q1, while E2-Q1 fusion
channels evoked constitutively active time-independent currents, again
similar to those induced by coexpression of E2 and Q1. Coexpression of
the E1-Q1 with E2 results in time-dependent currents which had a faster
deactivation time course and a more depolarized half-activation voltage,
compared to the E1-Q1 current. On the other hand, coexpression of E2Q1 and E1 exclusively produced voltage- and time-dependent current
without any constitutively active component, providing a functional
evidence to show that all E2-Q1 coassembles with E1. These results
suggest that E1 and E2 can be couple to a Q1 channel molecule forming
complexes with unique current properties.
Regulation of muscarinic K+ channel by
extracellular lysophosphotidylcholine via
G2A membrane receptor
Ding, Wei-Guang1; Toyoda, Futoshi1; Zankov, Dimitar1;
Wu, Jie1; Ueyama, Hisao2; matsuura, Hiroshi1 (1Dept.
Physiol., Shiga Univ. Med. Sci., Otsu, Japan; 2Dept. Biochem. Shiga
Univ. Med. Sci., Otsu, Japan)
In recent years, some of biological actions of lysophosphotidylcholine
(LPC) have been found to be mediated through a class of G protein-coupled receptors, termed G2A. The present study was designed to examine
the possible regulation of the muscarinic K+ current (IK,ACh) by LPC and
its associated signaling pathways in guinea-pig atrial myocytes, using
whole-cell patch-clamp method. Bath application of LPC (2 µM)
reversibly and almost completely (93.3%; n = 9) inhibited IK,ACh
preactivated by acetylcholine (ACh). On the other hand, LPC almost
irreversibly inhibited IK,ACh preactivated by intracellular loading of nonhydrolyzable GTP analogue GTPγS (84.2%; n = 6), suggesting an
involvement of G protein. The inhibitory action of LPC was partially,
but significantly, attenuated by pretreating myocytes with an anti-G2A
antibody and phospholipase C (PLC) blockers (compound 48/80 and
neomycin). Furthermore, the inhibitory effect of LPC was still
significantly reduced by exogenously adding phosphatidylinositol 4,5bisphosphate (PIP2, 50 µM) to the cell inside, and became nearly
irreversible when atrial myocytes were continuously exposed to
wortmannin (10 µM), which suppresses the resynthesis of PIP2. As
expected, LPC greatly reversed the shortening of action potential
duration evoked by ACh. Based on these results, we conclude that LPC
markedly inhibits IK,ACh through a mechanism involving an activation of
G protein-coupled G2A receptor causing a depletion of membrane PIP2
via PLC activation.
Intracellular and extracellular magnesium
concentration-dependent alteration of
electrical excitability in cardiac myocytes
Quantitative analysis of cardiac cell
volume regulation by experiments and
Takeda, Yukiyo; Shimaoka, Toru; Uchino, Tomoko;
Isomoto, Shojiro; Ono, Katsushige (Dept. Cardiovasc. Sci.,
Takeuchi, Ayako1,2; Tatsumi, Shuji2; Sarai, Nobuaki1,3;
Terashima, Keisuke1,4; Matsuoka, Satoshi1,2; Noma,
Akinori1,2 (1Kyoto Univ. Group in Leading Project for
Oita Univ. Sch. Med., Oita, Japan)
[Background] Regulation of intracellular and extracellular free magnesium (Mg2+) concentration is important to control the excitability in cardiac myocytes. [Objective] To assess the role of intracellular and
extracellular Mg2+ imbalance in cardiac myocytes associated with membrane potentials and ionic currents derangement. [Methods and Results]
Serum and intracellular Mg2+ concentrations were measured from normal (control) and the Mg2+ -deficient food feeded rats. The serum
[Mg2+]e and intracellular Mg2+ concentrations [Mg2+]i were significantly decreased from 6.9 ± 0.3 mg/dl and 2.3 ± 0.2 mg/dl to 4.4 ± 0.2 mg/dl
and 0.7 ± 0.1 mg/dl on Mg2+ -deficient diet on day 28, respectively. We
obtained ECG signals by a radio transmitter from control and the Mg2+deficient rats. Low [Mg2+]e significantly prolonged RR duration
(control, 181 ± 17 ms versus low [Mg2+]e, 158 ± 18 ms, p < 0.05) in
accordance with various types of cardiac arrhythmias such as
supraventricular extrasystole, atrioventricular blocks and so on. Action
potentials (AP) from rat ventricle myocytes were recorded by using
intracellular microelectrode technique. AP durations (APD90) in
myocytes obtained from Mg2+ -deficient rats were significantly
prolonged (control, 101.2 ± 3.2 ms versus low-Mg2+, 314.0 ± 11.8 ms, p
< 0.01). [Conclusion] Decreases in intracellular and extracellular Mg2+
concentrations cooperatively contribute toward arrhythmogenecity
presumably affecting intracellular Ca2+ concentrations.
Biosimulation, Kyoto, Japan; 2Dept. Physiol. and Biophys., Grad.
Sch. Med., Kyoto Univ., Kyoto, Japan; 3Dept. Nano-Medicine
Merger Education Unit, Grad. Sch. Med., Kyoto Univ., Kyoto,
Japan; 4Pharmacokinet. Res. Lab., Dainippon Sumitomo Pharma
Co., Ltd., Osaka, Japan)
In general, animal cells tend to swell if Na/K pump is blocked. However,
the volume of guinea-pig ventricular myocytes remained almost constant during 3 hours under 40 µM ouabain. The Na/K pump block
simulation using the Kyoto model showed that the cell swelling is
retarded in parallel to a decrease in the background membrane
conductances for Cl– and Na+, suggesting very low conductances for
these ions in actual ventricular cells. In addition, it is suggested by the
simulation that the active Ca2+ extrusion by sarcomlemmal Ca2+ pump
(PMCA) has an important role in the delayed cell swelling. The
transmembrane [Ca2+] gradient is partially maintained by PMCA, and
this makes the Na/Ca exchanger extrudes Na+ in reverse mode,
compensating for Na/K pump. In the experiment where the Cl– leak was
increased beyond these compensatory mechanisms by applying 5 µM
isopreterenol, cell swelling could be induced, but only after a delay of
about 55 min after ouabain application. The simulation revealed a
gradual membrane depolarization during the delay, and finally it reached
a voltage range of continuous opening of a significant fraction of L-type
Ca2+ channels (window current). A rapid accumulation of Ca2+
subsequently activates Ca2+-dependent Na+ conductance, which finally
initiates the rapid cell swelling.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Heart & circulation
Termination of Spiral-wave Reentry by
Regional Cooling in 2-dimensional Rabbit
Ventricular Myocardium
Activation of cardiac chloride current and
the electrocardiographic properties in
transverse aortic-banding mice
Yamazaki, Masatoshi1; Honjo, Haruo1; Ashihara,
Takashi2; Harada, Masahide1; Sakuma, Ichiro3;
Nakazawa, Kazuo4; Horie, Minoru2; Kamiya, Kaichiro1;
Kodama, Itsuo1 (1Res. Instit. Environ. Med., Nagoya Univ.,
Facult. Med., Saga Univ. Saga, Saga, Japan)
Nagoya, Japan; 2Shiga Univ. Med. Sci., Otsu, Japan; 3Grad. Sch.
Eng. Frontier Sci., Univ. Tokyo, Tokyo, Japan; 4Nat. Cardiovasc.
Center, Suita, Japan)
Spiral-wave reentry is a principal mechanism of life-threatening ventricular tachyarrhythmias. We investigated the effects of regional cooling
(RC) on the dynamics of spiral-waves. Methods and Results: Optical action potential signals were recorded from 2-dimensional ventricular myocardium (1 mm thick) of Langendorff-perfused rabbit hearts.
Ventricular tachycardias (VTs, >2 min) were induced by burst stimulation. RC (by 5-7°C) was applied to the left ventricular free wall using a
transparent cooling device (diameter, 10 mm). RC caused a marked
prolongation of action potential (by 25±4%) and a slight reduction of
conduction velocity (by 16±5%) in the affected region under constant
pacing (2.5Hz, n=10). Spiral-wave dynamics during VTs were
transformed by RC from stationary to chaotic meandering types. During
RC, the rotors moved irregularly around the cooled region, and often
collided with the anatomical boundary (atrioventricular groove)
resulting in spontaneous termination. The incidence of termination of
VTs within 15 s was increased from 2/18 (11%) without RC to 14/18
(78%) with RC. Conclusion: RC facilitates spontaneous termination of
spiral-wave reentry through the unpinning of rotors.
Yamamoto, Shintaro; Ehara, Tsuguhisa (Dep. Physiol.,
Cardiac hypertrophy is an adaptive response to chronic cardiac overload,
and causes an abnormal electrical activity leading to arrhythmia. It is important to understand the electrophysiological mechanisms underlying
the development of this condition. Therefore we analyzed the electrocardiogram (ECG) in hypertrophic heart of mice persistently pressure-overloaded by transverse aortic constriction (TAC). In pre-operated or shamoperated (SO) mice, the first upstroke in QRS-complex (designed to as
"a") is followed by a fast second upstroke ("b") and sometimes a slower
downstroke ("c"). In TAC mice, "b" is often followed by another slow
upstroke ("b'") before "c", and a prolongation of Qc interval was revealed. These results suggest that ECG is sensitive enough to confirm
development of cardiac hypertrophy in TAC mice. In addition, the
whole-cell patch clamp recordings showed a prolongation of the action
potential duration (APD) and a basal activation of chloride (Cl–) current
in single myocytes freshly isolated from left ventricle of TAC mice. The
Cl– current may reflect a persistent activation of volume-regulated Cl–
current (ICl,vol) in isotonic condition, because of the time-dependent inactivation at higher positive potentials, the outwardly rectifier currentvoltage relationships and the inhibitory effect of 4,4'-Diisothiocyanatostilbene-2,2'-disulphonic acid. The basal activation of ICl,vol may be responsible for prolongation of APD and Qc interval in TAC mice.
Effects of Pacemaker Currents on Creation
and Modulation of Pacemaker Activity in
Human Ventricular Myocytes: a theoretical
study with applications to engineering of
biological pacemaker
Role of ICFTR in positive inotropic action of
isoproterenol in cardiac myocyte: a
simulation study
Kurata, Yasutaka1; Matsuda, Hiroyuki2; Hisatome,
Ichiro3; Shibamoto, Toshishige1 (1Kanazawa Medical
Univ. Group in Leading Project for Biosimulation, Kyoto, Japan;
2Dept. Physiol. and Biophys., Grad. Sch. Med., Kyoto Univ., Kyoto,
Japan; 3Discovery Res. Lab., Shionogi & Co., Ltd., Osaka, Japan;
4Pharmacokinet. Res. Lab., Dainippon Sumitomo Pharma Co., Ltd.,
Osaka, Japan)
University, Ishikawa, Japan; 2Kyoto Univ. Group in Leading Project
for Biosimulation, Kyoto, Japan; 3Div. Regener. Med. Therap.,
Tottori Univ. Grad. Sch. Med. Sci., Yonago, Japan)
The cardiac biological pacemaker (BP) has been created by suppression
of the inward-rectifier K+ current (IK1) or overexpression of the hyperpolarization-activated current (Ih) in ventricular (Purkinje) or atrial myocytes, suggesting possible development of the functional BP as a
therapeutic alternative to the electronic pacemaker. In this study, we investigated the effects of incorporating regulatory inward currents (pacemaker currents) such as Ih, sustained inward current (Ist), and low
voltage-activated L-type Ca2+ channel current (ICa,LD) on 1) creation of
BP cells, 2) robustness of BP activity to electrotonic loads of adjacent
non-pacemaker cells, and 3) BP cell ability to drive the surrounding nonpacemaker cells. Bifurcation structures of single BP cell and coupledcell models for human ventricular myocytes (HVMs) were explored during changes in conductance of IK1, the regulatory inward currents, and
gap junction. Our findings suggest that 1) incorporating Ih and Ist (or Ih
and ICa,LD) facilitates BP generation during IK1 suppression, although it
does not lead to BP oscillation without IK1 inhibition, and that 2) incorporating Ist or ICa,LD significantly improve the structural stability of BP
cells to electrotonic loads and BP cell ability to drive adjacent HVMs.
Kuzumoto, Masanori1,3; Nakai, Hiroyuki1,3; Terashima,
Keisuke1,4; Matuoka, Satoshi1,2; Noma, Akinori1,2 (1Kyoto
β-adrenergic receptor agonists, such as isoproterenol, activate protein
kinase A (PKA) and induce positive inotropy in the ventricular myocyte.
Major mechanisms underlying the positive inotropy are enhanced
activities of the sarcolemmal L-type Ca2+ channel (ICaL) and the
sarcoplasmic reticulum Ca2+-ATPase via phosphorylation of
phospholamban. However, role of activation of cystic fibrosis
transmembrane conductance regulator channel (ICFTR) in the inotropic
effect has not been well understood.We have constructed a computer
model of β-adrenergic signaling network including PKA activation of
ICaL, phospholamban and ICFTR based on Saucerman et al., (2003, 2004)
and implemented it into Kyoto model (Matsuoka et al., 2004). When 1
µM isoproterenol was applied, the amplitudes of ICaL, Ca2+ transient, and
cell shortening were increased, and the action potential duration was
prolonged. Elimination of the activation of ICFTR resulted in a slight rise
of the plateau phase of action potential and a decrease in the amplitudes
of ICaL due to the decrease in driving force. Ca2+ transient was attenuated
by about 20%. The results demonstrate that the activation of ICFTR
induces compensatory increase in ICaL and facilitates indirectly the
positive inotropic action of isoproterenol.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Heart & circulation
Cardiac energetics calculated using a
comprehensive cardiac myocyte model.
A simple system for video-based
measurement of heart cell contraction
Takahata, Takayuki1,4; Kouchi, Yasuhiro1,4; Sarai,
Nobuaki1,3; Matsuoka, Satoshi1,2; Noma, Akinori1,2
Shioya, Takao; Ehara, Tsuguhisa (Dept. Physiology, Fac.
(1Kyoto Univ. Group in Leading Project for Biosimulation, Kyoto,
Japan; 2Dept. Physiol. And Biophys., Grad. Sch. Med., Kyoto Univ.,
Kyoto, Japan; 3Dept. Nano-Medicine Merger Education Unit, Grad.
Sch. Med., Kyoto Univ., Kyoto Japan; 4Central Research
Laboratories, Sysmex corporation, Kobe, Japan)
The analytical method of cardiac energetics, developed by H.Suga, has
been extensively used to evaluate the relationship between mechanical
contractility and energy consumption by the left ventricle both in experimental and in clinical studies. The end-systolic maximum elastance
(Emax) is measured to indicate contractility of the heart, and the mechanical energy is obtained by the pressure-volume area of a cardiac
contraction cycle (PVA), which well correlates with the oxygen consumption. Here, we aim at interpreting the cardiac energetics in terms of
the cellular mechanisms. We constructed a heart model of spherical
shape to apply the Laplace law in converting the wall tension to pressure.
The wall tension was calculated by the contraction model of Negroni and
Lascano (1996), which is driven by the Ca transient of the comprehensive cardiac myocyte model, Kyoto Model. The 'Laplace heart' was connected with preload and afterload, and the pressure-volume loop was
constructed. Our results of simulation under an aerobic condition show
that the Negroni & Lascano model well reconstructs the linear PVA - oxygen consumption curve. The PVA-independent oxygen consumption
could be well simulated by the ATP hydrolysis via the Na/K pump and
the Ca pump on the sarcoplasmic reticulum.
Med., Saga Univ, Saga, Japan)
Excitation and contraction are the two main functions of heart cells.
Therefore for studies of physiology and pathophysiology of heart cells,
measurement of cell contraction is as important as that of membrane current and action potentials. In addition, measurement of cell contraction
provides a simple way for assesing intracellular calcium transients.
However, the measurement of single-cell contraction has not been a popular experimental technique, because it requires very specialized instruments, and because the measurement of cell contraction from patchclamped cells was not quite trivial. In this report, we describe a simple
system for video-based measurement of heart cell contraction, which can
be used with patch-clamp instruments without difficulty. In this system,
motion of the cell is recorded with a CCD video camera attached to the
microscope, and the video images are digitized and analysed on a Windows-based PC using ScionImage public domain software. With the aid
of software image manipulation using Sobel filter, cell shortening could
easily be measured at 1/60 s time-resolution, from patch-clamped single
heart cells. Values of the measurements are stored in text-based data
files, which can easily be imported into pClamp or spreadsheets for further analysis. Contraction properties of patch-clamped mouse heart cells
is also provided as an example of cell analysis using this system.
Contractiel properties of cardiac muscle of
non-insulin-dependent daibetic mice:
Effect of insulin and free fatty acid.
Tameyasu, Tsukasa; Ido, Chiaki (St. Marianna Univ. Sch.
Cardiovascular effects of a Chinese herbal
medicine, Seabuckthorn, in the
spontaneously hypertensive stroke-prone
rat, SHRSP
Med., Kawasaki, Japan)
Koyama, Tomiyasu1; Taka, Akira2 (1Hokkaido University
Though non-insulin-dependent (type 2) diabetes which accounts for over
90% of all diabetic patients leads to congestive heart failure, contractile
property of cardiac muscle in type 2 diabetes has not been well characterized. We studied the contractile property of left ventricular papillary
muscle of model mouse of the type 2 diabetes, db/db mouse, and effect
of insulin and palmitate on it at 20 °C. There was no difference in the
time course of twitch between db/db mouse and the control except its
magnitude; the rested-state twitch force (Fmax) in the Krebs solution (10
mM glucose) was 21.2 mg/mm2 (n=6) in the db/bd mouse and 190.3 mg/
mm2 (n=6) in the control. Application of either insulin (80 i.u./l) or
palmitate (0.4 mM) increased Fmax in both the db/db mouse and the
control. Steady-state force decreased with increasing stimulus
frequency. Insulin and palmitate increased the steady-state force at a
frequency > 1 Hz in the bd/bd mouse and the control except that
palmitate decreased it at a frequency > 1 Hz in the db/db mouse. The rate
of short-term mechanical restitution (SMR) decreased with increasing
magnitude of the preceding twitch. Palmitate delayed the SMR, while
insulin had variable effect on it. There was no apparent difference in the
characteristics of the SMR between the db/db mouse and the control. We
characterized Ca2+ release/content in the cardiac sarcoplasmic reticulum
from the SMR with or without insulin and palmitate in the db/db mouse
and the control according to the rationale described previously
(Tameyasu et al, 2004).
(retired), Sapporo, Japan; 2Institute for Eco Technology, Sapporo,
Cardiovascular effects of sea buckthorn (Hippophae rhamnoides L.)
called simply "Saji" in China were studied in SHRSP. Saji is a spiky
shrub with yellow or orange berries.It has nodule rooting capability of
fixing nitrogen from the air. Chemical analyses of Saji berries revealed
that it contained vitamins C, B1, B2, E, F, K, P, provitamin A. The average protein content was 22% of the crude material, including polyphenols having high SOD activity. Further, it contained serotonin, cytosterol
and selenium as well as zinc. In the in vivo study 5 SHRSPs were fed
with rat chow containing powdered Saji fruits at the dose of 30mg/400g
for 60 days. As the control group 4 SHRSPs were fed with normal rat
chow. Then the arterial blood pressure and heart rate were measured on
the tail. The venous blood weas sampled under anesthesia and subjected
to biochemical analyses, and the heart was removed and frozen quickly
in liquid nitrogen for histochemical analyses. The left ventricle was
sliced horizontally in a crosssectional plane and studied by the double
staining methods as described previously for the analyses of the arteriolar, intermediate and venular capillary portions. The arterial blood pressure and heart rate decreased. HbA1c, total plasma cholesterol and
triglyceride decreased significantly. The arteriolar capillary portion
tended to decrease, indicating the release from the stress caused by the
hypertension.Thus the herbal medicine, Saji, is beneficial for cardiovascular and metabolic functions.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Heart & circulation
Propofol increases pulmonary vascular
resistance during acutely elevated
vasomotor tone
Edanaga, Mitsutaka1; Nakayama, Masayasu1; Kanaya,
Noriaki1; Hukao, Mitsuhiro2; Yamada, Yoichi2;
Tsutsuura, Masaaki2; Kobayashi, Takeshi2; Mizuno,
Satoshi2; Shiratori, Kaori2; Touse, Noritsugu2; Namiki,
Akiyoshi1 (1Department of Anesthesiology, Sapporo Medical
University School of Medicine, Sapporo, Japan; 2Department of
Cellular Physiology and Signal Transduction, Sapporo Medical
University School of Medicine, Sapporo, Japan)
We tested whether pulmonary vascular effects of propofol depend on the
level of vasomotor tone. We used the isolated perfused normal rat lung
and monocrotaline (MCT)-induced pulmonary hypertension (PH) rat
model. Rats were subcutaneously given MCT (PH group). On day 21 after injection, the rats were anesthetized, and the lungs were isolated and
perfused. Pulmonary perfusion pressure (PPP) was measured. Changes
in PPP during administration of propofol were compared between both
groups in the absence or presence of phenylephrine (PE). Propofol had
no effect on PPP in the absence of PE. Propofol caused dose-dependent
pulmonary vasoconstriction in the presence of PE. To study effects of
propofol on endothelial function or myofilament Ca2+ sensitivity, N-nitro-L-arginine methyl ester and indomethacin or an inhibitor of PKC,
bisindolylmaleimide I (BIS) and calphostin C were administrated before
PE treatment. BIS and Calphostin C significantly decreased propofol-induced PPP elevation in MCT rats. In contrast, indomethacin significantly decreased propofol-induced PPP elevation in normal rats. Propofol
may exert pulmonary vascular contractility through PKC activation in
MCT rats and through inhibition of cyclooxygenase pathway in normal
Oxidative stress index at the cell level in
hypertensive and/or hypercholesterolemic
Asahara, Hiroko; Endo, Kosuke; Tsujioka, Katsuhiko
(Dep. Physiol., Kawasaki Med. Sch., Okayama, Japan)
We investigated the effects of exercise on endothelial function in Dahl
salt-sensitive rats with risk factors for atherosclerosis, i.e. hypertension
and/or hypercholesterolemia. We randomly assigned 174 rats to four experimental diet groups: (MF) control diet; (S) high-salt diet; (C) highcholesterol diet; and (SC) combination of high-salt and high-cholesterol
diet. The rats took these diets ad libitum. Each group was further assigned to two exercise conditions: sedentary group (SD) and exercise
group (EX), swimming for 1 hour/day, 5 days/week. Chronic swimming
exercise and experimental diet were started at the same time and continued for 8 weeks. After physiological examination every 2 weeks, we observed the distribution of NO and superoxide production in endothelial
cells around aorto-renal bifurcation by using confocal laser-scanning microscopy. We evaluated the endothelial function with the oxidative stress
index, the ratio of superoxide production to NO production. Chronic
swimming exercise decreased the physiological risk for atherosclerosis,
growth rate of body weight in all diet groups significantly, blood pressure in MF and C significantly, tended to decrease trigriseride, and increased HDL in all diet groups significantly. There was no difference in
the oxidative stress index between artery portions, risk factors, and sedentary and exercise, but, decrease of NO production was related to atheroma prone portion. Therefore NO production may more contribute to the
localization of atherosclerosis than oxidative stress.
Responses of arterial pressure to
perturbations on a simulation model by a
neural network algorithm
Kemuriyama, Takehito; Hirakawa, Haruhisa; TandaiHiruma, Megumi; Nishida, Yasuhiro (Department of
Physiology, National Defense Medical College, Saitama, Japan)
We have been trying to calculate of arterial pressure (AP) on simulation
model by a neural network (NN) algorithm without approximate equations, to develop a new approach to estimate the relationship between AP
and other factors (renal sympathetic nerve activity (RSNA), heart rate
(HR) and respiration rate). Previously, we confirmed the simulated AP
consisted with measured AP by the NN model. In this study, we examined whether the simulated AP would consist with measured AP when
measured values of hypertensive rats were input to the learned NN by
those of normotensive rats. The AP simulation model was developed using Neural Network Toolbox of MATLAB (The Mathworks, Inc.). The
back propagation was selected as a learning algorithm of the layered NN.
AP, HR, RSNA and respiration rate were obtained from conscious
chronically instrumented rats. Those were used for the learning of NN
algorithm to establish the relationship between AP and other factors.
When measured values obtained from hypertensive rats were input to the
learned NN by those from normotensive rats, the simulated AP were
lower than measured AP. We confirmed that the relationship between AP
and other factors of hypertensive rats were different from those of normotensive rats by the NN model. These results suggest that AP of hypertensive rats was not only dependent on the relationship between HR,
RSNA and respiration rate.
Accumulation of colloidal particles in
rabbit popliteal lymph nodes in vivo
Ikomi, Fumitaka; Nagai, Takashi; Suzuki, Shigeru;
Mizuno, Risuke; Kawai, Yoshiko; Ohhashi, Toshio (Dept.
of Physiol., Shinshu Univ. Sch. of Med., Matsumoto, Japan)
Colloidal particles are widely used as tracer and vehicle of drugs which
target the lymph nodes. Little information, however, exists regarding
mechanisms of colloidal accumulation in the lymph nodes in vivo. Thus,
in this study, we have attempted to evaluate size-dependent mechanisms
for accumulation of colloidal particles in the lymph nodes in vivo. Male
Japan White rabbits were anesthetized with ketamine (20 mg/kg iv.) and
pentobarbital (20 mg/kg iv.). Centripetal and retrograde cannulations
were performed in one of the popliteal afferent and efferent lymph vessels, respectively. All other efferent lymphatics were ligated completely.
Labeled latex with fluorescence microspheres with 0.5, 1.1, 1.7 and 1.9
µm in diameter were injected into the afferent lymph vessel. Two
microgram of each particle was administered at one time and artificial
lymph fluid was injected through the same route at constant rate of 1.47
ml/h. Then, changes in concentration of the microspheres in the efferent
lymph vessel were determined by measuring the number of the particles
under a fluorescent microscope. 11.0 ± 6.4% of the 0.5 µm-microsphere
passed through the lymph node in 2.5 h. On the other hand, no
microsphere with 1.9 µm in diameter was observed in the efferent
lymph. The decreasing order of ability for accumulating in the lymph
node was as follows: 1.9 µm > 1.7 µm >1.1 µm > 0.5 µm-microsphere.
These results strongly suggest that size-dependent accumulating
mechanisms exist in the lymph node.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Heart & circulation
Uptake of micro- and nano-particles from
interstitial space into lymphatic system
Suzuki, Shigeru; Ikomi, Fumitaka; Nagai, Takashi;
Mizuno, Risuke; Ohhashi, Toshio (Dept. of Physiol., Shinshu
Electrical muscle stimulation effectively
improve popliteal venous flow on sitting
Morita, Hironobu; Abe, Chikara; Tanaka, Kunihiko (Dept.
Univ. Sch. of Med., Japan)
Physiol., Gifu Univ. Grad. Sch. Med., Gifu Japan)
Micro- and nano-particles are known to be taken up into initial lymphatics. The particles have been used for efficient delivery of drugs and diagnostic agents to lymphatic system. Little information, however, exists
regarding size-dependent mechanisms for the particle uptake. Thus, in
this study, we have attempted to evaluate effects of size on uptake of the
particles. Male Japan White rabbits were anesthetized with ketamine
chloride (20 mg/kg iv.) and pentobarbital sodium (20 mg/kg iv.). Retrograde cannulation was performed in one of the popliteal afferent lymph
vessels. Labeled latex with fluorescence microspheres with 0.5, 1.1, 2.0,
5.6 and 10.0 µm in diameter were injected subcutaneously at dorsal
portion of rabbit foot. Then, concentrations of the microspheres in the
efferent lymph vessel were determined by measuring the number of the
particles under a fluorescent microscope. The decreasing order of
particle concentration in efferent lymph was as follows: 0.5 µm > 1.1 µm
> 2.0 µm-particle. No particle with 5.6 and 10.0 µm in diameter was
observed in the efferent lymph. When mechanical massage was
administered on the injection site, both lymph flow rate and particle
concentration were markedly increased. These results strongly suggest
that size- and mechanical stimulation-dependent mechanisms exist in
transport of micro- and nano-particles from subcutaneous tissue into the
lymphatic system.
In sitting position, combined effects of movement restriction and hydrostatic pressure difference between the heart and the lower limb might enhance venous pooling/stasis, and then enhance development of deep
venous thrombosis. However, our understanding of the actual hemodynamic effects generated by sitting position in the venous circulation of
the lower limb remains surprisingly incomplete. Accordingly, the purpose of the present study was to examine this and the effect of electrical
muscle stimulation (EMS) on the popliteal venous flow was further examined. Fifteen healthy adult volunteers, none with a history of lower
limb surgery or thromboembolism, were recruited. One lower limb was
randomly selected for stimulation with the other serving as a control.
The sitting position significantly decreased maximum velocity (Vmax)
from 30.2 ± 5.7 to 6.5 ± 0.5 cm/s at 30 min and continuously suppressed
for 120 min. In contrast to the Vmax, cross sectional area was
significantly increased from 9.6 ± 1.2 to 53.1 ± 6.7 mm2. Due to the
combined effect of the decreased Vmax and the increased cross sectional
area, the flow volume was not affected by the sitting position (from
116.8 ± 20.7 to 115.9 ± 21.1 ml/min). The Vmax of the EMS leg was
significantly higher than that in the non-EMS leg throughout the 120 min
sitting period. There was no difference in the cross sectional area
between the EMS leg and the non-EMS leg, while the flow volume was
significantly higher in the EMS leg compared in the non-EMS leg. Thus,
lower limb venous stasis elicited by sitting position was improved by the
Lymph flow in vivo-resistance through the
lymphatic vessels with popliteal lymph
Nagai, Takashi; Ikomi, Fumitaka; Suzuki, Shigeru;
Ohhashi, Toshio (Dept. of Physiol., Shinshu Umiv. Sch. of Med.,
Effects of trypsin on effective permeability
of 77KDa dextran in isolated lymph vessels
Mizuno, Risuke1; Ono, Nobuyuki2; Kawai, Yoshiko1;
Ikomi, Fumitaka1; Ohhashi, Toshio1 (1Dept. Physiol. Shinshu
Matsumoto, Japan)
Univ. Sch. Med., Matsumoto, Japan; 2Dept. Electro. & Cont. Engine.
Nagano Nat. Coll. Tech., Nagano, Japan.)
The lymphatic vessels and lymph nodes serve a one-way drainage system which return fluid and protein from the interstitial space to blood
stream. Almost all of lymph passes through one or more lymph nodes in
which water is exchanged freely between lymph and blood compartments. Little information, however, exists regarding lymphdynamics
through the lymph nodes in vivo. Thus, in this study, we have attempted
to evaluate pressure-flow relationships in rabbit leg lymphatic system
and then estimate perfusion resistance through the lymph node. Male Japan White rabbits were anesthetized with ketamine chloride (20 mg/kg
iv.) and pentobarbital sodium (20 mg/kg iv.). Retrograde cannulation
was performed in one of the popliteal efferent lymph vessels at the groin.
All other efferent lymphatics were ligated completely. Then, centripetal
cannulation was performed in one of the popliteal afferent or efferent
lymph vessels near to the lymph node. Lymph infusion pressure-outflow
rate relationship was determined at the constant outflow pressure of -5
cmH2O. Lymph flow rate increased monotonically with infusion pressure. Calculated resistance at infusion pressure of 25 cmH2O was about
50 cmH2O/ml/h with the lymph node and about 15 cmH2O/ml/h without
the lymph node. From these results, resistance of the lymph node was
calculated as about 35 cmH2O/ml/h. Effect of water exchange between
lymph and blood compartments should be evaluated in future.
One of the major functions of the lymphatic system is to return plasma
proteins from the interstitial space to the blood stream. Previously, we
have demonstrated that small molecular FITC-dextran are permeable
from the intraluminal to extraluminal space of isolated lymph vessels
and that the endothelial cell surface structure plays a barrier role in effective permeability of medium size of FITC-dexran through the wall of
the lymph vessels (Am. J. Physiol. 289: H1676-H1682, 2005). In the
present study, we further investigated the effects of trypsin, which is useful enzyme for removal of lymphatic endothelial cells in vitro, on effective permeability of large size of FITC-dextran (77 KDa) in isolated
lymph vessels. Afferent lymph vessels were isolated from iliac lymph
nodes of Wistar rats. The isolated lymph vessels were cannulated with
glass-micropipettes and perfused with Krebs-bicarbonate solution with
or without 77 KDa FITC-dextran. Changes in the intensity of 77 KDa
FITC-dextran in an intraluminal space of the lymph vessels were measured by a video-microscope system and the concentration of 77 KDa
FITC-dextran were calculated before and after treatment with trypsin. 77
KDa FITC-dextran did not permeate the wall of lymph vessels before
and after the treatment with trypsin. These results suggest that the nonendothelial wall structure of lymph vessels may be a barrier for effective
permeability of a large size of FITC-dextran in vitro.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Heart & circulation
Development of in vitro assay system for
evaluating macromolecular permeability
through cultured rat lymphatic endothelial
Kawai, Yoshiko; Mizuno, Risuke; Ohhashi, Toshio
(Shinshu Univ. Sch. of Med., Matsumoto, Japan)
One of the important functions of lymph vessels and lymph nodes is to
return plasma proteins from the interstitial space in tissues. There exists
few or no report, except for one (Ono, et al: AJP H1676-H1682, 2005),
regarding for evaluation of hydrophilic permeability through the walls of
lymph vessels. No experiment in vitro model using cultured lymphatic
endothelial cells has been reported to elucidate macromolecular permeability through the cell layer. Therefore, we have attempted to develop
firstly in vitro assay system to study hydrophilic permeability and then
investigated the effects of TNF-α.Rat lymphatic endothelial cells
cultured from the thoracic ducts or afferent lymphatic vessels were
seeded onto the collagen-coated inserts of double chambers in-vitro
vascular permeability assay kit (CHENICON). The cell monolayers
were treated with or without TNF-α (10ng/ml) over night, and then
fluorescent-labeled dextrans (4, 12, and 77KD) were added on the top of
the cells coated on upper chamber to evaluate the hydrophilic
permeability. The extent of permeability was determined by measuring
fluorescent activity of the solution dropped down the lower chamber.
The pretreatment with TNF-α caused a significant increase of the
permeability of all FITC-dextran through the cultured endothelial cell
layer.These findings suggest that the in vitro assay system may be
suitable for hydrophilic permeability through cultured lymphatic
endothelial cell layer. Inflammatory cytokine, TNF-α, caused a
significant increase of permeability with FITC-dextran.
Vasodilator-induced spreading dilatation
requires arterial hyperpolarization
Takano, Hiromichi1; Garland, Chris2; Dora, Kim2;
Shibamoto, Toshishige1 (1Kanazawa Med. Univ., Uchinada,
Japan; 2Univ. Bath, Bath, UK)
August Krogh first reported that the local application of vasodilators
could stimulate extensive vasodilatation which spreads rapidly to distant
sites. Conducted responses of this type are referred to as spreading dilatation, a response which cannot be explained simply on the basis of diffusion of the locally applied dilator agent. The present study was
designed to test the hypothesis that local stimulation of hyperpolarization can be conducted longitudinally over significant distances to spread
dilatation in small mesenteric arteries. A segment of mouse mesenteric
artery was cannulated at each end with a glass pipette and then pressurized. In the presence of the NO synthase inhibitor, L-NAME, focal application of the PAR2 agonist, SLIGRL induced dilatation at the site of
application. This local dilatation was associated with simultaneous dilatation along the entire artery segment. Measurement of smooth muscle
membrane potential revealed local hyperpolarization to SLIGRL which
was also observed at distant sites. In contrast, focal application of forskolin did not induced any hyperpolarization, and although it evoked local
dilatation, this was not associated with any coordinated dilatation along
the isolated mesenteric artery. These results indicate that spreading dilatation responses in mesenteric resistance arteries are only evoked with
vasodilators which hyperpolarize the artery.
Correlation between compliance and high
frequency centroid of pressure waveform
in the abdominal aorta.
Role of vestibular system in controlling
arterial pressure during 60°head-up tilt in
conscious rats
Tanaka, kunihiko; Abe, Chikara; Morita, Hironobu (Grad.
Abe, Chikara; Tanaka, Kunihiko; Morita, Hironobu (Dep.
Sch. Med. Gifu Univ. Dep. Physiology)
Physiology, Grad. Sch. Med. Gifu Univ, Gifu, Japan)
According to Windkessel theory, diastolic pressure waveform is a function of the total peripheral resistance and the compliance of the artery.
Higher compliance makes the decrescence gentler, and the waveform
would be further different from the input pressure waveform made by the
cardiac contraction. Frequency analysis of the arterial pressure waveform shows fundamental frequency (F) derived from the heart rate and
harmonic frequency which is the integral multiple of the F. In the present
study, we measured abdominal aortic pressure, abdominal aortic flow,
and the diameter during infusion of Nitroprusside with the speed of 1,
0.6, 0.3 µg/kg/h. The compliance was calculated from the pulse pressure
and volume change calculated by the diameter and the length of the
aorta. The compliance was also calculated with first order of the
Windkessel theory. High frequency centroid (HFC), which is amplitudeweighted mean frequency of the third to seventh peaks of the harmonic
waveform was analyzed. HFC was normalized by F. Total harmonic
distortion (THD), which is an index of distortion from a pure sine wave
was also analyzed. Measured compliance and the calculated compliance
of the aorta were significantly correlated. Normalized HFC and both
measured compliance and the calculated compliance also showed
significant linear correlation. THD did not show significant correlation
between the compliance. These results suggest HFC can be an index of
the compliance or the aorta, and the compliance may be analyzed from
the aortic pressure waveform only.
Previous studies from our laboratory demonstrated that the vestibular
system has a significant role in controlling arterial pressure during gravitational stress. The vestibular system is thought to be stimulated not
only by gravitational change but also by postural change. Thus, the vestibular system might have a significant role in controlling arterial pressure during postural change. To examine this, arterial pressure was
measured during 60°head-up tilt in conscious rats with or without intact
vestibular system. Rats were divided into 3 groups: intact, sinoaortic
denervation (SAD), and SAD+vestibular lesion (VL). The posture
change did not alter arterial pressure in intact group. In SAD group,
however, arterial pressure was increased by the posture change. This
increase in arterial pressure was completely abolished by additional VL.
Thus, the vestibular system has a significant role in an increase in arterial
pressure during posture change in SAD group. The increased arterial
pressure induced by the vestibular system is buffered by baroreflex, thus
arterial pressure is well maintained during posture change in intact
J. Physiol. Sci., Vol. 56, Suppl., 2006
Heart & circulation
Inhibitory effect of deep ocean water on
mild hypertension in KHC rabbits
Role of nitric oxide in the ischemiareperfusion injury in mouse livers
Shin-ichiro, Katsuda1; Yasukawa, Takeshi2; Nakagawa,
Koji2; Miyake, Masao1; Yamasaki, Masao1; Hazama,
Akihiro1; Katahira, Kiyoaki3; Mohri, Motohiko4; Shimizu,
Tsuyoshi5 (1Dept. of Physiol., Fukushima Medical Univ. Sch. of
Shibamoto, Toshishige; Cui, Sen; Liu, Wei; Takano,
Hiromichi; Kurata, Yasutaka (Dept. Physiol., Kanazawa Med.
Med., Fukushima, Japan; 2Ako Kasei, Co., Ltd., Hyogo, Japan;
3Exp. Anim. Res. Ctr., Fukushima Medical Univ., Fukushima, Japan;
Marine Enterprises, LTD., Kanagawa, Japan; 5Shimizu
Inst. of Space Physiol., Nagano, Japan.)
We investigated effect of deep ocean water on hemodynamics in Kurosawa and Kusanagi-Hypercholesterolemic (KHC) rabbits. The refined
deep ocean water (RDOW) at a degree of hardness of 1,000 was freely
accessed to the KHC rabbits aged 4 months old for 6 months. Aortic
pressure and flow waves were simultaneously recorded under pentobarbital anesthesia. There were no significant differences in body weight
and food and water intakes. Systolic, diastolic, mean and pulse pressures, augmentation index of pressure waves and total peripheral vascular resistance were significantly lower in the RDOW group than in the
control group, whereas no significant differences were observed in mean
aortic flow and total percent lesioned area of the aorta between the two
groups. Though serum lipid levels, plasma renin and ACE activities and
angiotensin 1 and 2 levels tended to decrease after the feeding of RDOW
in the two groups, the difference in these parameters before and after the
feeding of RDOW was almost the same between the two groups. Serum
electrolyte levels except Mg2+ changed little during the intake of RDOW.
We can conclude that RDOW improved mild hypertension in KHC rabbits, which might be partly related to the slight but significant increase
in serum Mg2+ due to the intake of RDOW.
We determined the changes in hepatic sinusoidal pressure and liver
weight, and the effects of a NO synthase inhibitor, NG-nitro-L-arginine
methyl ester (L-NAME) in ischemia-reperfusion (I/R) injury of isolated
mouse livers perfused portally with diluted blood (Hct 3%). Following
pretreatment with L-NAME (100 µM) or D-NAME (100 µM), Ischemia
was induced at room temperature by occlusion of the inflow line of the
portal vein for 1 hour, followed by 1-hour reperfusion in a recirculating
manner. The sinusoidal pressure was assessed by the double vascular
occlusion pressure (Pdo). In the D-NAME group, immediately after
reperfusion, the portal pressure increased by 2.8±0.1(SE) mmHg, which
was accompanied by an increase in Pdo by 1.5±0.1 mmHg, suggesting
increases in pre- and post-sinusoidal resistances in a similar degree.
Liver weight increased 0.14±0.04 g after reperfusion followed by a
gradual return towards baseline. Liver injury, assessed by perfusate
levels of hepatic enzymes was observed at 60 min after reperfusion.
There were no significant differences in changes in any variables
between the D- and L-NAME groups. In conclusion, the increases in
both the hepatic vascular resistances and the sinusoidal pressure were
small in magnitude, resulting in absence of edematous changes in mouse
hepatic I/R, and nitric oxide does not play any significant roles in this
Long-term monitoring of pulmonary
arterial pressure in conscious,
unrestrained mice
Schwenke, Daryl O.1; Pearson, James T.2; Mori,
Hidezo1; Shirai, Mikiyasu3 (1National Cardiovascular Centre
Research Institute, Suita, Japan;
University, Melbourne,
Australia; 3Faculty of Health Sciences, Hiroshima International
The ability to genetically engineer specific gene knock-out mice has provided a powerful tool for investigating the pathogenesis of pulmonary
arterial hypertension (PAH). Yet, there have been no reports describing
the measurement of pulmonary arterial pressure (PAP) in conscious
mice; an essential requirement for monitoring dynamic changes associated with PAH. In this study we describe a new technique for long-term
measurement of PAP in conscious mice using telemetry. In five male
C57BL/6 mice (B.W. 25-30 g) the sensing catheter of a telemetric transmitter was inserted into the right ventricle and advanced into the pulmonary artery. The transmitter body was positioned within the abdominal
cavity or subcutaneously on the back. During recovery from surgery
mean PAP was recorded daily for one week. Subsequently, the PAP responses to acute hypoxia (8% O2 for 10 min) and L-NAME (50 mg/kg,
s.c.) were tested in three mice. By one-week post surgery, all mice had
fully recovered from surgery and baseline MPAP was stable at 14.9 ± 0.7
mmHg. Additionally, acute hypoxia and L-NAME provoked a 63% and
86% increase MPAP, respectively. In summary, this study has
demonstrated the ability to accurately measure PAP by telemetry in
conscious mice. One important application of this technique for future
studies is the possibility to assess the contribution of specific genes (i.e.
knock-out mice) for the development of pulmonary pathological
Univ., Uchinada Ishikawa, Japan)
Role of liver in anaphylactic hypotension of
anesthetized mice
Liu, Wei; Shibamoto, Toshishige; Cui, Sen; Takakno,
Hiromichi; Kurata, Yasutaka (Dept. Physiol., Kanazawa Med.
Univ., Uchinada Ishikawa, Japan)
We determined the roles of liver and splanchnic vascular bed in anaphylactic hypotension in anesthetized mice. In anesthetized mice sensitized
with ovalbumin (0.01 mg), an intravenous injection of 0.01 mg ovalbumin caused not only a decrease in systemic arterial pressure (Psa) from
92±1.6 (SE) to 39±2.6 mmHg but also an increase in portal venous
pressure (Ppv) which persisted for 40 min after the antigen injection. The
elimination of the splanchnic vascular beds, by the occlusions of the
celiac and mesenteric arteries, combined with total hepatectomy
attenuated anaphylactic hypotension. In addition, a head-down tilt
maneuver, which could facilitate venous return in the splanchnic organs,
significantly attenuated the decrease in Psa induced by an antigen
injection. These results suggest that liver and splanchnic vascular beds
are involved in anaphylactic hypotension presumably due to
anaphylactic contraction-induced portal hypertension, which induced
splanchnic congestion resulting in a decrease in circulating blood
volume and thus systemic arterial hypotension.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Heart & circulation
L-NAME potentiates anaphylactic
presinusoidal venoconstriction in perfused
rat livers
Sympathetic nerve response to
endogenous hemeoxygenase inhibition in
conscious rats
Cui, Sen; Shibamoto, Toshishige; Liu, Wei; Takano,
Hiromichi; Kurata, Yasutaka (Dept. Physiol., Kanazawa Med.
Hirakawa, Haruhisa; Kemuriyama, Takehito; Hiruma,
Megumi; Nishida, Yasuhiro (Dept. Physiol. II, Natl. Defense
Univ., Uchinada Ishikawa, Japan)
Med. Coll., Saitama, Japan)
Effects of a NO synthase inhibitor,
methyl ester (LNAME), on anaphylaxis-induced venoconstriction were examined in
isolated rat liver perfused with blood of various hematocrit (Hct) to clarify the role of nitric oxide (NO) in anaphylactic venoconstriction in rat
livers.The rats were sensitized with ovalbumin (1 mg), and two weeks
later, the liver was excised and perfused portally and recirculatingly at a
constant flow with blood at Hct of 0, 5, 16, and 22%. We measured the
viscosity of perfusing blood. Using the double occlusion technique to estimate the hepatic sinusoidal pressure (Pdo), presinusoidal resistance
(Rpre) and postsinusoidal resistance (Rhv) were calculated. The antigen,
ovalbumin (0.1 mg), was injected into the reservoir 10 min after pretreatment with L-NAME (100 µM) or D-NAME (100 µM). The viscosity, a
determinant of vascular resistance and shear-stress, increased in a Hctdependent manner. L-NAME pretreatment increased exclusively basal
Rpre in liver perfused at Hct 22%. The antigen caused hepatic
venoconstriction as characterized by predominant presinusoidal
constriction in all antigen administered livers. L-NAME pretreatment
potentiated the antigen-induced venoconstriction, as compared with the
D-NAME pretreatment, by increasing Rpre, but not Rpost. These
finding suggests that hepatic anaphylaxis increased production of NO,
venoconstriction in isolated perfused rat livers.
Carbon monoxide is formed in the process of degradating heme from
biliverdin by heme oxygenase (HO) in the various tissues, including central nervous system (CNS). Previous studies suggested that inhibition of
HO activity increased arterial pressure (AP) mediated by the autonomic
nervous system. The present study was designed to investigate the sympathetic nerve response to inhibition of HO activity by a direct renal
nerve recording in conscious rats. Zinc deuteroporphyrin 2, 4-bis glycol
(ZnDPBG), an inhibitor of HO activity, was administered ip in chronically instrumented Wistar rats: 8 intact, 8 atropine-treated, and 7 sinoaortic denervated (SAD). ZnDPBG induced significant increases in mean
AP (MAP) from 95.9 ± 1.6 to 116.9 ± 4.7 mmHg and renal sympathetic
nerve activity (RSNA) from 100.0 to 186.7 ± 19.2%, but no significant
change in heart rate (HR) in intact rats. In atropine-treated rats, ZnDPBG
also induced significant increases in MAP from 96.7 ± 1.5 to 110.6 ± 2.0
mmHg and RSNA from 100.0 to 184.7 ± 22.5%, but induced no change
in HR. In SAD rats, ZnDPBG induced significant increases in MAP
from 91.4 ± 5.7 to 118.8 ± 8.5 mmHg, HR from 356.8 ± 10.4 to 409.1 ±
14.4 beats/min, and RSNA from 100.0 to 211.6 ± 18.9%. The present
study suggested that inhibition of HO activity caused sympathoexcitation via a direct action on CNS, resulted in an increase in AP.
Further study is required to investigate the underlying mechanism of HR
response to HO inhibition.
Autonomic control based on heart rate
variability in congenital heart diseases with
increased pulmonary blood flow
Spinally mediated articulo-cardiac
sympathetic reflex in anesthetized rats
Matsuura, Hideaki1; Hata, Tadayoshi2; Shindo,
Yoshiaki3; Nomura, Hiroko3; Nagaoka, Shunji3 (1Fujita
(1Lab. Physiol., Health Science Univ., Yamanashi, Japan; 2Sch.
Veterinary Sci., Univ. Miyazaki, Miyazaki, Japan; 3Lab. Anatomy,
Health Science Univ., Yamanashi, Japan)
Health Univ.Hosp.Clin.Lab,Aich,Japan; 2Fujita Health Univ.School
of Health Science.Clinical Pathophysiology,Aich,Japan; 3Fujita
Health Univ.School of Health Science.Physiology,Aich,Japan)
Objective Ventriculoseptal defect;VSD and atrial septal defect;ASD exhibit increased pulmonary blood flow through the left-to-right shunt. In
patients with these pathological states arising from volume and/or pressure overload in the right heart can result in sympathovagal imbalance.
We investigated the correlation between the HRV and respiratory frequency, and shunt ratio (Qp/Qs) measured by the Doppler echocardiography.Subjective and methodsWe enrolled 66 patients. Diagnoses
were: VSD (n=21) and ASD (n=45). Mean age was 5.6 ± 3.3 years. ECG
and respiratory waves were recorded during the Doppler UCG test. We
performed HRV analysis to calculate LF, HF, TF, respiratory frequency
(RF), LF/HF, RF/TF, and LF/RF. Qp/Qs was also calculated and
quantified by multivariate analysis with HRV parameters. Results1) Qp/
Qs and LF/HF exhibited correlation in both ASD and VSD groups. 2)
The ASD group showed a positive correlation between Qp/Qs and LF/
RF. However, in the VSD group the correlation was negative. 3) Qp/Qs
correlated negatively with RF/TF in the ASD group, however, the
correlation was positive in the VSD group.ConclusionsASD and VSD
showed different effects of respiratory vagal activity on HRV. We
believe that a reason is that volume and low pressure overload to the
right atrium in ASD inhibits respiratory vagal innervation of the sinus
Suzuki, Atsuko1; Nakayama, Tomohiro2; Ito, Ryuzo3
It has been proven that noxious articular stimulation of hindlimb produce
reflex increase in heart rate, in anesthetized animals, to be a reflex response whose reflex center is in the brain and efferent arc is a cardiac
sympathetic nerve. Using central nervous system (CNS)-intact and
acutely spinalized anesthetized rats, the present study aimed to examine
the possibility of whether articular stimulation could influence heart rate
via sympathetic nerve at the spinal level. In CNS-intact rats, noxious articular movement of both knee and elbow joint produced an increase in
the cardiac sympathetic nerve activity and heart rate. In acutely spinalized rats, noxious movement of elbow joint produced large increases in
the cardiac sympathetic nerve activity and heart rate, while noxious
movement of knee joint induced no increase in the cardiac sympathetic
nerve activity and only a marginal increase in heart rate. As the marginal
heart rate response following knee joint stimulation in spinalized rats
was abolished after adrenalectomy, the responses were suggested to be
induced by catecholamine secreted from the adrenal grand. It is concluded that the spinal cord is capable of producing propriospinally the reflex
increases in heart rate via reflex activation of the cardiac sympathetic efferent nerve following stimulation to the elbow joint stimulation whose
afferent information enters the spinal cord at the same segments or segments overlapping the cardiac sympathetic outflow.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Differencial effect of short term exercise
training on the cardiovagal baroreflex
sensitivity and carotid arterial compliance
in older subject
Komine, Hidehiko; Hayashi, Koichiro; Sugawara, Jun;
Yoshizawa, Mutsuko; Yokoi, Takashi (Natl Inst Adv Ind Sci
& Technol, Tsukuba, Ibaraki, Japan)
Previous studies have reported that carotid arterial compliance increased
by habitual exercise, so that the cardiovagal baroreflex sensitivity increased. This concept is based on the anatomical fact that the arterial
baroreceptor is a stretch sensitive receptor, a part of which is located in
the carotid sinus. However, we previously reported that neural component of baroreflex sensitivity estimated by R-R interval corresponding
end-systolic lumen diameter was greater in physically active young men
than that in sedentary, but mechanical component of baroreflex sensitivity estimated by end-systolic lumen diameter corresponding systolic
blood pressure was not different between active and sedentary group. We
hypothesized that short term exercise training increase arterial baroreflex sensitivity due to the neural alteration but not increase arterial compliance because alteration in neural property will occur faster than that
in mechanical property of blood vessel wall. To examine this hypothesis,
we estimated baroreflex sensitivity and carotid arterial compliance in
elderly subjects before exercise training, and at 2, 6, and 12 weeks after
training. Arterial baroreflex sensitivity increased at 2 weeks after training but arterial compliance did not increase at this time point. This result
suggests that arterial baroreflex sensitivity increase by short term exercise training, which is probably due to alteration in "neural" arc of the
arterial baroreflex.
The carotid body of spontaneously
hypertensive rats
Kusakabe, Tatsumi1; Kawakami, Tadashi2; Takenaka,
Toshifumi1; Hayashida, Yoshiaki3 (1Dept. Sport Med. Sci.,
Kokushikan Univ., Tokyo, Japan; 2Dept. Physiol., Kitasato Univ.
Sch. Med., Sagamihara, Japan; 3Internat. Buddist Univ., Osaka,
Morphological characteristics in the carotid bodies of spontaneously hypertensive rats (SHR) and those of age-matched normotensive Wistar
rats (NWR) as well as age-matched genetically comparable Wistar rats
(WKY) were examined. The distribution and abundance of four different
regulatory neuropeptides, substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), and neuropeptide
Y (NPY) in the carotid bodies of three strains of rat were also examined.
The carotid bodies of SHR were greater than those of NWR and WKY.
The values in the long axis of the carotid bodies of SHR were significantly 1.3 times larger than those of NWR and WKY. In the carotid bodies
of SHR, the percentage of relatively large vessels were similar to that in
the carotid bodies of WKY, although the carotid bodies themselves were
significantly larger than in WKY. In the carotid bodies of NWR and
WKY, the density of NPY-immunoreactive varicose fibers were more
numerous than that of VIP, SP, and CGRP fibers. These immunoreactive
fibers were mainly associated with the vasculature and the clusters of
glomus cells. The density of VIP varicose fibers in the carotid bodies of
SHR were smaller than that in the carotid bodies of WKY, although that
of SP, CGRP, and NPY fibers was similar to that in the carotid bodies of
NWR and WKY. The present results suggest that the mechanisms of carotid body enlargement in hypertensive rats are different from those in
hypoxic rats.
Distribution and axonal projection of
pontine respiratory neurons in the rat
Ezure, Kazuhisa; Tanaka, Ikuko (Dept. Neurobiol., Tokyo
Metropol. Inst. Neurosci., Fuchu, Tokyo, Japan)
The area around the parabrachial nucleus (PB) including the KollikerFuse (KF) nucles is deeply involved in respiratory control, but not
enough information is available about the respiratory neurons of this area. In Nembutal-anesthetized, paralyzed, and artificially ventilated rats
with intact vagal nerves, we recorded from more than 300 respiratory
neurons in the PB and KF areas. The respiratory neurons were densely
distributed in the KF nucleus and sparsely in the medial parabrachial nucleus (PBM) and its vicinity. Only few neurons were purely inspiratory
(I) and most "inspiratory" neurons had more or less expiratory (E) activity, exhibiting the property of E-I phase-spanning neurons. By far the
less number of I-E phase-spanning neurons were recorded, and their firing was labile and weak. The E neurons exhibited augmenting, decrementing or whole-phase expiratory firing patterns. Activity of the
respiratory neurons was variously modulated by lung volume, but we
could not find non-respiratory neurons whose activity was modulated
thoroughly by lung volume. The majority of the respiratory neurons examined by antidromic stimulation had medullary axons, some having axons descending the midline medulla toward the spinal cord. Many E and
"inspiratory" neurons projected to the nucleus tractus solitarii (NTS).
Some "inspiratory" neurons projected to the hypoglossal nucleus. It was
suggested that PB and KF neurons project to motor output nuclei, such
as the nucleus ambiguus and the hypoglossal nucleus, as well as to the
rhythm-related structures, such as the Botzinger complex and the NTS.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Hypoxic ventilatory response in the light
and dark periods in unanesthetized mice
lacking histamine type-1 receptors
Does cardiorespiratory synchronization
show competitive aspect of
cardiorespiratory modulation?
Ohshima, Yasuyoshi; Izumizaki, Masahiko; Ishiguro,
Takashi; Iwase, Michiko; Kanamaru, Mitsuko; Homma,
Ikuo (Dept. 2nd. Physiol., Showa Univ. Sch. of Med., Tokyo,Japan)
System Eng. Fac. Eng. Yamagata Univ. Yonezawa, Japan)
Saitoh, Tsuyoshi; Nishidate, Izumi; Niizeki, Kyuichi (Bio-
The effects of the circadian light/dark cycle on ventilatory responses to
chemical stimuli have rarely been studied in experimental animals, despite evidence that the cycle may be a factor in respiratory results. We
measured the ventilatory response to hypoxia (HVR) in unanesthetized
wild-type and histamine type-1 receptors knockout (H1RKO) mice in
the light and dark periods with a whole-body, single-chamber plethysmograph. Animals were subjected to a 10-min hypoxic exposure (7% O2
and 3% CO2 in N2) after acclimatization to the chamber for 90 min. In
both groups of mice, minute ventilation increased in response to the hypoxia and declined gradually after the peak response regardless of when
HVR was determined. However, we found differences in the HVR between wild-type and H1RKO mice. In wild-type mice, the minute ventilation response was higher in the dark period than in the light period,
which was due to differences in the tidal volume response rather than the
respiratory frequency response. Conversely, in H1RKO mice, minute
ventilation responses did not differ between the two periods, which were
similar to the response of wild-type mice determined in the dark period.
In summary, the circadian light/dark cycle altered the HVR in wild-type
mice, whereas, in H1RKO mice, the cycle difference in the HVR disappeared. These results suggest that H1R contributes to the circadian light/
dark cycle differences in the HVR in unanesthetized mice.
Respiratory modulation of the heart rate (i.e.,respiratory sinus arrhythmia:RSA) and phase synchronization of respiratory and cardiac rhythms
are manifestation of cardiorespiratory coordination. However, the question of whether they represent two competing entities of interaction remains unresolved. The present study was designed to characterize the
synchronization and modulation between patterns of breathing and RSA
during normoxia and hypoxia under resting condition. For this purpose,
we recorded ventilation and electrocardiogram in 9 subjects while
breathing was paced either 6,8,10, or 12 breaths/min for 5 min each. The
instantaneous phases were calculated for RSA curve and respiratory patterns using Hilbert transform and then relative phase differences were
obtained. The cardiorespiratory synchronization and modulation were
quantified by the index based on conditional probability of the phase difference and by the amplitude of RSA power calculated using a fast Fourier transform, respectively. The synchronization index decreased
significantly at 12 breath/min compared with that observed at lower
breathing frequency, while the modulation index (i.e., RSA power) decreased almost linearly with increasing breathing frequency. There was
a significant positive correlation between the indexes of the synchronization and modulation (p<0.01). The observed relation was not altered
by hypoxia, suggesting that the coordination may not depend on functional modulations. Our analysis indicates that the cardiorespiratory synchronization can coexist with the modulation.
Momentary rise in breathing rate during
light sleep in mice and humans
Effects of hypercapnia on respiratory
neurons of rat medullary raphe nuclei.
Sato, Shinichi1; Yamada, Katsuya1,3; Kanbayashi,
Takashi2; Kondo, Hideaki2; Shimizu, Tetsuo2; Inagaki,
Nobuya1,4 (1Dept. Physiol., Akita Univ. Sch. Med., Akita, Japan;
Hosogai, Masae; Matsuo, Satoshi (Div. Adaptation Physiol.,
Neuropsychiatry, Akita Univ. Sch. Med., Akita, Japan; 3Dept.
Physiol., Hirosaki Univ. Sch. Med., and CREST, Aomori, Japan;
4Dept. Diabetes and Clinical Nutrition, Kyoto Univ. Grad. Sch.
Med., and CREST, Kyoto, Japan)
Breathing activity during sleep is closely related to sleep stage. Indeed,
breathing rate (BR) during rapid eye movement (REM) sleep show much
larger fluctuation than that during deep sleep. In the present study,
breathing movement of freely moving C57B6/J mice were analyzed during sleep by piezoelectric device placed on the floor of the cage in a noninvasive manner (PCT/2003/001109), while animal behavior was monitored by an infrared camera. We found that mice momentarily exhibited
an extraordinary large rise in BR by up to 14 breaths/sec during sleep.
Such rise in BR was detected in all four mice tested, especially after atonia and shortly before awakening (mean BR = 10.5±1.2 breaths/sec,
which is 4.3±1.3 times the BR in stable deep sleep; mean duration =
0.4±0.1 sec). In humans, interestingly, a similar momentary increase in
BR by up to 116 breaths/min (2.4-3.7 times the BR in deep sleep) was
detected during REM sleep in normal volunteers tested. No such
increase was detected in stages 3 and 4. In addition, more prolonged
increase in BR also was observed during REM/light sleep in human
subjects. Further studies may clarify the correlation between the central
regulation mechanisms of respiration and sleep stages.
Facult. Med., Univ. Tottori, Yonago, Japan)
Numerous studies have suggested that caudal raphe nuclei may play
some roles in respiration. However, details of the role of the raphe nuclei
in controlling of respiration have not been clarified. We have previously
reported that respiratory neurons are present in medullary raphe nuclei.
The present study was undertaken to examine the effects of hypercapnia
on the respiratory neurons of medullary raphe nuclei. The experiments
were performed on decerebrate, paralyzed, vagotomized and artificial
ventirated rat. Extracellular recordings were made from neurons having
firing patterns related to phrenic nerve discharge in the midline medullary tegmentum. We recorded mainly Inspiratory (I) throughout neurons
which firerd throughout the I phase in the raphe obscurus and pallidus.
They were tested changes in neuronal discharges when the ventilator
was stopped (during 8-24 sec; end-tidal CO2 levels were raised from
5±0.4 to 8±0.3%). Most of the neurons examined responded to
hypercapnea in the neuronal discharge. But some neurons examined did
not respond. These results suggest that the midline caudal raphe nuclei
are involved in central chemoreception.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Proposal of the fine anatomical model for
the central respiratory chemoreceptor
Okada, Yasumasa1; Kuwnana, Shun-ichi2; Oku,
Yoshitaka3; Oyamada, Yoshitaka4; Chen, Zibin5 (1Dept.
Med. Keio Univ. Tsukigase Rehab. Ctr. Shizuoka, Japan; 2Dept.
Physiol. Teikyo Univ., Tokyo, Japan; 3Dept. Physiol. Hyogo Col.
Med., Nishinomiya, Japan; 4Dept. Resp. Med, Tokyo Med. Ctr.,
Tokyo, Japan; 5Dept. Biochem. Analyt. Pharmacol.
GlaxoSmithKline, RTP, NC USA)
We have studied the fine anatomical architecture of the central respiratory chemoreceptor using electrophysiological, optical and histological
techniques. We have identified medullary chemosensitive sites in the
surface parts of the rostral ventrolateral medulla, raphe pallidus and
parapyramidal regions. Pontine regions (locus coeruleus and A5 region)
were also chemosensitive. We found two types of CO2-excitable cells
(Type I and II cells) in the superficial ventral medulla. A Type I cell is
smaller in size, located within the marginal glial layer, and intrinsically
CO2-excitable. A cluster of Type I cells surround fine penetrating vessels, and a large surface vessel covers this region. A Type II cell is larger,
located at a depth of a few hundred micrometer from the medullary surface, and excited transsynaptically by CO2. A single Type II cell forms
dendro-somatic synapses with multiple Type I cells within the marginal
glial layer, and sends axonal branches to multiple neurons of the ventral
respiratory group (VRG). We propose a cell-vessel architecture model
for the central respiratory chemoreceptor. Type I cells are primary
chemosensors. In response to increased CO2 in perivascular tissue, Type
I cells secrete neurotransmitter, and excite Type II cells. Excited Type II
cells relay and amplify this information by innervating multiple VRG
Kanamaru, Mitsuko; Iwase, Michiko; Homma, Ikuo (Dept.
of Physiol., Showa Univ. Sch. of Med., Tokyo Japan)
We have reported that serotonin (5-HT) type 2 receptors in the dorsomedial medulla oblongata (DMM) influence ventilation and airway resistance in mice. In the present study, the role of 5-HT2 receptors in the
DMM in respiration to hypoxia was investigated. Each male mouse was
anesthetized with pentobarbital sodium i.p. for insertion of a microdialysis probe into the DMM. The mouse was placed into a double chamber
plethysmograph to obtain two respiratory flow curves to calculate respiratory variables, while extracellular fluid was collected at 1.2 microL/
minute every 5 minutes. Extracellular fluid via the microdialysis probe
was analyzed with an ECD-HPLC. Artificial cerebrospinal fluid (aCSF)
or 1 x 10–5M LY-53857 (a 5-HT2 receptor antagonist) was perfused via
the microdialysis probe in the DMM with air or hypoxic gas (7% O2 in
N2) inhalation. Changes in respiratory variables and the 5-HT concentration in the DMM were analyzed every 5 minutes. Respiratory variables
during hypoxia in a LY-53857-perfused group were not different from
those in an aCSF-perfused group. Post-hypoxia frequency in the LY53857-perfused group was decreased compared to that in the aCSF-perfused group, while the 5-HT concentration in the DMM was significantly increased under hypoxia in both the aCSF-perfused group and the LY53857-perfussed group. We discuss whether or not the post-hypoxia frequency decline with LY-53857 perfusion in the DMM is a result of respiratory hypoxic excitation with a more detailed analysis of respiration
Characteristics of GABAergic respiratory
neurons in the ventrolateral medulla:
studies in GAD67-GFP knock-in neonatal
Kuwana, Shun-ichi1; Okada, Yasumasa2; Kuribayashi,
Junya3; Yanagawa, Yuchio4,5; Obata, Kunihiko6 (1Dept.
Physiol., Teikyo Univ. Sch. Med., Tokyo, Japan; 2Dept. Med., Keio
Univ. Tsukigase Rehab. Ctr., Izu, Japan; 3Dept. Anesth., Keio Univ.
Sch. Med., Tokyo, Japan; 4Dept. Genet. and Behav. Neurosci.,
Gunma Univ. Grad. Sch. Med., Maebashi, Japan; 5SORST, Jpn. Sci.
and Tech. Corp., Kawaguchi, Japan; 6Obata Res. Unit, RIKEN
Brain Sci. Inst., Wako, Japan)
We investigated electrophysiological and morphological characteristics
of GABAergic neurons in the pre-Boetzinger complex (PBC, the caudal
part of the rostral ventrolateral medulla), the putative kernel site for respiratory rhythm generation. We used GAD67-GFP knock-in neonatal
mice, which enabled us to identify GABAergic neurons in a living condition. We recorded respiratory activity from the hypoglossal nerve in
medullary transverse slices that contained the PBC and whole-cell recorded activities of GFP-positive neurons in the PBC. Six out of 32 GFPpositive neurons were inspiratory. All of the remaining neurons were
non-respiratory. In addition, 6 GFP-negative inspiratory neurons were
recorded in the PBC. The electrophysiological properties of GFP-positive inspiratory neurons included high membrane resistance and mild adaptation of spike frequency in response to depolarizing current pulses.
The somata of GFP-positive inspiratory neurons were smaller than those
of GFP-negative inspiratory neurons. These results indicate that
GABAergic inhibition by inspiratory neurons with particular properties
in the PBC is involved in neural respiratory control.
Effects on hypoxic respiratory responses
of 5-HT2 receptors in the dorsomedial
medulla oblongata in mice
Dopaminergic modulation on the
respiratory neuronal network using
isolated brainstem-spinal cord preparation
from neonatal rat
Arata, Akiko; Fujii, Morimitsu (Lab. for Memory & Learning,
RIKEN-BSI, Wako, Saitama, Japan)
Here we report that unique 'switching' roles of dopamine in regulating
the respiratory rhythm generation. We employed medulla-spinal cord
block preparation which contains intact respiratory rhythm generator. In
this preparation, respiratory rhythm generator consists mainly Pre-Inspiratory (Pre-I) neurons and inspiratory (I) neurons. I neurons are premotor neurons and Pre-I neurons trigger I neuronal firing as a pacemaker. Optical imaging with voltage-sensitive dye revealed that application
of dopamine selectively disrupted phasic Pre-I neuronal firing and
slightly enhanced I neuronal firing. This dopaminergic effect for Pre-I
neuron was mimicked by dopamine D4 receptor agonist, PD168077 application and the enhanced effect of I neuronal firing was mediated with
D1 receptor. Dopamine depolarized Pre-I neuronal membrane potential
significantly but not on I neurons under TTX perfusion. Finally, dopamine depressed I neuronal PSPs which are probably originated from Pre-I
neurons. Dopamine selectively disrupts synchronized phasic Pre-I neuronal firing through dopamine D4 receptor, and enhances I neuronal network through dopamine D1 receptor. Dopamine has opposite effects for
respiratory network though different type of dopamine receptors. It
seems to be the switching involuntary respiration to voluntary respiration.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Deletion of histamine type I receptors
affects circadian pattern of ventilation.
Ishiguro, Takashi; Iwase, Michiko; Kanamaru, Mitsuko;
Izumizaki, Masahiko; Ohshima, Yasuyoshi; Homma,
Ikuo (Department of 2nd physiology, Showa University School of.
Medcine, Tokyo 142-8555, Japan)
Ventilation changes depending on circadian light-dark cycle. Histaminergic neurons are involved in the circadian regulation in the brain, but a
role of circadian pattern of ventilation is unknown. We examined ventilation and metabolism for 24 h in histamine type 1 receptor knockout
(H1RKO) and wild-type mice. Mice were measured for ventilation by a
whole-body plethysmograph and metabolism by a magnetic-type mass
spectrometry, and consciousness was characterized by electroencephalogram. In both genotypes, minute ventilation (VE) and metabolic rate
were greater during the dark period (20:00 to 8:00) than during the light
period (8:00 to 20:00). H1RKO mice during the light period were greater
in VE with increased tidal volume and greater in VCO2 without changes
in VO2 than wild-type mice. The increased VE during the light period
was responsible for the increased CO2 production with an identical ratio
of VE to VCO2. However, H1RKO mice decreased the ratio compared to
that of wild-type mice during the dark period, which was responsible for
an increase of delta/theta ratio calculated from power spectrum density
of electroencephalogram. Thus, deletion of H1 receptor affects patterns
of ventilation accompanied with changes in metabolic substrates and
arousal state of the dark period. Results suggest that histamine modulates
ventilatory pattern during the light and the dark periods via H1 receptors.
Habutobin inhibits the tyrosinephosphorylation of FAK at an early stage of
collagen-induced platelet aggregation
Nakamura, Mariko; Tengan, Hiroatu; Takara, Shigeru;
Yoshioka, Miwa; Hanashiro, Kazuhiko; Sunagawa,
Masanori; Kosugi, Tadayoshi (1st Dept of Physiol, Sch of Med,
Univ of the Ryukyus, Okinawa, Japan)
Habutobin, thrombin-like enzyme that converts rabbit fibrinogen to fibrin, was purified from T. flavoviridis . Habutobin bound to β3 subunit
of integrin αIIbβ3, and then habutobin inhibited the collagen-induced
aggregation, in previously study. We investigated the effect of habutobin
for intracellular signaling through tyrosine(Tyr)-phosphorylation of
protein occurring in the collagen-induced aggregation. Rabbit platelet
was washed with ACD-A and HEPES-Tyrode's sol (Ca2+, Mg2+ free)
and the washed platelet was suspended in the Tyrode's sol containing
Mg2+ (1 mM). From results of Western blot with anti-phosphotyrosine
antibody(4G10), Tyr-phosphorylation of several intracellular proteins
increased dramatically during collagen-induced aggregation. Tyrphosphorylated proteins were observed in 55-60, 72, 85 and 116 kD at
90 sec after the addition of collagen. In the presence of habutobin,
although Tyr-phosphorylated proteins was not observed at 90 sec after
the addition of collagen, they were observed in 60, 72 and 116 kD at 3
min after the addition of collagen. In addition, in the presence of
habutobin, FAK Tyr-phosphorylation was inhibited at an early stage of
collagen-induced aggregation, but it was not inhibited at a late stage of
collagen-induced aggregation. From the present study, it was suggested
that the binding of habutobin to β3 subunit of integrin αIIbβ3 resulted to
inhibit FAK Tyr-phosphorylation, and habutobin inhibits the β3
signaling, that is, outside-in signaling.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Kidney & body fluids
Intracellular pH dependent regulation of
neuronal nitric oxide synthase activity in
cultured mouse macula densa cells
Kawada, Hideaki1; Yasuoka, Yukiko1; Kokubo, Kenichi2;
Kobayashi, Hirosuke2; Kuroshima, Tomomi3; Fukuda,
Hidekazu4; Hirose, Shigehisa4; Kawahara, Katsumasa1
(1Dept. Physiol., Kitasato Univ. Sch. Med., Kanagawa, Japan;
2Dept. Clin. Engineering, Sch. Allied Health Sci., Kitasato Univ.,
Kanagawa, Japan; 3Dept Biosci, Kitasato Univ Sch Sci, Kanagawa,
Japan; 4Dept. Biol. Sci., Tokyo Inst. Tech., Kanagawa, Japan)
Kidney & body fluids
Kidney macula densa cells sense the chloride concentration of the fluid
in the adjacent lumen and control glomerular filtration rate. We have
used a functionally intact macula densa cell line (NE-MD) established
from immortalized renal cells in culture. NE-MD cells specifically express neuronal nitric oxide synthase (nNOS) regulated by either low
NaCl intake or furosemide (an inhibitor of Na+-K+-2Cl– symporter). We
have examined whether L-arginine-induced NO production is pH dependent or not in NE-MD cells, by using a NO-sensitive electrode. The NO
production was low in control, but increased when NE-MD cells were
treated with furosemide (12 µM) for 2 hrs. When furosemide-treated
NE-MD cells were incubated in the presence of 100 µM amiloride (an
inhibitor of Na+/H+ exchanger), L-arginine-induced NO production was
unaffected. However, the NO production significantly decreased by 42%
when the cells were placed in a solution containing amiloride. Similar
results were obtained when NE-MD cells were incubated in the low pH
solution (pH=7.1). These results strongly suggest that furosemideinduced expression of nNOS protein is not sensitive to acidosis, but its
activity is pH sensitive. This may partly account for polyuria in
abnormalities of electrolyte and acid-base balance.
Effects of angiotensin II and nicotine on rat
subfornical organ neurons
Effect of CO2 on intracellular pH in
perfused bullfrog proximal tubules
Ono, Kentaro; Kai, Aya; Nakamura, Taiji; Honda, Eiko;
Inenaga, Kiyotoshi (Kyushu Dental. Col., Fukuoka, Japan)
Kubota, Takahiro; Mori, Yoshiaki; Inui, Takaki; Yamaji,
Jyunko; Yoshida, Ryotaro (Dept. of Physiol., Osaka Med.
The subfornical organ (SFO) plays important roles for drinking behavior
and cardiovascular regulation. It is well known that excitation of SFO
neurons by angiotensin II (ANG) induces the two behavioral responses,
but that by nicotine (NIC) induces only cardiovascular responses. Therefore, there may be present subpopulations of SFO neurons showing different sensitivity between ANG and NIC. In the present study, we
verified that intracerebroventricular (i.c.v.) injections of ANG at 400 ng
induced large water intake in rats, but that of NIC at 50 µg induced small
water intake. The i.c.v. injection of NIC induced cardiovascular
responses increased c-Fos expression in the SFO. In slice preparation,
we compared with the responses of ANG and NIC on 101 SFO neurons
by using an extracellular recording system. ANG and NIC respectively
increased spontaneous firing frequencies in 67 and 62 SFO neurons.
Although 45 ANG-sensitive neurons had NIC-sensitivity, the remaining
22 neurons did not. Recently, we have reported cell classification of
dissociated SFO neurons, F- and S-types, and that a half of F-type and
all S-type neurons have ANG-sensitivity. A patch-clamp recording
showed that although almost F-type had NIC-sensitivity (n = 19/21), Stype did not (n = 7/8). From these results, we hypothesize that classified
S-type cells have ANG-sensitivity without NIC-sensitivity and are
related to drinking behavior, but not to cardiovascular responses, and
their function is different from that of F-type cells.
Coll., Takatsuki, Osaka, Japan)
We investigated the effect of basolateral CO2 on the cytosolic pH (pHi)
regulation in the proximal tubule of perfused bullfrog kidney by using
H+-selective microelectrode. Furthermore, in sliced kidney, we monitored the changes in acridine orange fluorescence in acid vesicles of
proximal tubule cells during the elevation of basolateral CO2 with or
without H+-pump inhibitors. Elevating baolateral CO2 from 1.5 to 5% at
constant HCO3– concentration induced an initial slight decrease followed by a sustained increase in pHi with 10 mV hyperpolarization of
basolateral membrane. In the presence of 10–6 M bafilomycin A1
(BAFA) or 10–7 M concanamycin A (CNCA), a specific H+-pump inhibitor, elevating basolateral CO2 produced no increase in pHi, but a decrease in pHi with a depolarization of basolateral membrane. The
increase in acridine orange intensity in acid vesicles was inhibited by the
perfusion of 10–6 M BAFA during the elevation of CO2 in the perfusion
fluid. The peritubular perfusion of 10–6 M BAFA suppressed the alkalinaization of pHi with 20 mM NH3/NH4+ application to peritubular perfusion fluid. These results indicate that intracellular H+ transport in the
acid vesicles as well as Na+/H+ exchanger or Na+-HCO3– cotransporter
in the cell membrane play an important role of the pHi regulation in the
proximal tubule of bullfrog kidney.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Kidney & body fluids
Galanin inhibits spontaneous firing of rat
subfornical organ neurons
Kai, Aya1; Ono, Kentaro1; Kawano, Hitoshi2; Honda,
Eiko1; Nakanishi, Osamu1; Inenaga, Kiyotoshi1 (1Kyushu
Dental Col., Fukuoka, Japan; 2Sch. Med. Univ. Saga, Saga, Japan)
Activation by angiontensin II (ANGII) of subfornical organ (SFO) neurons induces water intake and vasopressin release. On the other hand,
central and peripheral administrations of galanin (GAL) have been reported to inhibit water intake and vasopressin release in recent in vivo
studies. We anatomically, molecularbiologically and electrophysiologically investigated existences of GAL-receptors, and GAL-induced responses in SFO neurons of rats. Immuno-electron microscopic
observation revealed existence of GAL-containing synaptic vesicles in
the SFO. Conventional RT-PCR analysis demonstrated that there were
present all mRNAs of three known GAL receptor subtypes, GALR1,
GALR2 and GALR3, in the SFO tissues. In the extracellular recordings,
application of GAL inhibited spontaneous firing rate in more than half
of SFO neurons, even after synaptic blockade by a low [Ca2+] and high
[Mg2+] solution. The inhibitory responses were dose-dependent. Seventy-two percent of GAL-sensitive neurons were also responsive to ANGII. ANGII increased the firing rate. These results suggest that GAL
functions as an inhibitory neurotransmitter or neuromodulater in the
SFO. Furthermore, the electrophysiological results offer a plausible explanation for the in vivo-observed opposite effects of ANGII and GAL
on water intake and vasopressin release.
Analysis of ANP- and cGMP-activated
cation channels in the frog urinary bladder
Yamada, Toshiki; Matsuda, Koukei; Uchiyama, Minoru
(Dept. Life & Environmental Sci., Grad. Sch. Sci. & Eng., Univ.
Toyama, Toyama, Japan)
It has been known that atrial natriuretic peptide (ANP) inhibits Na+ reabsorption in renal tubules of the mammalian kidney. We, however, recently reported that ANP and cGMP stimulate amiloride-blockable Na+
absorption through PKA-dependent pathway in the urinary bladder of
the Japanese tree frog, Hyla japonica. We also suggested that there is a
possibility of expression of cyclic nucleotide-gated channels in the frog
urinary bladder using Ussing chamber voltage-clamp and whole cell
patch-clamp techniques (Yamada et al., 2005). In the present study, we
examined to identify characteristics of the channels activated with ANP
and cGMP by using a single channel recording in epithelial cells of urinary bladder. In the recordings of cell-attached patch with NaCl in a
patch pipette, a variety of ion channels were revealed the activity with
conductance ranging from 5 to 30 pS. Ion channels with conductance for
inward current of 5-7 pS were examined because the channels have the
same properties, such as conductance and slow opening and closing kinetics with the epithelial Na channels in the toad urinary bladder. When
Cl– in the pipette solution was replaced by gluconate, the conductance of
the channel did not change. This shows the channel carries cations but
anions. Addition of 10–4 M 8-Br-cGMP to the bath solution significantly
stimulated the inward current in the cell-attached patches. These results
suggest that ANP- and cGMP-dependent increases in Na+ absorption are
mediated by 5-7 pS cation channels in the epitherlial cells of the frog urinary bladder.
Modulation of two types of apical ion
channels in cultured mouse renal
collecting tubule cells
Kubokawa, Manabu; Komagiri, You; Nakamura,
Kazuyoshi (Dept. Physiol. II, Sch. Med. Iwate Med. Univ.,
Morioka, Japan)
Ion channels in the apical membrane of cultured mouse renal collecting
tubule cells were investigated using the patch-clamp technique. At least
two types of ion channels were observed with cell-attached patches under control conditions. One was an inwardly rectifying small conductance (20-30 pS) K channel (SK channel) and the other was an
intermediate conductance (40-50 pS) non-selective cation channel (NC
channel). Activity of the SK channel was relatively high, whereas that of
NC channels was usually low in the control condition. Addition of membrane-permeant cAMP analogue, 8Br-cAMP (100 µM), stimulated the
activity of SK channels but did not affect that of NC channels in insideout patches. In the presence of 1mM Ca2+, addition of Ca2+-ionophore,
ionomycin (1 µM), resulted in marked enhancement of NC channel
activity and reduction of SK channel activity. In inside-out patches, SK
channels required cytosolic ATP (1 mM) to maintain their activity,
which was not affected by cytosolic Ca2+. On the other hand, NC channel
was activated directly by elevation of cytosolic Ca2+, but not by ATP in
inside-out patches. These results suggest that regulatory mechanisms of
these channels are quite different, which may play a role in the
electrolyte transport along the collecting duct under various conditions.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Gastrointestinal functions
Effects of four drugs those affect
gastrointestinal motility on proximal
gastric tone in conscious dogs
Furukawa, Naohiro1; Hatano, Mizue1; Shimatani,
Tomohiko3; Kusunoki, Hiroaki2; Honda, Keisuke2;
Tanaka, Toshiaki2; Haruma, Ken2; Tsujioka, Katsuhiko1
(1Dept. Physiol., Kawasaki Med. Sch., Kurashiki, Japan; 2Dept. Int.
Med., Kawasaki Med. Sch., Kurashiki, Japan; 3Dept. General Med.,
Hiroshima Univ. Hosp., Hiroshima, Japan)
Gastrointestinal functions
Aim: Mosapride, selective 5-HT4 receptor agonist, and itopride, selective D2 receptor antagonist, were known to enhance motility of the distal
stomach and facilitate gastric empting. On the other hand, sumatriptan,
selective 5-HT1 receptor agonist, and paroxetine, 5-HT reuptake inhibitor, were reported to induce a relaxation in the proximal stomach. In this
study, effects of these four drugs on proximal gastric tone were researched using a barostat in conscious dogs with a gastric fistula. Methods: The effects of the drugs and those vehicles on the proximal gastric
volume at a constant intragastric tone were studied using a barostat. Simultaneously, proximal and distal gastric contractility, and duodenal
contractility was measured using three force transducers. Furthermore,
stepwise isobaric gastric distensions were performed before and after the
administration of the drugs. The same experiments were done in fasting
phase and in postprandial phase. Results & Conclusion: Effects of the
drugs on proximal gastric tone were more clearly seen in postprandial
phase than fasting phase, and sometimes differed from those on distal
gastric and duodenal contractility. Such a measurement of proximal gastric tone using a barostat in animal experiments seems to be useful for
estimation of effectiveness of some drug on gastrointestinal disorders.
The effects of central neuropeptide Y on
gastric relaxation of the proximal stomach.
Decrease in gastric acid secretory capacity
in aged mice
Shirota, Keisuke; Kobashi, Motoi; Xuan, Song-Xu;
Mitoh, Yoshihiro; Matsuo, Ryuji (Okayama Univ. Grad. Sch.
Kanai, Setsuko; Hosoya, Hiroko; Ohta, Minoru;
Miyasaka, Kyouko (Dept.Clin.Phusiol., Tokyo Metro. Inst.
Med. Dent. Pharma, Okayama, Japan)
Gerontol., Tokyo,Japan)
Potent appetite-stimulating effects of neuropeptide Y (NPY) are well
known. The present study undertook to demonstrate that the role of NPY
in gastric relaxation of the proximal stomach was examined in anaesthetized rats. Intragastric pressure (IGP) was measured using a balloon situated in the proximal part of the stomach. The administration of NPY
into the fourth ventricle induced relaxation of the proximal stomach in a
dose-dependent manner. The administration of an Y1 receptor agonist
[Leu31, Pro34] NPY induced a larger relaxation than NPY. The administration of an Y2 receptor agonist (NPY13-36) did not induce significant
changes in motility. Microinjection of [Leu31, Pro34] NPY into the caudal
part of the dorsal vagal complex (DVC) induced relaxation of the proximal stomach. In contrast, similar injections into the intermediate part of
the DVC increased IGP of the proximal stomach. The administration of
NPY into the fourth ventricle did not induce relaxation after bilateral injections of the Y1 receptor antagonist (1229U91) into the caudal DVC.
Bilateral vagotomy below the diaphragm abolished the relaxation induced by the administration of NPY into the fourth ventricle. The intravenous injection of atropine methyl nitrate reduced the magnitude of
relaxation induced by the administration of NPY. These results indicate
that NPY induces relaxation in the proximal stomach via Y1 receptors
situated in the DVC. This work was supported by a Grant-in-Aid for Scientific Research from the Japanese Society for the Promotion of Science
to MK.
The gastrointestinal (GI) tract demonstrates a number of changes that accompany advanced age. In the present study, we investigated gastric acid
secretion in young and old mice in response to chemical stimulation and
mechanical stimulation. Gastric acid secretion was measured in anesthetized mice. Proton pump (H+,K+ ATP ase) is a good maker of the parietal
cell function, therefore, protein expression of proton pump was determined by Western blotting. After 60 min basal collection, acid secretion
was stimulated by histamine (500 and 1000 mg/kg) or carbachol (10 and
20 mg/kg). To investigate the response to mechanical stimulation, the
stomach was distended by an intragastric injection of isotonic saline
(0.5, 1.0, 1.5 and 2.0 ml). 1) Administration of 2 doses of histamine produced a dose-dependent increase in acid secretion in young mice, whereas a higher dose of histamine could not produce a significant increase in
acid secretion in old mice. 2) Stimulatory effect of carbachol on gastric
acid secretion did not differ between young and old mice, although the
mean values tended to be lower in old animals than in the young. 3) The
response to mechanical stimulation tended to be lower in old animals. 4)
Protein expression of H+, K+ ATPase was significantly lower in old mice
than the young. The present study showed that gastric acid secretion was
different between young and old mice concerning to various kinds of
stimulations. The decrease in the secretory function of the stomach in the
old mice is partly associated with a decrease in parietal cells (H+,K+ ATPase ).
J. Physiol. Sci., Vol. 56, Suppl., 2006
Muscle physiology
Effects of exercises on cardiac and gastric
parasympathetic nerve activities in young
Sakakibara, Yoshikazu1; Hasunuma, Masashi1; Kishi,
Takuhiko1; Nagasaka, Mou2; Tanaka, Michiko2 (1Dept.
Effect of intracellular Cl– concentration on
acetylcholine-induced response in
endothelial cells of the guinea-pig
mesenteric artery
Psychol. Inf., Kanazawa Inst. Technol., Ishikawa, Japan; 2Miyazaki
prefectural Nursing Univ., Japan)
Yamamoto, Yoshimichi1,2; Suzuki, Hikaru2 (1Nagoya City
Univ. Sch. Nursing, Nagoya, Japan; 2Grad. Sch. Med. Sci., Nagoya
City Univ., Nagoya, Japan)
Thirteen young male subjects first lied in the supine position on bed with
eyes opened for 10 min, followed by 10 min exercise of a cycle ergometer with a load randomly chosen among three different ones inducible
heart rates as high as 15, 30, or 60% of the predicted maximal heart rate,
and again took a supine position as during the first rest for 40 min. ECG
and EGG were measured by using bio-amplifiers during all the experimental period, AD converted at the rate of 1kHz and stored into PC. RRintervals obtained from ECG were Fourier transformed by every 64, serially from the top to the end of the file with doubling about its 50%. Cardiac sympathetic (CS) as well as parasympathetic nerve activities (CPS)
were assumed from the power in high-frequency band (HF), and the relative magnitude of the power in low-frequency band (LF) to HF, respectively. EGG re-sampled by 10 Hz from the row EGG datum was Fourier
transformed by every 512, serially from the top to the end, with doubling
about its 50%. The power summed in the range between 2.4 and 3.6 cpm
was assumed to be gastric parasympathetic nerve activities (GPS). CPS
tended to increase only during 10-20 min district after 30% exercise
(ex30). GPS was significantly increased during 10-15 min district
(p<0.05) in the post-ex30 period. Either CPS or GPS after 60% exercise
was significantly depressed. These results suggested that moderate exercise could augment parasympathetic nerve activities, particularly in the
gastric region.
In vascular endothelial cells acetylcholine (ACh) increases [Ca2+]i which
in turn activates charybdotoxin-sensitive IKCa, apamin-sensitive SKCa
and ClCa channels. As a result, ACh induces a membrane hyperpolarization, but the response is sometimes transient and followed by the membrane depolarization. To investigate the possible mechanism for this
variation, a sheet of endothelial cells was isolated from a guinea-pig mesenteric artery and perforated whole-cell clamp experiments using amphotericin B were performed. The intracellular Cl– concentration was
modified by using either low-Cl– (20 mM) or high-Cl– (150 mM) pipette
solutions. In the current clamp mode, application of ACh (0.5 µM)
induced a large and sustained hyperpolarization when [Cl–]i was low,
while the response was small and transient when [Cl–]i was high. In the
voltage clamp mode in low [Cl–]i condition, the reversal potential of
ACh-induced current was -54.1 mV and it was changed to -27.8 mV
after the K conductance was blocked by charybdotoxin and apamin. On
the other hand, the reversal potential was changed from -57.8 mV to 76.0 mV by blocking Cl conductance with DIDS. When [Cl–]i was high,
the reversal potential was -22.0 mV and it was changed to -0.6 mV after
the K channels were blocked. These results indicate that enhanced Ca2+activated Cl– current modifies ACh-induced hyperpolarization to be
small and transient when the [Cl–]i is increased in some pathological
Contribution of each amino acid residue to
the dual effects of a synthetic peptide of
actin binding region of heat shock protein
20 on the contraction of skinned smooth
Yoshino, Yasumasa; Watanabe, Masaru (Dept. Physiol.
Tokyo Med. Univ, Tokyo , Japan)
Muscle physiology
Heat shock protein 20 (HSP20) has actin binding capacity and its amino
acid sequence of actin binding region (residues 110-121; GFVAREFHRRYR) is highly homogenous to the inhibitory region of skeletal muscle
troponin I (residues 104-115). Our previous study showed that, in TitonX-100 skinned muscle preparations from guinea pig taenia caeci, a synthetic peptide of the actin binding region of HSP20 (HSP20p) had both a
suppressing effect on the maximal Ca2+ induced force and an enhancing
effect on the Ca2+ sensitivity for the force (Yoshino et al., ). In the present
study, to evaluate the contribution of each amino acid residue of HSP20p
to the dual effects of HSP20p on the skinned taenia, we compared the effects of 11 HSP20p analogues, which consisted of single glycine replacement, on the Ca2+-induced contraction. Every residue of HSP20p was
necessary to achieve maximum inhibition of the Ca2+-induced contraction. On the other hand, replacing F111, V112, A113, E115, F116 or
R121 for glycine resulted in losing enhancing effects on the Ca2+ sensitivity for the force. These results suggest that, in phasic smooth muscles,
the pathways through which HSP20p modulated the maximal Ca2+ induced force and Ca2+ sensitivity for the force appears to be different.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Muscle physiology
Interaction between P2 antagonists and
Ca2+-dependent K+ channel antagonists in
the contraction of the guinea-pig vas
Contribution of each amino acid residue to
the force suppressing effect of cardiac
troponin I inhibitory peptide on the
contraction of skinned smooth muscles
Sakai, Saeko; Tosaka, Tsuneo (Tokyo Med. Univ., Tokyo,
Watanabe, Masaru; Yoshino, Yasumasa; Saifuding,
Musha (Tokyo Med. University, Tokyo, Japan)
Contractions evoked by electrical stimulation of 50 pulses with 40 Hz to
the guinea-pig vas deferens consist of L- and T-type Ca2+ channel-mediated adrenergic (α1) component and L-type Ca2+ channel-mediated
purinergic component. SK and BK channels closely located L- and Ttype Ca2+ channels greatly affect on the contraction, especially α1
adrenergic component. The α1 component consisting of the early and
late components was obtained by the treatment of P2 antagonist. P2
antagonist suramin (300 µM) not only antagonizes P2x purinoceptors
but also activates SK channels via P2Y2 purinoceptors (Ming et al).
PPADS (30 µM)-treated vas deferens revealed large early α1 component
suggesting that PPADS itself might inactivate SK channels. α,βMethylene ATP (desensitization of P2x purinoceptors, 10 µM) did not
affect directly K+ channels. SK channel antagonist apamin (100 nM)
amplified both early- and late-α1 components. BK channel antagonist
iberiotoxin (100 nM) amplified the late-α1 component without affecting
on the early-α1 component. On the contrary, another BK channel
antagonist CTX (100 nM) amplified not only the late-α1 component but
also the early-α1 component. CTX seemed to deprive the action of
PPADS to antagonize P2x purinoceptors. The results suggest that α,βmethylene ATP was suitable for antagonizing P2x purinoceptors, and
that apamin and iberiotoxin were more suitable for antagonizing SK and
BK channels, respectively.
TnIp, a synthetic peptide originating from an actin tropomyosin binding
region of rabbit cardiac troponin I [residues 136-147; GKFKRPTLRRVR], had biphasic effects on the relaxation of skinned smooth muscle,
as accelerating the initial phase and slowing the following latter phase of
the relaxation, resulted from accelerating fast cross-bridge dissociation
and also transformation from fast to slow (latch) cross-bridges (Watanabe et al., 2004). To evaluate the contribution of each amino acid residue of TnIp to its biphasic effects on the relaxation, we compared the
effects of 11 TnIp analogues, which consisted of single glycine replacement, on the relaxation time course by lowering Ca2+ concentration in
contracting skinned taenia caeci from guinea pig. Replacing K137, F138,
K139, R140, T142, L143, R144, or R145 for glycine resulted in loss of the accelerating effect of TnIp on the fast cross-bridge dissociation, seeming
that the entire residues of TnIp might be necessary for acceleration of the
cross-bridge dissociation by TnIp. On the other hand, the enhancing effect of TnIp on the translation from fast cross-bridges to latch bridges
was kept except only when R147 was substituted for glycine. R147 seems
to be a key residue for regulation of latch bridge formation by TnIp.
Effects of hypoxia on intracellular
mechanisms of smooth muscle cells
isolated from the guinea-pig stomach
Nakamura, Eri; Yokoi, Tsuyoshi; Suzuki, Hikaru
(Dept.Reg. Cell Physiol., Nagoya City Univ. Grad. Sch. Med. Sci.,
Nagoya, Japan)
Gastric smooth muscle cells generate slow waves spontaneously, and
their activities may be originated from interstitial cells of Cajal. Experiments were carried out to investigate the effects of hypoxia on the activities of smooth muscle cells isolated from the guinea-pig stomach
antrum by recording intracellular membrane potentials using conventional microelectrode methods, and also by measuring intracellular
Ca2+concentration ([Ca2+]i) with fura-2 fluorescence. Hypoxic condition
was produced by bubbling Krebs solution with N2-gass containing 5%
CO2. Hypoxia increased the frequency of slow waves with no significant
alteration to the membrane potential, and these were associated with the
increase in frequency and decreased in amplitude of Ca2+-transients. In
the presence of 10 µM cyclopiazonic acid (CPA, an inhibitor of Ca2+ATPase at internal Ca stores), the increase in frequency of slow waves
in response to hypoxia was abolished. In the presence of 1 mM KCN (a
blocker of oxidative phosphorylation), the frequency of slow waves was
decreased, with associated reduction of the amplitude. In the presence of
KCN, hypoxia again increased the frequency of slow waves. These
results suggest that hypoxia-induced increase in the frequency of slow
waves is associated with the elevated release of Ca2+ from internal CPAsensitive stores, possibly in the pacemaker cells. As the hypoxia-induced
increase in the frequency is not altered by KCN, no significant
contribution of mitochondrial factor in pacemaker cells is considered.
Functional overloading facilitates the
regeneration of injured skeletal muscles
Morioka, Shigeta1; Naito, Toshihito1; Kojima, Atsushi1;
Goto, Katsumasa1; Akema, Tatsuo1; Sugiura, Takao2;
Ohira, Yoshinobu3; Yoshioka, Toshitada4 (1Dept.
Physiol.,St. Marianna Univ. Sch. Med., Kawasaki, Japan; 2Facult.
Edu, Yamaguchi Univ., Yamaguchi, Japan; 3Grad. Sch. Med., Osaka
Univ., Osaka, Japan; 4Hirosaki Gakuin Univ., Aomori, Japan)
Muscle satellite cells have been considered to play an important role in
postnatal growth and the regeneration of skeletal muscle. Recently it has
been suggested that the activation of muscle satellite cells is also associated with muscle hypertrophy. Therefore, the regenerative process of injured muscles may be facilitated by the activation of muscle satellite
cells induced by various hypertrophic stimuli. The purpose of this study
was to investigate the effects of overloading on the regenerative process
of injured skeletal muscle in mice. Male mice (C57BL/6J) were divided
randomly into four groups: (1) cage control (CC), (2) cardiotoxin-injected (CX), (3) functional-overloaded (FO) and (4) CX+FO groups. In the
groups of FO with and without CX, overloading on soleus of both hindlimbs was performed by cutting the distal tendons of both plantaris and
gastrocnemius muscles. Two weeks after the surgery, cardiotoxin was injected into soleus muscles of both limbs in CX and CX+FO groups. Soleus muscles were dissected 14, 28 and 35 days after cardiotoxininjection. Responses of muscular protein contents and Pax7-positive
muscle satellite cells during the regeneration were analyzed. Evidences
suggest that functional overloading may facilitate the regeneration of injured skeletal muscles.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Muscle physiology
Characteristics of stem cells derived from
interstitial spaces of skeletal muscle:
Behavior and differentiation potential in
Characteristics of stem cells derived from
interstitial spaces of skeletal muscle:
Differentiation potential after
transplantation to non-muscle tissues
Tono, Kayoko1; Okada, Yoshinori1,2; Akatsuka, Akira1,2;
Tamaki, Tetsuro1 (1Muscle Physiology and Cell Biology Unit,
Okada, Yoshinori1; Tono, Kayoko2; Akatsuka, akira1,2;
Tamaki, Tetsuro2 (1TRSC, Tokai Univ. Sch. Med., Kanagawa,
Dept. Regenerative Medicine, Tokai Univ. Sch. Med., Kanagawa,
Japan; 2TRSC, Tokai Univ. Sch. Med., Kanagawa, Japan)
Japan; 2Muscle Physiology and Cell Biology Unit, Dept.
Regenerative Medicine, Tokai Univ. Sch. Med., Kanagawa, Japan)
Recently, we have identified multipotent stem cell populations residing
in the interstitial spaces of skeletal muscle. We characterized these cells
using FACS on the basis of cell surface antigen expression, and sorted
them as a CD34+/45- and CD34-/45- cell fractions from enzymatically
isolated cells. Cells in the CD34+/45- fraction (designated Sk-34 cells)
were >94% positive for Sca-1 and mostly negative (<3% positive) for
CD14, 31, 49, 144, c-kit and FLK-1showing that Sk-34 cells are not
committed endothelial progenitors. However, Sk-34 cells formed colonies in clonal cell culture, and CFU displayed the potential to differentiate into adipocytes, endothelial, and myogenic cells. We also identified
cells in the CD34-/45- (designated Sk-DN cells) fraction as a putative
cell population that includes further immature stem cells; that can form
clonal sphere-like colonies in a collagen based cell culture with bFGF
and EGF; and that exhibits the potential to differentiate into myogenic
and endothelial cells. In addition, the Sk-34 and Sk-DN cells included
only a few side-population (SP) cells and mostly composed by mainpopulation cells (1:1000). These findings demonstrated that multipotent
stem cells (Sk-34 and Sk-DN cells) residing in the interstitial spaces of
skeletal muscle, and potentially contribute to postnatal myogenesis and
vasculogenesis following muscle growth and/or muscle hypertrophy,
and there were not SP cells.
We have shown that multipotent stem cell populations residing in the interstitial spaces of skeletal muscle (Sk-34 and Sk-DN cells) can give rise
to myogenic, vascular (pericytes, vascular smooth muscle and endothelial cells), and neural (Schwann cells) cells, as well as contributed to the
synchronized reconstitution of blood vessels, muscle fibers, and peripheral nerves, with significant recovery of both mass and contractile function after transplantation. Intrinsic plasticity of these cells when there
were transplanted into non-skeletal muscle tissue environment was investigated. The renal capsule, spinal cord and heart were selected as nonskeletal muscle tissues. In the renal capsule, these cells could also give
rise to muscle fibers, nerve fibers, and blood vessels as well as in the
skeletal muscle tissue. In the spinal cord, however, MAP-2 positive neural cells and blood vessels were observed. Interestingly, in the cardiac
muscle tissue milieu, these skeletal muscle derived stem cells could differentiate into mono-nucleated cardiac muscle cells with an apparent
desmosome junctions between donor-recipient cells, and/or donor-donor
cells. Donor cell derived blood vessels were also observed. These results
suggest that Sk-34 and Sk-DN cells can give rise to myogenic, neural
and vascular cell lineage intrinsically, and these capacities can be affected by the different tissue milieus.
Characteristics of stem cells derived from
interstitial spaces of skeletal muscle:
Effect of transplantation to the sever
muscle damage model and differentiation
potential in vivo
Tamaki, Tetsuro1; Okada, Yosinori1,2; Tono, Kayoko1;
Akatsuka, Akira1,2 (1Muscle Physiology and Cell Biology Unit,
Dept. Regenerative Medicine, Tokai Univ. Sch. Med., Kanagawa,
Japan; 2TRSC, Tokai Univ. Sch. Med., Kanagawa, Japan)
We have shown that stem cell populations residing in the interstitial
spaces of skeletal muscle can give rise to myogenic-endothelial cell lineages, and were designated Sk-34 (CD34+/45-) and Sk-DN (CD34-/45) cells. Potential therapeutic use of these cells, such as the functional significance of the transplanted tissue, and vasculogenesis, myogenesis was
investigated in detail. For this purpose, we developed a severe-damage
model of mouse tibialis anterior muscle with a large deficit of nerve fibers, muscle fibers, and blood vessels. Freshly isolated and cultured Sk34 and Sk-DN cells were transplanted directly into damaged portion of
the muscle. Results showed that, after transplantation, implanted cells
give rise to myogenic, vascular (pericytes, vascular smooth muscle and
endothelial cells), and neural (Schwann cells) cells, as well as contributed to the synchronized reconstitution of blood vessels, muscle fibers, and
peripheral nerves, with significant recovery of both mass and contractile
function after transplantation. Intrinsic plasticity of these cells was also
revealed by fluorescence in situ hybridization (FISH) analysis for the
transplanted muscle detecting the Y chromosome. As well, there were no
donor-derived Sk-34 and Sk-DN cells in the muscle of lethally irradiated
bone marrow-transplanted animals, indicating that the Sk-34 cells were
not derived from bone marrow.
Improvement of therapeutic
neovascularization using skeletal musclederived stem cells
Iwaguro, Hideki1; Tono, Kayoko1; Okada, Yoshinori2;
Kobori, Michiru1; Ito, Rie1; Masuda, Haruchika1;
Asahara, Takayuki1; Tamaki, Tetsuro1 (1Dept. of
Regenerative Medicine, Tokai Univ. Sch. Med. Kanagawa, Japan;
Tokai Univ. Sch. Med. Kanagawa, Japan)
We have found multipotent stem cell populations residing in the interstitial spaces of skeletal muscle (designated Sk-34 and Sk-DN cells) can
give rise to myogenic, vascular (pericytes, vascular smooth muscle and
endothelial cells), and neural (Schwann cells) cells, as well as contributed to the synchronized reconstitution of blood vessels, muscle fibers, and
peripheral nerves. To expecting vasculogenic capacity of these cells, we
investigated the hypothesis that Sk-34 (CD34+/45-) cells may play an
important role for the ischemic tissues as a "tissue specific vasculogenic
cells" and may be contribute to vasculogenesis. For this purpose, the Sk34 cells, obtained from the muscles of 3-6 week-old GFP mice, were administrated intramuscularly into the nude mice with hindlimb ischemic
models (n=4 each), and same amount of physiological saline was administrated for control group. Two weeks after transplantation, the Sk-34
cells transplanted group demonstrated significantly less toe necrosis
(p<0.05), and enhanced recovery of peripheral perfusion measured by
Laser Doppler (p<0.05) compared to control group. Moreover, increase
in donor derived (GFP positive) CD31 positive cells and/or vessels can
be seen in treated animals compared to control by immunohistochemical
analysis. These findings indicate that this cell population represents an
accessible cell source that can be used therapeutically to improve postnatal neovascularization.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Muscle physiology
Roles of slow-fibers in recovery of skeletal
muscles from crush injury
Ishimine, Hisako1,2; Fukuda, Jun2; Matsui, Takemi1;
Seo, Akihiko1 (1Dept. of Management Systems Engineering,
Tokyo Metropolitan University, Tokyo, Japan; 2Dept. of Physiol.,
Natl. Defense Med. College, Saitama, Japan)
Skeletal muscle crush injuries occur in an incident when the limb is
trapped with a compressing force. Little is known about the recovering
mechanisms of injured muscles. To study these, we developed a new apparatus. For induction of crush injury, a pressure of 4.25 kg/cm2 was applied for 2 h on the left lower limb of the rat. We examined changes in
their histological and physiological properties. We report that slow fibers
play important roles in recovery of skeletal muscles from such injury. A
follow-up survey was done for 8 weeks on 12 species of hind limb muscles; their wet-weight, number of fibers, diameter and area of fibers were
measured. Slow fibers were identified by immunohistological staining.
Muscle function was analyzed from footprint. Soon after the crush, wetweight of muscles increased by 10-20% in 1-2 days (acute edematous
phase), decreased by 30% during 2 days to 2 weeks (atrophic phase) and
then increased gradually for 8 weeks (recovery phase). Diameter and
area of muscle fibers, which reduced in the first 2 weeks, started to increase gradually in the recovery phase. At 8 weeks, muscle weight exceeded 100% of the control (opposite-side muscles) although their fibers
were thinner than the control. Immunohistology revealed that slow fibers
showed a large increment in number in the recovery phase; it indicates
that they play a major role in recovery from the injury.
Udaka, Jun1; Ohmori, Shintaro2; Ishiwata, Shin'ichi2;
Ohtsuki, Iwao1; Kurihara, Satoshi1; Fukuda, Norio1
(1Dept.Physiol(II)., Jikei Univ.Sch.Med., Tokyo, Japan; 2Dept.of
Physics, Waseda Univ., Tokyo, Japan)
We investigated the effects of hindlimb immobilization (HI) on contractile properties of single skinned soleus fibers in rats. HI (6 weeks) resulted in reduced wet weight of soleus muscle (~40%). In immobilized
fibers, maximal Ca2+-actiavated force was reduced by ~40% and the
force-pCa curve was shifted to the lower pCa side by ~0.15 pCa units
(sarcomere length, 2.20 µm). Our EM observation revealed thinner
myofbrils in immobilized muscle, with the width of Z-line similar in
control and immobilized muscles during relaxation and contraction (pCa
4.5). We reconstituted thin filaments of control and immobilized fibers
with the identical troponin complex (from rabbit psoas muscle). It was
found that Ca2+ sensitivity was still lower in immobilized fibers by ~0.15
pCa units after troponin reconstitution, suggesting that troponin isoform
switches, if at all, can not account for functional changes of immobilized
fibers. We then induced Ca2+-independent active force by lowering the
MgATP concentration (= -log [MgATP], from 5 to 7) to investigate
whether or not the decreases in Ca2+-activated force are the result of
reduced cross-bridge formation. We found that in immobilized fibers,
maximal Ca2+-independent active force was reduced by ~40%, with a
leftward shift of the force-pMgATP curve. These results suggest that
cross-bridge recruitment is suppressed in immobilized muscle, via e.g.,
structural changes of the sarcomere, resulting in reduced active force
Sex difference and influence of menstrual
cycle on the muscle pressure pain
threshold and delayed onset muscle
Ando, Akemi1; Taguchi, Toru2; Mizumura, Kazue2
(1Dept.Home Economics. Aichi Gakusen Univ. Okazaki,
Aichi,Japan; 2Dept. Neural. Reg. Rese. Inst. Environ. Med. Nagoya
Univ. Nagoya, Aichi, Japan)
Many reports have shown that there are sex differences in pain sensitivity, and that hormonal condition also modulates pain sensitivity. Because
muscle pain sensitivity was not studied yet in Japanese, we examined sex
differences and differences in different phases of menstrual cycle in
muscle pain sensitivity of young Japanese. Healthy subjects (10 males,
10 females in follicular phase, and 10 females in luteal phase) were recruited from students of Aichi Gakusen University and provided informed consent. The age ranged between 18 and 22 years old.
Experimental muscle pain was induced in the biceps brachii muscle by
eccentric exercise (ECC). A weight was placed around the wrist of undominant arm, and the subjects were asked to flex and extend the arm.
Pressure pain threshold of the biceps brachii muscle (PPT), elbow joint
angles that the subjects could flex or extend without pain, blood pressure, hemoglobin and skin fold thickness were measured. The females
showed a significantly lower basal PPT, maximal blood pressure, and
blood hemoglobin content, and their skin fold was thicker than the
males. The PPT and blood hemoglobin in luteal phase were significantly
lower than the follicular phase. PPT decreased 1 day after ECC and returned to the pre-exercise value 4 days after ECC. This time course in the
development of delayed onset of muscle soreness was not different
among groups. Present data showed clear sex and menstrual cycle differences in muscle pain threshold.
The Effect of structural change on single
soleus fiber after hindlimb immobilization
in rats
Passive tension of cardiac and skeletal
muscle with a reference to the domain
structure of connectin.
Kimura, Masako1; Kimura, Sumiko2; Takemori, Shigeru1
(1Dept. Physiol., Jikei Univ. Sch. Med,, Tokyo, Japan; 2Dept. Biol.,
Facul. Sci., Chiba Univ., Chiba, Japan)
Titin/connectin is a giant spring-like protein that is responsible for passive tension generation of striated muscle. We observed the passive tension of the bundles of myofibrils from rabbit cardiac and psoas muscle,
and that from chicken breast muscle at various sarcomere lengths (SL)
to compared it with the domain structures of connectin predicted from
the cloned cDNAs. The bundles of the myofibrils of rabbit cardiac muscle were prepared mechanically from the homogenized muscle tissue in
the relaxing solution. The specimen was held by a pair of sharp glass
capillaries, and tension was estimated from their bending. SLs were observed microscopically. The passive tension of rabbit psoas and chicken
breast muscle was measured with tension transducers. Moderate extension of the specimens developed a small passive tension that rapidly decayed to a steady level. Significant tension development began with a
stretch beyond particular SL characteristic for each muscle type. That is,
rabbit cardiac myofibrils began to develop tension at the shortest SL
(around 2.0 µm), and chicken breast and rabbit psoas muscle began to
develop tension at longer SLs (at around 2.3 and above 2.6 µm,
respectively). On the other hand, the predicted length of PEVK region of
connectin was human cardiac < chicken breast < human psoas < human
soleus muscle. Assuming the connectin of rabbit to be similar to human,
these data strongly suggests that the length of PEVK region correlates
closely with the passive tension development at short SL.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Muscle physiology
Molecular dynamics study on mutant
troponin related to cardiomyopathy
Yamaguchi, Maki; Otsuka, Yumiko (The Jikei Univ. Sch. Med.
Effect of troponin exchange on lengthdependent activation in porcine ventricular
Tokyo, Japan)
Fukuda, Norio; Ohtsuki, Iwao; Kurihara, Satoshi (Dept.
A mutant troponin T of which glutamate is replaced with aspartate
(Glu244Asp) is one of the causes of familial hypertrophic cardiomyopathy (HCM). Incorporation of this mutant to skinned fibers has been reported to increase calcium sensitivity as well as maximal tension
(Nakaura et al. 1999). However, mechanism has not been elucidated.
Therefore, we constructed a model structure of this mutant troponin by
introducing the mutation to the crystal structure of human cardiac troponin (TIC complex) obtained from Protein Data Bank (ID number 1J1E).
Molecular dynamics simulation of the wild and the mutant structure was
carried out in water at 310 K to estimate a dynamic structure of the mutant troponin and search a possible mechanism for the enhanced tension
development. Dynamics was calculated by the use of software Amber
ver.7. Iteration was done in TIP3 water with 0.5 or 1 fs time step in periodic condition at constant temperature (310 K). It was found that the
electrostatic interaction between T244 and I111 which linked troponin T
and I in the wild was lost in the mutant. Furthermore, when a terminal
residue of troponin I was pulled toward an actin molecule, troponin T
seemed to be easier to follow troponin I in the mutant than that in the
wild. This may be involved in the enhanced tension development in the
mutant myofilament.
Physiol., Jikei Univ., Tokyo, Japan)
At the basis of the Frank-Starling mechanism of the heart is the intrinsic
ability of the contractile system to produce active force in response to
stretch. It has been reported that length-dependent activation is, at least
in part, modulated via interfilament lattice spacing reduction due to titinbased passive force (e.g., Fukuda et al., J. Physiol. 553, 147-154, 2003).
In the current study, we examined whether or not length-dependent activation is modulated at the thin filament level. We used skinned porcine
ventricular muscle that had been treated with 1% (w/v) Triton X-100. An
increase in sarcomere length (SL) from 1.9 to 2.3 µm exerted a marked
increase in Ca2+ sensitivity with a concomitant increase in passive force.
The SL-dependent increase in Ca2+ sensitivity was markedly attenuated
by perfusing preparations with rigor solution containing exogenous fast
skeletal troponin (T-I-C complex; rabbit psoas muscle), with little or no
effects on passive force. The magnitude of SL dependency was similar
to what was observed in rabbit psoas muscle. Our SDS-PAGE analyses
showed that endogenous cardiac troponin subunits were replaced with
skeletal counterparts by ~100% and that titin was not degraded upon
troponin exchange. These results suggest that, presumably downstream
of titin-based lattice spacing reduction, length-dependent activation is
modulated at the thin filament level.
Crossbridge dynamics during length
clamp after preload shortening in rat
papillary muscle
Plasmalemmal estrogen receptor (ER)
regulates ERα expression via PKC/MAPK
Toyota, Hiroko1; Okuyama, Hiroshi1; Mohri, Satoshi2;
Shimizu, Juichiro3; Miyasaka, Takehiro2; Tsujioka,
Katsuhiko1; Yagi, Naoto4 (1Dept. Physiol. Kawasaki Med. Sch.
Hatae, Junna1; Takami, Noboru2; Inoue, Ryuji1 (1Sch.
Kurashiki, Japan; 2Dept. Cardiovasc. Physiol., Okayama Univ.
Grad. Sch. Medicine and Dentistry Okayama, Japan; 3Dept.
Physiol. II, Nara Med. Univ. Kashihara, Japan; 4SPring-8/JASRI
Sayo-gun, Japan)
The intensity ratio of (1,0)/(1,1) equatorial x-ray diffraction indicates the
mass transfer from myosin filament to actin filament. This ratio decreases during isometric contraction. During a very low load shortening, the
amount of changes in this ratio reduced. To examine this reduced ratio
carefully, the length clamp method was introduced in rat papillary muscle. When the shortening was stopped at any moment during a preload
shortening, the isometric force was developed in accordance with the residual activity of crossbridges (length clamp). During the length clamp,
both the shortening length and the force were located between those of
preload and isometric contractions. Similarly the intensity ratio was also
located between those of preload and isometric contractions. After the
clamp of length, the time course of intensity ratio quickly separated from
that of preload contraction. If the number of crossbridges actually reduced during the preload shortening, the time course of intensity ratio
during length clamp was expected to follow that of preload shortening
because of the latency in crossbridge formation. These results suggest
that the number of crossbridges will not decrease during the preload
shortening, and that not so small number of cross-bridges that can contribute the force development are left even under the low load shortening.
Med. Univ. Fukuoka, Fukuoka, Japan; 2Radioisotope Raboratory)
We investigated cellular signaling cascades linked to estrogen-induced
ER synthes is in mouse C2C12 myoblasts. By immunoblotting, ER was
detected in the post-nuclear fraction with a corresponding molecular
weight of 66 kDa. The amount of ER protein was dose-dependently (10–
12-10–5 M increased after treatment with 17β-estradiol for 24 hours. 17αestradiol (10–8M), the stereoisomer of 17β-estradiol, and a BSA-17βestradiol-conjugate which is incapable of penetrating the plasma
membrane, mimicked the increasing effects on ER. The level of ER
expression was reduced by an ERK1/2 inhibitor, PD 98059(10µM), or a
specific p38 inhibitor, SB 203580 (10µM) regardless of the presence of
estradiol. Using 35S-methionine for immunoprecipitation, newlysynthesized ER was increased by 17β-estradiol. Novo-synthesis of ER
was further increased by the protein kinase C (PKC) activator, TPA
(1µM). These results suggest that ERs located at the plasma membrane
of mouse skeletal myoblast cell are a target of estrogen's actions. The
estrogen-stimulated ER synthesis involves the PKC/MAPK signaling
system, thereby presumably regulating the proliferation process.
However, somewhat contra-intuitively, up-regulation of estrogen
receptor was also observed in the mouse skeletal muscle treated with
antiestrogens, such as tamoxifen and ICI182780.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Muscle physiology
Regulation of gastrointestinal pacemaker
activity via type 3 serotonin receptors
Liu, Hong-Nian1; Susumu, Ohya2; Yuji, Nishizawa,3;
Yuji, Imaizumi2; SHinsuke, Nakayama1 (1Dept. Cell
Physiol., Nagoya Univ. Grad. Sch. Med. Nagoya, 466-8550 Japan.;
2Dept. Mol. Cell. Pharmacol., Grad. Sch., Nagoya City Univ.,
Nagoya 467-8603, Japan; 3Dept. Anat. Cell Biol., Nagoya Univ.,
Grad. Sch. Med., Nagoya 466-8550, Japan)
We assessed the involvement of serotonin receptors (5-HT-R) on pacemaker Ca2+ activity in gastrointestinal (GI). Small tissues of 100-300
mm diameter (cell cluster preparations) were prepared from the stomach
and small intestine muscle (including the enteric neurones) of mice by
enzymatic and mechanical treatments. After 2 to 4 days of culture, the
fluo-3 AM (acetoxymethly ester of Fluo-3) was loaded to measure the
intracellular Ca 2+ concentration ([Ca2+]i). In the presence of nifedipine,
we measured ([Ca2+]i) oscillations in c-Kit-immunopositive pacemaker
cells (= interstitial cells of Cajal: ICCs) and examined the effects of several drugs: LY-278584, 2-Methylserotonin maleatesalt, GR113808, Fluoxeline, SK&F96365, etc. RT-PCR and immunohistochemical
examinations were carried out to characterize expression of serotonin receptor subtype in pacemaker cells. We conclude that endogenous 5-HT
plays a crucial role in generating and maintaining pacemaker Ca2+ activity in ICCs via type 3 serotonin receptors (5-HT 3) under basal conditions, and a potentiates Ca2+ responses to electrical stimuli by facilitating
cholinergic neurons. Our results may provide a new therapeutic target in
diseases associated with abnormal GI motility, and add an important
member (i.e. ICCs) to assess the brain-gut (The Second Brain) interaction.
Effects of synthetic peptides orinating from
small heat shock proteins on the skinned
carotid artery from guinea pig
Hashimoto, Ryo1,2; Konishi, Masato1; Miki, Tamotsu2;
Haraoka, Jo2; Watanabe, Masaru1 (1Tokyo Med. University,
Tokyo, Japan; 2Tokyo Medical University, Tokyo Japan)
Heat shock protein 20 (HSP20) has actin binding capacity and its amino
acid sequence of actin binding region (residue 110-121; GFVAREFHRRYR) is highly homogenous to the inhibitory region of skeletal muscle
troponin I (residue 104-115; TnIp). Previous study by Rembold et al
(2000) showed that, in carotid artery, HSP20 monomer might directly interfere with actin-myosin resulting in the smooth muscle relaxation. In
phasic skinned taenia caeci, however, a synthetic peptide of HSP201102+
121 enhanced Ca -induced force development except under the condition of high concentrations of Ca2+, so increased in the Ca2+-sensitivity
for the force (Yoshino et al., 2003). To determine whether HSP20p also
enhance Ca2+-induced contraction in tonic skinned carotid artery as well
as in taenia, we studied the effects of HSP20p on contractile properties
of beta escin skinned muscle preparations from guinea pig carotid artery.
In skinned carotid artery, HSP20p suppressed Ca2+-induced contraction
with little effects on the Ca2+-sensitivity for the force. Also a synthetic
peptide originating from HSP27 actin binding region (residues 131-142;
GYISRCFTRKYT; HSP27p) suppressed Ca2+-induced contraction.
These results suggest that, different from the case of phasic smooth muscles, HSP20p might mainly affect thin filament and interfere with actinmyosin interaction causing muscle relaxation in tonic carotid artery.
Identification and spatio-temporal analysis
of the rat pelvic pacemaker region using a
macro zoom microscope and voltagesensitive dye
Yamashtia, Toshikazu1; Kokubun, Shinichiro1,2; Konishi,
Masato3; Nakayama, Shinsuke4 (1Dept. Physiol., Nihon
Univ. Sch. Med., Tokyo, Japan; 2Advanced Medical Reserch Center,
Nihon Univ. Sch. Med., Tokyo, Japan; 3Dept. Physiol., Tokyo
Medical College, Tokyo, Japan; 4Dept. Cell Physiol., Sch. Med.,
Nagoya Univ., Nagoya, Japan)
As we reported in the last meeting in Sendai, we established in situ Ca2+
imaging technique of the rat renal pelvis using a macro zoom microscope
(Olympus MVX 10). The clear view and smooth zooming operation with
this technique enabled us to search upstream of Ca2+ transient and to
identify the pacemaker region easily. With higher magnification of the
region, we could observe that not only one cell but several cells increased their intracellular Ca2+ concentration simultaneously. Interestingly their propagating pathway were slightly different every time.
Spontaneous Ca2+ rises in the other part of renal pelvis rarely occurred;
they never propagated to the downstream. Contrary many spontaneous
but asynchronous Ca2+ rises were observed in the presence of low concentration of heptanol, a gap junction blocker. Using the same technique
we could also successfully record image of di-4-ANEPPS, a voltagesensitive dye. The initial depolarization occurred at the exactly same
place as the Ca2+ rise. These results might suggest that the smooth muscle cells connected via gap junctions and with synchronous Ca2+ rise
played an important role in the pacemaker mechanisms.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Ionic channels & receptors
Differences in the sensitivity of the TTXresistant Na+ channel to the PKCβ inhibitor
LY333531 in small dorsal root ganglion
neurons of control and diabetic rats
Hayase, Fumiko1; Matsuura, Hiroshi2; Toyoda, Futoshi2;
Kitada, Kanako1; Sanada, Mitsuru1; Omatsu-Kanbe,
Mariko2; Yasuda, Hitoshi3 (1Div.Neurol.,Dept.Medicine,Shiga
Univ.med.Sci.,Otsu,Shiga,Japan; 2Dept.Physiol.,Shiga
Univ.Med.Sci.,Otsu,Shiga,Japan; 3Dept.Community Life
Ionic channels & receptors
Experimental evidence has been presented to suggest that the protein kinase Cβ inhibitor LY333531 is effective at alleviating diabetic
hyperalgesia to some extent. The present study was designed to examine
the acute action of LY333531 (0.01-1µM) on the tetrodotoxin (TTX)resistant Na+ current (INa) in small (<25 mm in soma diameter) dorsal
root ganglion (DRG) neurons in control and streptozocin (STZ)-induced
diabetic rats, using the whole-cell patch-clamp method. The cell
membrane was initially hyperpolarized from a holding potential of -70
mV to -120 mV for 20 ms and then depolarized to various test potentials
ranging from -50 to +50 mV, in the presence of TTX (0.1 mM) and the
appropriate blockers for Ca2+ and K+ currents. INa was measured as a
transient inward current during depolarizing steps. The maximal density
of INa was significantly increased in diabetic rats compared with control
(50.5 pApF–1 vs 32.3 –1). INa recorded from diabetic rats was found to
exhibit a significantly higher sensitivity to inhibition by LY333531
compared with control (IC50, 6 nM vs 30 nM). Thus, our results provide
experimental evidence to show that the sensitivity of INa to LY333531 is
substantially enhanced by diabetic state, which suggests that INa is
considerably inhibited by LY333531 at clinically relevant
concentrations of <100 nM in diabetic state.
Effect of 8-bromo-cAMP on the
tetrodotoxin-resistant sodium (Nav 1.8)
current in rat small-diameter nodose
ganglion neurons
Matsumoto, Shigeji; Yoshida, Shinki; Saiki, Chikako;
Takeda, Mamoru (Dept.Physiol., Sch.Dent. at Tokyo, Nippon
Dental Univ., Tokyo, Japan)
We examined whether 8-bromo-cAMP (8-Br-cAMP)-induced modification of tetrodotoxin-resistant (TTX-R) sodium current (INa) in neonatal
rat nodose ganglion neurons is mediated by the activation of protein kinase A (PKA) and/or protein kinase C (PKC). In 8-Br-cAMP applications ranging from 0.001 to 1.0 mM, 8-Br-cAMP at 0.1 mM showed a
maximal increase in the peak TTX-R Na+ (Nav1.8) current and produced
a hyperpolarizing shift in the conductance-voltage (G-V) curve. The
PKC inhibitor bisindolylmaleimide Ro-31-8425 (Ro-31-8425, 0.5 µM)
decreased the peak Nav 1.8 current. The Ro-31-8425-induced
modulation of the GV1/2 baseline (a percent change in G at baseline V1/2)
was not affected by additional 8-Br-cAMP application (0.1 mM). The
application of a PKC activator, phorbol 12-myristate 13-acetate (PMA,
0.1 µM), increased the Nav 1.8 current, and this increase was not
significantly affected by additional 0.1 mM 8-Br-cAMP application.
Intracellular application of a PKA inhibitor, protein kinase inhibitor
(PKI, 0.1 mM), greatly attenuated the 8-Br-cAMP-induced increase in
the peak Nav 1.8 current but caused a significant increase in the slope
factor of both activation and inactivation curves. The PKI application
lowered the G max to below the control level. These results suggest that
the 8-Br-cAMP-induced increase in Nav 1.8 currents may be mediated
by activation of PKC.
Prostaglandins have no detectable effect
on the tetrodotoxin-resistant sodium
currents mediated by Nav1.8 and Nav1.9 in
small neurons from mouse dorsal root
Zheng, Taixing; Kakimura, Jun-ichi; Matsutomi,
Tomoya; Nakamoto, Chizumi; Ogata, Nobukuni
(Department of Neurophysiology, Gaduate School of Biomedical
Sciences, Hiroshima University, Hiroshima, Japan)
One possible mechanism that may contribute to the inflammatory hyperalgesia, an inflammation-induced sensitization of primary afferent neurons, is the modulation of tetrodotoxin-resistant (TTX-R) voltage-gated
Na currents by inflammatory mediators such as prostaglandins (PGs).
Evidence for this compelling idea is mainly based on the electrophysiological finding that the TTX-R Na current, which is expressed preferentially in small primary afferent neurons and likely to be involved in
nociception, is augmented by inflammatory mediators. However, the detailed mechanism underlying this observation has not been fully investigated. We re-investigated the effect of PGE2 (and also PGI2) on the
heterogeneous TTX-R Na currents mediated by Nav1.8 and Nav1.9 in
mouse dorsal root ganglion neurons, using conventional and nystatinperforated whole-cell patch-clamp recordings on wild-type and Nav1.8null mutant DRG neurons. Unexpectedly, PGE2 (and also PGI2) had no
detectable effect on these TTX-R Na currents, raising a question regarding the well-known modulatory role of PGs on TTX-R Na currents in inflammatory hyperalgesia.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Ionic channels & receptors
CaMKII phosphorylates the C-terminal tail
of Cav1.2 Ca2+ channel and modulates
interaction of the channel with calmodulin
Saud, Zahangir
Minobe, Etsuko1; Han, Dong-Yun1,2;
A. ; Hao, Li-Ying1; Kameyama, Masaki1
(1Grad.Sch.of Med.& Dent.Sci.Kagoshima Univ,kagoshima,Japan;
Sch. Med. Univ, China, Shenyang, China)
Simulation study of the current through the
L-type Ca2+ channels
Suzuki, Shingo1; Findlay, Ian2; Kurachi, Yoshihisa3
(1Clinical Genome Informatics Centre, Kobe Univ. Sch. Med.;
Univ.; 3Osaka Univ. Sch. Med.)
Cav1.2 Ca2+ channel is suggested to be modulated by calmodulin (CaM)
and Ca2+/CaM-dependent protein kinase II (CaMKII), that are proposed
to underlie Ca2+-dependent inactivation and facilitation of channel activity. To explore phosphorylation sites for CaMKII, three GST-fusion fragment peptides derived from the C-terminal tail of guinea-pig Cav1.2,
CT-1 (amino acids number 1509-1791), CT-2 (1777-2003) and CT-3
(1944-2169) were examined in vitro. Only CT-1 was consistently phosphorylated by CaMKII. By using mutated CT-1, the phosphorylation site
was suggested to be threonine residue at position 1603. In pull-down assay, CT-1 but not CT-2 nor CT-3 was found to interact with CaM at both
low and high [Ca2+] conditions. CT-1 treated with CaMKII showed a
higher affinity for CaM than that treated with alkaline phosphatase.
These results suggest that interaction between CT-1 and CaM is modulated by phosphorylation mediated by CaMKII, and that they are consistent with the hypothesis that both CaM and CaMKII are involved in
maintaining basal activity of the channel and in Ca2+-dependent inactivation and facilitation of the channel.
L-type Ca2+ channel currents have the voltage-dependent inactivation
(VDI) and Ca2+ dependent inactivation (CDI) processes. These inactivation processes regulate the the amount of Ca2+ influx into cardiac myocytes. Under the β-adrenergic stimulation, the CDI dominate the decay
of Ca2+ current since the VDI is slow (I. Findlay, J. Physiol., 2002). In
this study, we have modeled the VDI and CDI under the control and the
β-adrenergic stimulation (100nM isoproterenol). We have developed the
VDI model by using the experimental time course of VDI estimated
from the outward-going K+ current through the Ca2+ channel in the
absence of extracellular Ca2+. The current dacay calculated by using the
pure VDI was close to the experimental Ba2+ current decay which
possessed minimal CDI. After that, we modeled the CDI satisfying the
experimental current. The inactivation rate was expressed as a function
of the Ca2+ current. In this formula, two components CDI was adopted
to reproduce the strong bi-phasic decay for the β-adrenergic stimulation.
The Ca2+ current dependent K+ current through the Ca2+ channel are
introduced into this model to reproduce the experimental reversal
potential. The calculated time course of the inward Ca2+ current was in
excellent agreement with the experimental results. Therefore we have
successfully established the inactivation model of the L-type Ca2+
channel in consistent with the experimental results.
Calcium- and dose-dependent effects of
calmodulin on activity of Cav1.2 Ca2+
channels in guinea-pig ventricular
Wang, Wu-Yang1; Xu, Jian-Jun1; Kameyama, Masaki1
(1Grad.Sch.of Med.&Dent.Sci.Kagoshima Univ,kagoshima,Japan;
2Pharm. Sch. Med. Unv. China, Shenyang, China)
We have previously reported that calmodulin (CaM) + ATP can reverse
rundown of Cav1.2 L-type Ca2+ channels observed in the inside-out
patch mode. In this study we have examined Ca2+- and dose-dependent
effects of CaM on activity of the Ca2+ channels in guinea-pig ventricular
myocytes. Application of CaM (0.1-14 µM) + ATP (3 mM) to the
intracellular side of the channels within 1 min after patch excision
resulted in a bell-shaped dose-dependent effect with a maximum effect
at 2-4 µM CaM at low [Ca2+] condition (Ca2+-free to 80 nM). This
relation between CaM and channel activity may be related to Ca2+dependent facilitation and inactivation of the channel. This bell-shaped
curve for the dose-dependent effect of CaM on channel activity was
significantly shifted toward left (lower concentration) by increasing
[Ca2+]. These results suggest that CaM plays a crucial role in regulation
of the Cav1.2 Ca2+ channels, and that not only [Ca2+] but also CaM
concentration are important factors for functional modulation of the Ca2+
Store-operated Ca2+ entry activated by
hyperpolarization in rat submandibular
acinar cells
Yoshida, Hideyo; Nakahari, Takashi (Dept. Physiol., Osaka
Med. College, 2-7 Daigaku-cho, Takatsuki, Japan)
Store-operated Ca2+ entry activated by hyperpolarization in rat submandibular acinar cellsYoshida, H. and Nakahari, T.Department of Physiology, Osaka Medical College, 2-7 Daigaku-cho, Takatsuki 569-8686,
JapanIn rat submandibular cells, ACh evoked a biphasic increase in
[Ca2+]i, that is, an initial transient phase followed by a sustained phase.
The initial transient phase is induced by a Ca2+ release from the intracellular stores and the sustained phase is maintained by a Ca2+ influx from
the extracellular fluid, which is the so-called "store-operated Ca2+ entry". Store-operated Ca2+ entry were stimulated using 4 µM thapsigargin,
sarco(end)plasmic reticulum Ca2+ ATPase inhibitor, in Ca2+-free
solution followed by superfusion with control (Ca2+-containing)
solution. This protocol resulted in a [Ca2+]i increase, which was inhibited
in the presence of 1 µM Gd3+. A restoration from 150 mM K+
solution(Ca2+-containing) to control solution also evoked an [Ca2+]i
increase during thapsigargin stimulation, and an [Ca2+]i increase
following reintroduction of Ca2+ during thapsigargin stimulation was
completely inhibited by addition of 1 µM Gd3+. Although a restoration
from 7.5 mM K+ solution to control solution did not evoke an [Ca2+]i
increase during thapsigargin stimulation, addition of tetraethyl
ammonium into 7.5 mM K+ solution evoked an [Ca2+]i increase.
Consequently, Ca2+ entry pathways is activated by the store-depletion
and hyperpolarization.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Ionic channels & receptors
BK channels in
store control
Yamashita, Masayuki; Sugioka, Miho; Ogawa, Yoichi
Analyses of heteromultimeric assembly of
Kir2.1 and Kir3.4 inward rectifier K+
channel subunits
(Nara Med. Univ. Kashihara, Japan)
Kubo, Yoshihiro1; Ishihara, Keiko2 (1Div. Biophys. and
release from intracellular
stores terminates after a rapid release of a fraction of releasable Ca2+ (called "quantal" release). To explain the quantal nature, it has been hypothesized that a decrease in
luminal Ca2+ attenuates Ca2+ efflux. However, the mechanism remains
an enigma. We show that voltage- and Ca2+-activated potassium channels in Ca2+ store control Ca2+ release. The potassium channel was identified as BK-type by patch-clamp recordings from an enlarged nuclear
envelope in the nucleus-attached mode and by immunolabeling. The
store BK channel was activated by positive shifts in luminal potential
and luminal Ca2+ increases. The closing or blockage of store BK channels developed lumen-negative potentials and suppressed Ca2+ release.
Ca2+ uptake by store Ca2+ pumps would reactivate the store BK channels
regeneratively with K+ entry to allow repetitive Ca2+ release. Indeed, the
luminal potential oscillated bistably ~45 mV in amplitude as revealed
with an organelle-specific voltage-sensitive dye [DiOC5(3)]. Our study
suggests that Ca2+ release-induced closings of store BK channels cause
a lumen-negative potential towards the equilibrium potential for Ca2+ to
attenuate Ca2+ efflux.
Neurobiol., Dept. Mol. Physiol., Natl. Inst. for Physiol. Sci.,
Okazaki, Aichi, Japan; 2Dept. Physiol., Facult. Med., Saga Univ.,
Nabeshima, Saga, Japan)
Facilitation of HERG channel can be
induced by various HERG blockers
Iwata, Miki1; Hosaka, Yukio2; Kurachi, Yoshihisa1
(1Dept.Pharmacol., Osaka Univ.Sch.Med.,Osaka, Japan;
2Dept.Cardio.,Nigata City Hospital,Nigata,Japan)
Blockers of cardiac I kr channels encoded by HERG gene is reported as
the drug clinically effective for lethal ventricular arrhythmias. However
the kinetic property of a HERG channel by these blockers is not yet studied in detail. We attempted to find the characteristics.
By using the standard two-microelectrode voltage clamp technique on
HERG channels expressed in Xenopus oocytes, HERG blocker
(nifekalant, quinidine, carvedilol, E-4031, and dofetilide) blocked
HERG channels in a use-dependent manner . However, some characters
were found under the block effect. Nifekalant, quinidine or carvedilol
increased HERG channel current at low voltages only in the presence of
a previous strong depolarizing pulse, and so this pulse could separate the
facilitation effect from the block effect. On the other hand, with the
facilitation effect, they caused a significant negative shift in the voltagedependence of activation. In the case of E-4031 etc, the facilitation effect
was not found. Moreover, some mutant HERG channel blocked by
quinidine induced stabilization of the closed state of channel. These
results reveal that each HERG blocker has a different mechanism of
block effect, and has not only block effect but also other effects.
Kir2 subfamily members of inward rectifier K+ channel are known to coassemble to form heteromultimers, and it is also known for Kir3. Here
we examined whether Kir2.1 (IRK1) and Kir3.4 (GIRK4) belonging to
different subfamilies can assemble each other or not. First, we examined
the association by co-immunoprecipitation experiments using FLAG or
myc tagged constructs co-transfected in HEK 293 cells. We observed
"GIRK4-FLAG and IRK1-myc" as well as "IRK1-FLAG and GIRK4myc" co-immunoprecipitated at a comparable level with a positive control pair, IRK1-FLAG/IRK1-myc. This clear co-immunoprecipitation
was not observed in a negative control pair, P2X2 receptor-FLAG/
GIRK4-myc. As a next step, we anlayzed electrophysiologically using
Xenopus oocyte expression system the effect of co-injection of GIRK4
or GIRK4/GIRK1 cRNA on IRK1 current. We could not record a clear
emergence of current with unique features which reflects formation of
functional heteromultimers, but observed a decrease in the amplitude of
IRK1 current, suggesting a suppression effect possibly by hetero-multimerization. Finally, we carried out FRET analysis of IRK1-CFP/GIRK4YFP pair and GIRK1-CFP/IRK1-YFP pair expressed in CHO cells, and
obtained preliminary data supporting association of these two subunits.
Taken together, these results suggest that IRK1 and GIRK4 subunits
have a capability to form heteromultimers in heterologous expression
Analyses of the voltage and ATPdependent "gating" of ATP receptor
Fujiwara, Yuichiro1; Kubo, Yoshihiro1,2 (1Div Biophys
Neurobiol, Dept Mol Physiol, Natl Inst Physiol Sci, Okazaki,
P2X receptors are ligand-gated cation channels activated by extracellular ATP. The P2X2 channel current at the steady-state after ATP application is known to have voltage-dependence, i.e. - it shows inward
rectification, and a gradual increase in the inward current is observed
upon hyperpolarization. We analyzed this "activation" phase quantitatively under two-electrode voltage clamp using Xenopus oocytes expression system, and also approached its structural background by mutating
a glycine residue (G344) in the 2nd-transmembrane helix (2nd-TM), a
putative kink for the "gating". We observed that the inward current of
G344A mutant increased instantaneously upon hyperpolarization without a gradual increase. On the contrary, G344P mutant showed a slower
"activation" than that of WT. We also analyzed the conductance-voltage
relationship by measuring the tail current, and observed that the halfmaximal voltage of "activation" of G344P was shifted to the hyperpolarized potential in comparison with that of WT. The mutation did not affect
the ATP dose-response relationships significantly. By the glycine scanning mutageneses on the G344A mutant background, we observed a recovery of the "activation" phase by introducing a glycine residue to the
middle region of 2nd-TM. Taken together, we speculate that the flexibility of G344 in the 2nd-TM contributes to the voltage dependent "gating"
of P2X2 channel, which could be caused by an intrinsic or extrinsic
mechanism to the channel molecule.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Ionic channels & receptors
Two opposing roles of 4AP-sensitive
current in spike-initiation and invasion in
mesencephalic trigeminal neurons
Saito, Mitsuru1; Murai, Yoshinaka2; Sato, Hajime1;
Takada, Masahiko3; Kang, Youngnam1 (1Dept. Neurosci.,
Osaka Univ. Grad. Sch. Dent., Osaka, Japan; 2Dept. Physiol., Sch.
Med., Kurume Univ., Fukuoka, Japan; 3Div. Syst. Neurosci, Tokyo
Metro. Inst. Neurosci., Tokyo, Japan)
The axon initial segment plays important roles in spike-initiation and invasion of axonal spikes into the soma. Among PSNs, those in the MTN
can exceptionally initiate spikes in response to synaptic inputs, consequently displaying two kinds of spikes, one caused by invasion of an axonal spike arising from the sensory receptor and the other initiated by
somatic inputs. We addressed where spikes are initiated in MTN neurons
and whether there are any differences between initiated and invaded
soma spikes (S-spikes). Simultaneous patch-clamp recordings from the
soma and axon hillock revealed a spike-backpropagation from the initiation site in the stem axon to the soma in response to somatic current
pulse, which brought about the delayed emergence of S-spikes after the
offset of the current pulse. These initiated S-spikes were smaller in amplitude than invaded ones generated by stimulation of the stem axon;
however, 4AP (<=0.5mM) eliminated the amplitude difference. Furthermore, 4AP markedly shortened the delay in spike-initiation without affecting the latency to spike-invasion, whereas it prolonged the refractory
period of invaded S-spikes without affecting that of presumed axonal
spikes markedly. These observations suggest that 4AP-sensitive K+ currents exert two opposing effects on S-spikes depending on their origins;
suppression of spike-initiation and facilitation of spike-invasion at higher frequencies.
Determination of a critical amino acid
residue associated with calmodulindependent kinase II (CAMKII)-mediated
activation of vascular receptor-operated
Ca2+ entry channel TRPC6
Takahashi, Shinichi1; Geshi, Naomi1,2; Mori, Yasuo3; Ito,
Yushi2; Inoue, Ryuji1 (1Dept. Physiol., Sch. Med. Fukuoka Univ.;
2Dept. Pharmacol., Grad. Sch. Med., Kyushu Univ.; 3Lab. Mol.
Biol., Dept. Syn. Chem. & Biol. Chem, Grad. Sch. Engineer., Kyoto
The molecular background for CaMKII-mediated regulation of a murine
transient receptor potential protein homologue TRPC6 was explored by
mutation analysis of CAMKII phosphorylation motifs (RXX(S/T)),
which were found on the N-terminal (NT) and transmembrane (TM) (but
not C-terminal) regions of wild-type TRPC6 and its chimera T776 with
the NT and TM domains of TRPC7. Substitution of the last serine or
threonine in these motifs with alanine revealed that, out of eight and five
candidate sequences, only the mutations T487A in wild type TRPC6 and
T433A in T776 respectively can strongly attenuate Ba2+ influx or inward
cationic current evoked by muscarinic receptor stimulation with 100µM
carbachol. Assuming a smilar membrane topology suggested by a recent
structural analysis of TRPC1, T487 in TRPC6 and T433 in T776 may be
located on a long intracellular stretch between the second and third TM
domains (II-III loop) of each channel. Thus, considering the absolute
requirement of a putative calmodulin/IP3 receptor binding site (CIRB)
for TRPC6 channel activation, it is conceivable that close spatial
arrangements of CIRB and the II-III loop might allow effective
phosphorylation of T487 via the actions of CAMKII, thereby increasing
the availability of TRPC6 channel for opening in response to subsequent
receptor stimulation.
Regulation of the Kir2.1 potassium channel
current by intracellular pH
Role of TRPV4 in control of body
temperature under heat radiation
Yan, Ding-Hong; Ehara, Tsuguhisa; Ishihara, Keiko
Mochizuki-Oda, Noriko1,2; Kusuno, Tomoyuki1; Hanada,
Tsunehisa1; Tominaga, Makoto3; Tominaga, Tomoko3;
Suzuki, Makoto4; Yamada, Hisao2; Yamada, Hironari1
(Department of Physiology, Faculty of Medicine, Saga University,
Saga, Japan)
The Kir2.1 channel currents show a strong inward rectification under the
whole-cell recordings because of a voltage-dependent block of the channel by intracellular polyamines and Mg2+. Here we examined the effects
of the intracellular pH on the Kir2.1 channel current using a 293T cell
expression system. When the inside-out patch membrane was exposed to
the intracellular solution of pH 7.2 containing no polyamines and Mg2+,
the currents still showed a relatively strong inward rectification, but the
closing of the channel in response to depolarizing voltage steps usually
required several tens of seconds. Acidification of the intracellular solution from pH 7.2 to 6.8 and 6.4 induced a voltage-dependent decay of the
outward currents during depolarizing voltage steps and increased the
slow time-dependent component of the inward tail currents on hyperpolarization. Lowering of the intracellular pH from 7.2 to 6.0 and 5.6 also
decreased the amplitude of the inward currents. In the presence of 0.1-5
µM spermine or 1-10 µM spermidine, a decrease in pH changed the
amplitude of the outward currents with a complex voltage-dependence.
Analyses of the inward tail currents suggested that the contribution of the
pH-induced gating to the decrease of the outward currents increased as
the intracellular pH was decreased.
(1Synchrotron Light Life Science Center, Ritsumeikan Univ. Kusatsu,
Shiga, Japan; 2Dept. Anat.and Cell Sci. Kansai Med. Univ.
Moriguchi, Osaka, Japan; 3NIPS, Okazaki, Aichi, Japan; 4Dept.
Pharmacol.Jichi Med. Sch. Minamikawachi, Tochigi, Japan)
TRPV family, identified as thermosensitive, Ca2+-permeable channels,
consists of six subtypes. TRPV3 and 4 were described as heat transducers operative at moderately warm temperatures (>34 °C), whereas
TRPV1 is activated by temperature above 42 °C. In natural environment,
infrared light is detected as thermal radiation through skin. TRPV3 and
4 were reported to express in keratinocytes. They might, therefore,
respond to infrared radiation and transfer the thermal signal to CNS. In
this study, we examined the role of TRPV4 in regulation of body
temperature by using transgenic mice defecting in TRPV4 protein. As
thermal stimulus, infrared laser irradiation (λ=830 nm, 150 or 300 mW,
15 min) was applied to the back skin of the mouse, and temperatures of
both skin surface and rectum were monitored. In wild type mouse, laser
radiation which caused the increase in skin temperature up to 55 °C did
not induce the change in body temperature. In TRPV4-knockout mice,
however, moderate thermal stimulus, which increased the skin
temperature less than 43 °C, resulted in the increase in the body
temperature during the laser irradiation suggesting the loss of autonomic
temperature regulation. The processing of moderate thermal radiation
may partly depend on the TRPV4 expressed in skin cells.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Ionic channels & receptors
Mechanical stress activates TRPM7
channels expressed in HEK293T cells
Identification of TRPM8 Ion Permeation
Numata, Tomohiro; Shimizu, Takahiro; Okada,
Yasunobu (Dept. Cell Physiol., Natl. Inst. Physiol. Sci., Okazaki,
Yamanaka, Keiji1; Hosokawa, Hiroshi1; Matsumura,
Kiyoshi2; Kobayashi, Shigeo1 (1Dept. Intelligence Science and
Technology, Grad. Sch. Informatics, Kyoto Univ., Kyoto, Japan;
2Dept. Information Science and Technology, Osaka Institute of
Technology, Osaka, Japan)
Mechanical stress activates TRPM7 channels expressed in HEK293T
cells Numata, Tomohiro; Shimizu, Takahiro; Okada, Yasunobu (Dept.
Cell Physiol., Natl. Inst. Physiol. Sci., Okazaki, Japan)Stretch-activated
cation channels play an essential role in sensing and transducing external
mechanical stresses in living cells. In the previous meeting we reported
that TRPM7 channels endogenously expressed in human epithelial HeLa
cells are activated by membrane stretch or osmotic cell swelling. However, it has not been known whether TRPM7 shows mechanosensitivity
when heterologously expressed. HEK293T cells overexpressed with
TRPM7 exhibited whole-cell currents typical of TRPM7, such as outward rectification, conductivity to Ca2+, and sensitivity to Mg2+ and ruthenium red. In addition, TRPM7 currents were augmented by following
three kinds of mechanical stimuli: shear stress imposed by perfusion of
extracellular solution, membrane stretch produced by patch membrane
suction, cell swelling due to hypotonic stimulation. We thus conclude
that the TRPM7 channel can be activated by mechanical stress in the heterologous expression system.
TRPM8 was cloned as the family of thermally activated TRP channels.
TRPM8 is cooling activated channel involved in cold sensation. However, it is unclear which amino acid residues are critical for ion permeation
as the pore.
We hypothesized that the pore is made by three consecutive hydrophobic
amino acid residues between TM5 and TM6, highly conserved among
different species or related TRP channels. WIF (898-900) were residues
consistent with above hypothesis. We examined the involvement of this
region on ion permeation by constructing TRPM8 mutant WIF898AAA,
in which corresponding amino acid residues were replaced by alanine.
Ion permeation was investigated with Fura-2 calcium imaging method.
Menthol or cooling (from 30 °C to 15 °C) elevated intracellular calcium
ion concentration in wild type TRPM8 -expressing cells but not in
WIF898AAA -expressing cells.
We conclude that the conserved three consecutive hydrophobic amino
acid residues between TM5 and TM6 (WIF) are critical for ion
permeation of TRPM8.
Regulation of TRPV4 activity by the PDZLIM protein
Higashi, Tomohiro1,2; Numazaki, Mitsuko3; Tominaga,
Tomoko1,2; Kuroda, shun'ichi4; Tominaga, Makoto1,2
(1Dept. Physiol. Sci. Grad. Univ. Adv. Stud., Okazaki, Japan;
2Okazaki Inst. Int. Biosci. Okazaki, Aichi, Japan; 3Dept. Anesthesiol.
Univ. Tsukuba Sch. Med. tsukuba, Japan; 4Inst of Sci. and Ind. Res,
Osaka Univ, Ibaraki, Osaka, Japan.)
TRPV4, a member of TRPV subfamily, is a nonselective cation channel
that is activated by hypotonic stimulus, warm temperatures (about 2534°C) or chemical compounds such as 4α-PDD, and is expressed in
various tissues. To investigate TRPV4 function in the epithelial tissue,
we screened a cDNA library from epithelial cells to identify TRPV4
interacting protein using a yeast two-hybrid system. Sequence analysis
revealed that one of the positive clones encodes a protein containing
PDZ and LIM domains. We found that the N terminal region of TRPV4
bound to LIM domains of the PDZ-LIM protein. When both TRPV4 and
the PDZ-LIM protein were co-expressed in HEK293 cells, patch-clamp
analysis showed that 4α-PDD-evoked currents were larger than those
observed in cells expressing TRPV4 alone. Increase in the 4α-PDDevoked currents was not observed in the cells expressing TRPV4 and the
PDZ-LIM protein lacking LIM domains. These results suggest that the
PDZ-LIM protein regulates TRPV4 activity by physical binding through
LIM domains.
Transient receptor potential A1 is nonselective cation channel, activated by
Sawada, Yosuke1; Hosokawa, Hiroshi1; Matsumura,
Kiyoshi2; Kobayashi, Shigeo1 (1Dept. Intelligence Science and
Technology, Grad. Sch. Informatics, Kyoto Univ., Kyoto,Japan;
2Dept. Info. Sci. and Tec., Osaka Institute of Technology, Osaka,
In TRP (Transient Receptor Potential) cation channels family, cooling
activated channels, TRPM8 and TRPA1, were identified in peripheral
sensory neurons. However, it is still controversial whether TRPA1 is activated by cooling. Story et al [Cell,2003] reported that TRPA1 is activated by cooling below 17oC. In contrast, Jordt et al [Nature,2004]
reported that cooling did not activate TRPA1. Here, we investigated thermal sensitivity of TRPA1 by Fura-2 microfulorimetry and patch-clamp
recordings in TRPA1-expressing HEK293 cells.
1: Cooling increased intracellular calcium levels.
2: In whole-cell voltage-clamp recording (-60mV), cooling below
threshold evoked inward current. Threshold temperature was 17.5 ± 2.7
3: In inside-out single-channel recording, cooling induced activities of
ion channels with non-selective cation channel properties. This indicated
that channel activities occurred without intracellular soluble component.
Single channel currents showed inward rectification. Single channel
conductance was 74.1 ±18.8pS
We conclude that TRPA1 is non-selective cation channel, activated by
J. Physiol. Sci., Vol. 56, Suppl., 2006
Ionic channels & receptors
Roles of two putative coiled-coil domains
in the cytoplasmic C-terminal region of
KCNQ channels
Yoshihiro1,2 (1NIPS, Okazaki, Japan;
JST, Kawaguchi, Japan)
KCNQ channels have two putative coiled-coil domains in the cytoplasmic C-terminal region. These two domains, also known as A-domain
and B-domain, are well conserved among KCNQ channel family and are
recognized as subunit interaction domains. We have previously shown
that serine residues in A-domain may be phosphorylated when PKC
shifts the voltage-dependence of KCNQ channels. Therefore, we hypothesized that the stability of coiled-coil domain might determine the
voltage-dependence of KCNQ channels. The amino acid sequence of the
coiled-coil domain is characterized by heptad repeats (a-b-c-d-e-f-g) in
which positions a and d are occupied by hydrophobic amino acid residues. To decrease the stability of the coiled-coil domain, we introduced
double glutamine (Q) mutations at positions a and d of each heptad repeat. We analyzed electrophysiological properties of four double
glutamine mutants for A-domain (I551Q/Y554Q, H558Q/M561Q,
L565Q/I568Q, V572Q/I575Q) and three for B-domain (L606Q/V609Q,
V613Q/M616Q, L620Q/L623Q). Three of them (I551Q/Y554Q,
L565Q/I568Q, L620Q/L623Q) did not express detectable current.
Among mutants showing functional expression, V572Q/I575Q showed
drastic changes in its voltage-dependence: V1/2 of the G-V curve was
negatively shifted (-65.1 ± 1.1 mV vs. -39.0 ± 0.7 mV) and the apparent
valence of charge movement (z) became smaller (1.54 ± 0.04 vs. 4.08 ±
0.07). The results of V572Q/I575Q suggest A-domain may also be
responsible for the voltage-dependence other than the subunit
Role Of Cytoplasmic Domain In VoltageDependency Of Zebrafish VSP
Hossain, Mohammmad1,2; Sasaki, Mari1,2; Okamura,
Yasushi1,2 (1Section of Developmental Neurophysiology, Okazaki
Institute for Integrative Biosciences, NINS, Aichi, Japan; 2The
Graduate University of Advanced Studies)
A protein called Ci-VSP (Voltage Sensor containing Phosphatase) has
recently been reported (Murata et al, Nature 2005). VSPs have ion channel like transmembrane segments from S1 to S4 as the voltage sensor domain and the C-terminal cytoplasmic domain. It exhibits phosphatase
activity in a voltage dependent manner. We have previously compared
zebrafish ortholog of VSP (Z-VSP) with Ci-VSP and found that Z-VSP
shows more robust charge movements in mammalian heterologous expression than Ci-VSP. To understand voltage-sensing mechanisms of
VSP, we focused on the double mutant of Z-VSP in which two arginine
residues were inserted into the S4 (DM). This shows negative shift of the
Q-V curve (threshold was around -40 mV). Furthermore, DM showed
biphasic profile of the Q-V curve that could not be fitted by a single Boltzmann equation: the movement of the voltage senor saturates around at
100 mV but then it increases as the membrane potential is more depolarized. Such biphasic profile of the Q-V curve did not depend on the phosphatase activity as examined from C302S mutant. This exhibited the
threshold of charge movement even more negative (around -60 mV) and
the simple Q-V curve that can be fitted with the single Boltzmann equation. These results suggest that the C-terminal domain but not its enzyme
activity affects charge movement probably through exerting some constraint on the movement of the VS domain.
Voltage dependence of the adaptation in
MscS occurs independent of the charged
residues in the transmembrane domain
Maxi-anion channels in rat cardiomyocyte
sarcolemma are heterogeneously
Nomura, Takeshi1; Yoshimura, Kenjiro1,2; Sokabe,
Masahiro1,3 (1ICORP/SORST, Cell-Mechanosensing Project, JST,
Dutta, Amal K.1; Okada, Yasunobu1; Korchev, Yuri E2;
Shevchuk, Andrew2; Sabirov, Ravshan Z1 (1Dept. Cell
Nagoya, Japan; 2Struc. Biosci., Grad. Sch. Life Environ. Sci., Univ.
Tsukuba, Ibaraki, Japan; 3Dept. Physiol., Nagoya Univ. Grad. Sch.
Med., Nagoya, Japan)
Physiol., Natl. Inst. Physiol. Sci., Okazaki, Japan; 2Div. Med., MRC
Clin. Sci. Centr., Fac. Med., Imperial Coll. Sci., Tech. Med., London,
United Kingdom)
MscS (mechanosensitive channel of small conductance) is ubiquitously
found among bacteria and has been proposed to play an important role
in osmoregulation. Although the MscS gating is regulated by membranestretch and voltage, little is known how MscS senses membrane potential. Three arginine residues (Arg-46, Arg-74, and Arg-88) in the transmembrane domain have been proposed to serve as voltage sensors. To
examine whether some of these three residues constitute voltage sensors,
we neutralized the charge of each residue by substitution with asparagine
(R46N and R74N) or glutamine (R88Q). Mechanical threshold for the
opening of the expressed wild-type MscS did not change with voltage in
the range from -40 to +100 mV. Replacement of the arginine residues
with asparagine or glutamine did not alter the threshold. By contrast, inactivation process of wild-type MscS was strongly affected by voltage.
At the pipette potential of -40 to -80 mV the current of the wild-type
MscS rapidly declined whereas the current at -20 to +60 mV sustained
for a longer time, as reported previously (Akitake et al., 2005). The voltage dependence of the inactivation rate of all mutants tested, was almost
indistinguishable from that of the wild-type MscS. These findings indicate that the voltage dependence of the inactivation occurs independently of the positive charges of Arg-46, Arg-74, and Arg-88.
ATP conductive maxi-anion channels are functionally expressed in rat
cardiomyocytes and activated in hypotonic, hypoxic or ischemic conditions. In the present study, using a newly developed Scanning Ion Conductance Microscopy (SICM) technique combined with patch-clamp,
we studied the spatial distribution of maxi-anion channels over the surface of sarcolemma of primary cultured neonatal rat cardiomyocytes and
isolated adult cardiomyocytes. In primary cultured rat cardiomyocytes,
maxi-anion channels were found to be predominantly expressed around
the center of the cell body compared to the cell extensions. Using a P2Xreceptor-based biosensor technique, it was found that the local concentration of ATP released in response to hypotonic stress was higher
around the cell center compared to that at cell extensions. In isolated
adult rat cardiomyocytes, maxi-anion channel activity was observed
when a fine-tipped patch pipettes were giga-sealed at the grooved areas
of sarcolemma using a conventional patch-clamp method. After taking a
3-D image of isolated cell by SICM technique, we specifically patched
T-tubules, Z-grooves and scallop crest areas. We found that maxi-anion
channels are predominantly expressed in T-tubules and Z-groove areas,
but not in scallop crests, in adult cardiomyocytes. The results obtained
indicate that maxi-anion channels are heterogeneously expressed over
the surface of the rat cardiac sarcolemma.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Ionic channels & receptors
Effects of opioid- and cannabinoidreceptor agonists on calcium channels in
rat nucleus tractus solitarius
Specific molecular actions of sarcolemmal
phosopholipid metabolites on cardiac
ryanodine receptors
Endoh, Takayuki; Suzuki, Takashi (Dept. Phys. Tokyo Dent.
Yasukochi, Midori1; Inoue, Ryuji2; Uehara, Akira2
Coll. Japan)
(1Human Biology Med. Univ. Fukuoka, Fukuoka, Japan; 2Physiol.
Med. Univ. Fukuoka, Fukuoka, Japan)
The profile of opioid- and cannabinoid receptors in neurons of the nucleus tractus solitarius (NTS) have been studied using the whole cell configuration of the patch clamp technique. Experiments with selective
agonists and antagonists of opioid, opioid-receptor-like-1 (ORL-1) receptor and cannabinoid receptors indicated that µ-opioid, κ-opioid,
ORL-1 and CB1, but not δ-opioid, receptors inhibit VDCCs currents in
NTS. Application of DAMGO (µ-opioid receptor agonist), Orphanin FQ
(ORL-1 receptor agonist) and WIN55,122 (CB1 receptor agonist)
caused inhibition of VDCCs currents in a concentration-dependent
manner with an IC50 of 390 nM, 220 nM and 2.2 µM, respectively.
Intracellular dialysis of the Gαi-protein antibody attenuated DAMGO-,
Orphanin FQ- and WIN55,122-induced inhibition of IBa. Both
pretreatment with adenylate cyclase inhibitor and intracellular dialysis
of the protein kinase A (PKA) inhibitor attenuated WIN55,122-induced
inhibition of IBa, but not DAMGO- and Orphanin FQ-induced inhibition.
Mainly N- and P/Q-type VDCCs were inhibited by both DAMGO and
Orphanin FQ, while L-type VDCCs were inhibited by WIN55,122.
These results suggest that µ- and κ-opioid receptors and ORL-1 receptor
inhibit N- and P/Q-type VDCCs via Gαi-proteins β γsubunits, whereas
CB1 receptors inhibit L-type VDCCs via Gαi-proteins involving PKA in
We examined with a lipid bilayer method how the single channel currents of cardiac RyR channels are modified by pathophysiological metabolites from the sarcolemmmal membrane phospholipids. During the
apoptosis and the hyperlipidemia, sphingosylphosphatidylcholine (SPC)
is metabolized from sphingomyelin (SM) of a minor sarcolemmal phospholipid. (1-1) The cytoplasmic-side addition of SPC blocked the RyR
channels at the µM level, while the SR luminal-side addition of SPC did
not affect. (1-2) SPC unaltered the membrane capacitance. Thus, SPC
could exert a specific effect via an intermolecular binding to the
cytoplasmic domain of the RyR molecule, although SPC belongs to the
lipid. Kinetics of a long-lived blocking state of the SPC-modified
channels is characterized by an extremely low dissociation rate constant.
During the cardiac ischemia, lysophosphatidylcholine (LPC) is
produced from phosphatidylcholine (PC) of a major sarcolemmal
phospholipid. (2-1) Both cytoplasmic-side and SR luminal-side
additions of LPC activated the RyR channels at the µM level. (2-2) LPC
increased the membrane capacitance. In contrast to SPC effects, LPC
could thus exert an indirect effect via a fusion of LPC into the membrane
lipids on the RyR channel. Here we propose that a second messenger
metabolited from SM of sarcolemmmal membrane phospholipids
specifically regulated in vivo cardiac RyR channel activities.
Functional interactions between P2X
receptors and GABA- or Glycine-receptor
in rat area postrema neurons
The mechanism of intracellular Ca2+
oscillation during P2Y2 receptor activation
in rat bone marrow stromal cells
Sorimachi, Masaru1; Akaike, Norio2; Wakamori, Minoru1
Ichikawa, Jun; Gemba, Hisae (Dept. Physiol., Kansai Med.
(1Grad.Sch.Med.Dent.Univ.Kagoshima,Japan; 2Kumamoto Health
Sci. Univ., Kumamoto, Japan)
Univ., Moriguchi, Japan)
We previously reported the negative interaction between excitatory P2X
receptor (P2XR)- and nicotinic ACh receptor-channels in rat area postrema (AP) neurons. We now investigated whether there was also the interaction between excitatory P2XR and inhibitory GABAA- or glycinereceptors (GlyR). At a holding potential of -70mV, the amplitude of the
GABA- or Gly-induced current was significantly reduced in the presence of ATP, and there was an inverse correlation between the amplitudes of these responses. On the other hand, the ATP-induced current in
the presence of GABA or Gly was only slightly reduced. As the GABAor Gly-induced current desensitizes faster than the ATP-induced current,
the weaker inhibition of the ATP-induced current by GABA or Gly could
be due to the reduced amplitude of the GABA- or Gly-induced current at
the time of ATP application. In fact, the current caused by the concomitant applications of ATP and GABA or Gly was smaller than the predicted sum of the individual currents.These results suggest that the negative
interactions between the different receptors modify the strength of excitatory or inhibitory neurotransmission when plural transmitters are simultaneously released from presynaptic nerve terminals.
Rat bone marrow stromal cells express G protein-coupled purinergic receptor (P2Y2 receptor). We have investigated intracellular Ca2+ signals
in these cells using fura-2 AM and found that UTP, an agonist of P2Y2
receptor, induced not only Ca2+ rise but also Ca2+ oscillation. Removal
of extracellular Ca2+ diminished UTP-induced Ca2+ oscillation. This fact
indicates that the Ca2+ oscillation involves Ca2+ entry from extracellular
space. SKF96365, a blocker for store-operated Ca2+ entry channel suppressed UTP-induced Ca2+ oscillation. Carbenoxolone (CBX), a gap
junction blocker, also suppressed Ca2+ oscillation at higher concentration than 50µM, but enhanced at 10µM in some cells. At 10µM, CBX
enhanced the peak amplitude of UTP-induced Ca2+ rise and kept high
Ca2+ concentration until CBX was washed out. L-type voltagedependent Ca2+ channel blockers, nifedipine or verapamil did not affect
UTP-induced Ca2+ oscillation. These results suggest that UTP-induced
Ca2+ oscillation may be regulated by a complex mechanism including
Ca2+ entry through store-operated Ca2+ channel at plasma membrane and
Ca2+-transport system via gap junction.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Ionic channels & receptors
Vasopressin activates orexin neurons
through a V1a receptor
Regulation of IKr potassium current by α1
receptor in HL-1 cells
Ichiki, Kanako1; Yamanaka, Akihiro1,2; Tsunematsu,
Tomomi1; Sakurai, Takeshi1,2; Goto, Katsutoshi1 (1Grad.
Zankov, Dimitar1; Toyoda, Futoshi1; Tei, Ikoh1;
Matsuura, Hiroshi1; Horie, Minoru2 (1Dept. Physiol., Shiga
Sch. Comp. hum. Sci. Univ. Tsukuba, Tsukuba, Japan; 2ERATO
Yanagisawa Orphan Receptor Pro. JST)
Univ. of Med. Sci., Otsu, Japan; 2Dept. Internal Medicine, Shiga
Univ. of Med. Sci.,Otsu, Japan)
Orexin A and B are a pair of neuropeptides which are implicated in the
regulation of sleep-wakefulness and energy homeostasis. The regulatory
mechanism of orexin neurons is poorly understood so far. In this study,
we studied the effects of various neuropeptides on the activity of orexin
neurons by calcium imaging using transgenic mice in which orexin neurons specifically express calcium sensing protein (Yellow Cameleon
2.1). We screened 21 neuropeptides and found that arginine-vasopressin
(AVP), cholecystokinin-8s and oxytocin triggered a robust, concentration-dependent calcium increase in orexin neurons.
Long QT syndrome (LQTS) results from mutations of several genes encoding cardiac ion channels. It was reported that in LQTS2, form associated with dysfunction of the rapid component of IK (IKr), acute auditory
stimuli could trigger the symptoms thus suggesting fast neural control of
IKr. The aim of this study was by means of whole-cell patch-clamp method to investigate acute regulation of native IKr by α1-adrenergic receptor
(AR) in HL-1 cardiomyocytes.In the cells transiently transfected with
AR, bath-application of 30 µmol phenylephrine (PHE) reversibly
decreased IKr density by 29.4%, shifted activation curve (Vh from -17.6
to -9.2 mV) and accelerated deactivation. These effects remained in the
presence of protein kinase C (PKC) inhibitor bisindolylmaleimide (200
nmol). In non-transfected cells 30 µmol PHE did not affect IKr. In HL-1
cells expressing muscarinic M1-receptor (known to be coupled to Gq PLC pathway as AR), 10 µmol acetylcholine (Ach) suppressed IKr even
more (37.2%).To confirm involvement of membrane PIP2 breakdown in
IKr modulation, HL-1 cells cotransfected with PH (PLCδ pleckstrin
homology domain) -GFP and AR or M1-receptor were used for confocal
microscopy. 30 µmol PHE or 10 µmol Ach induced translocation of PHGFP fluorescence from the cell membrane to citosol, which was not
observed in the cells transfected with PH-GFP alone.AR stimulation in
HL-1 cells acutely suppressed IKr by depletion of membrane PIP2 and
was not dependent on PKC. This effect could explain onset of symptoms
in the LQTS2 patients.
We revealed the intracellular mechanisms and the subtype of AVP
receptors involved in the AVP-induced activation of orexin neurons. The
V1a AVP receptor antagonist, SR49059, inhibited AVP-induced
activation of orexin neurons in a concentration-dependent manner,
whereas the V1b and V2 receptor antagonists (SSR149415 and
SR121463) had little effect. Removing extracellular calcium eliminated
the AVP-induced increase in intracellular calcium concentration.
These results suggested that the V1a receptor is involved in the AVPinduced activation of orexin neurons. This AVPergic excitatory input to
orexin neurons might have an important role in the physiological
regulation of sleep-wakefulness.
Interdomain interaction within type 1
ryanodine receptor is involved in
dysfunction of Ca2+ release channel in
malignant hyperthermia
Hideto3; Kurebayashi, Nagomi1; Hara, Hiroshi4;
Wakebe, Kikuo4; Ikemoto, Noriaki5; Ogawa, Yasuo1
(1Juntendo Univ. Sch. Med., Tokyo, Japan; 2Nagoya City Univ. Grad.
Sch. Med. Sci., Nagoya, Japan; 3Showa Univ. Sch. Med., Tokyo,
Japan; 4Miyazaki St., NLBC, Miyazaki, Japan.; 5BBRI, MA, USA)
Malignant hyperthermia (MH) is an inherited disorder caused by a missense mutation of type 1 ryanodine receptor (RyR1) of skeletal muscle.
We have recently showed that Ca2+-induced Ca2+ release (CICR) activity
of RyR1 in the SR is selectively stabilized by a probable interdomain interaction between the N-terminal and the central regions of RyR1 where
many of mutated sites for MH are clustered (Murayama et al., Am. J.
Physiol. 288; C1222-C1230, 2005). According to our hypothesis, a mutation within these regions will weaken the interdomain interaction, resulting in an increased CICR activity. We are presenting here further
evidence to support this hypothesis using SR vesicles from skeletal muscles of wild type and MH pigs carrying the N-terminal Arg615Cys mutation in RyR1. Furthermore, we will show results using RyR1 channels
carrying several human MH mutations including those in the C-terminal
region that are stably expressed in HEK293 cells.
Muscarinic receptor-operated cation
channels as calcium entry pathways in
bovine ciliary muscle
Miyazu, Motoi1; Yasui, Fuminori1; Ohhinata, Hiroshi1;
Takai, Yoshiko2; Takai, Akira1 (1Dept. Physiol., Asahikawa
Med. Coll., Asahikawa, Japan; 2Dept. Ophthalmol., Sch. Med.
Nagoya Univ. Nagoya, Japan)
In the ciliary muscle, tonic contraction requires a sustained influx of
Ca2+ through the cell membrane. However, little has been known about
the routes for the Ca2+ entry in this tissue that lacks voltage-gated Ca2+
channels. Recently we have shown by whole-cell voltage clamp experiments that in bovine ciliary muscle cells (BMCs) there are two types of
non-selective cation channels (NSCCs) with widely different unitary
conductances (35 pS and 100 fS), which are opened by muscarinic stimulation with carbachol (CCh). Here we examined effects of inhibitors of
the NSCCs, La3+ and Gd3+, on CCh-induced changes of the intracellular
Ca2+ concentration [Ca2+]i. BMCs cultured for 18~48 hours in a serumfree media were used. The [Ca2+]i was monitored by a Fluo-4 fluorescence method. Application of CCh to the BMCs in normal Krebs solution caused an initial phasic increase in the [Ca2+]i followed by a plateau
which was abolished by La3+ or Gd3+ (10-100 µM) as well as by removal
of external Ca2+. The CCh-induced elevation of [Ca2+]i was also
completely inhibited by 100 nM of atropine or 4-DAMP. These results
support the idea that the muscarinic receptor-operated NSCCs serve as
entry pathway of Ca2+ during the sustained phase of contraction. We also
conducted immunofluorescence microscopy of the plasma membrane of
BCMs and thereby detected transient receptor potential (TRP) channel
homologues (TRPC1, TRPC3, TRPC4 and TRPC6), which are now
regarded as possible molecular candidates for receptor-operated NSCCs.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Ionic channels & receptors
Functional involvement of
tempromandibular joint P2X receptor in the
jaw reflex activities in rats
Role of negatively charged residues of the
transmembrane segments of the voltagesensitive phosphatase, Ci-VSP
Watanabe, Tatsuhisa1; Tsuboi, Yoshiyuki2; Iwata,
Koichi1 (1Nihon Univ. oral surgery , Tokyo,Japan; 2Univ. Toronto,
Murata, Yoshimichi; Okamura, Yasushi (Section of
Toronto, Canada)
Developmental Neurophysiology, Okazaki Center for Integrative
Biosciences, National Institutes of Natural Sciences, Aichi, Japan)
Purinergic receptor mechanisms have recently been implicated in peripheral (Rong et al 2000) and central (Hu et al 2002, Chiang et al 2005)
nociceptive processing. Of the 7 iontotropic purinergic receptor subtypes
(P2X receptor family), the expression of the P2X3 receptor is reported
to be much higher than that of the other P2x receptor subtypes in trigeminal ganglia (Xiang et al 1998) and it has recently been found in TMJ tissues (Ichikawa et al 2004, Shinoda et al 2005). To clarify further the role
of P2X receptors in TMJ-related functions, the application of P2X receptor agonist to the TMJ elicits nociceptive behaviors (Oliveira et al 2005,
Shinoda et al 2005). The first aim of the present study was to test if the
application of a P2X receptor agonist to the rat TMJ induces reflex activity in the jaw muscles and if this excitatory effect can be blocked by
peripheral application of P2X receptor antagonist. The second aim was
to test if blockade of peripheral NMDA receptors can influence the reflex jaw muscle activity evoked by ATP agonist.
We previously reported an ascidian protein Ci-VSP which has a transmembrane voltage sensor motif with significant homology to voltagegated channels and a phosphatase domain just downstream of the transmembrane region. We showed that the voltage sensor functionally couples with the phosphatase domain (Nature, 2005). However, it remains
unknown whether similar mechanisms for voltage sensing of voltagegated channels operate in Ci-VSP.There are conserved negative-charged
residues in S2 and S3 regions of both Ci-VSP and voltage-gated ion
channels. These residues are known to contribute to the gating charge of
the channels. We have previously shown that a mutation which neutralized the negative-charged residue in S2 altered voltage dependency of its
gating current. In this mutant, voltage-dependence of the coupling was
shifted in the same direction as the change of the Q-V curve. In this
work, we systematically mutated negatively charged residues in S1-S3
regions of Ci-VSP. cRNAs encoding these mutants were expressed in
Xenopus oocytes and the two-electrode voltage clamp recording was
performed to record the gating current of these mutants. In addition,
these mutants were co-expressed with GIRK2 channels and changes of
the phosphatase activity with membrane potentials were detected by
monitoring changes of ion currents through GIRK2 channels. We also
analyzed the voltage dependence of phosphatase activity of the mutant
constructs of which the properties of voltage sensor movement are altered.
Conformational changes in the
cytoplasmic domain of KcsA potassium
channel upon gating
Iwamoto, Masayuki1,3; Shimizu, Hirofumi1,3; Inoue,
Fumiko1,3; Konno, Takashi1,3; Nihei, Amiko4; Sasaki,
Yuji C.2,3; Oiki, Shigetoshi1,3 (1Dept. Mol. Physiol. Biophys.,
Facult. Med. Sci., Univ. Fukui, Fukui, Japan; 2JASRI/SPring-8,
Mikazuki, Japan; 3CREST/JST; 4Seiko Instruments Inc., Matsudo,
For the cytoplasmic domain (CD) of potassium channels, crucial roles
for mediating intracellular stimuli and assembling subunits have been investigated. KcsA channel, with only 160 amino acid residues, possesses
intracellular stretches in the C-terminus, which forms CD in the tetrameric channel. KcsA channels are activated by intracellular acidic pH,
the mechanism of pH-sensing remains unsolved. Also only predicted
structure is available for the CD. Present study investigated surface
structure of the CD in KcsA channel by developing a novel approach.
Single cystein was introduced into various parts of the channel and specific reaction between introduced cystein and a flat gold surface was
evaluated by surface plasmon resonance signals. All mutations did not
alter single-channel properties, such as single-channel current-voltage
curves and the gating characteristics. In contrast to the closed channel at
pH 7.5, various sites in the CD became exposed to the surface when
channels were activated (pHi = 4.0). These observations indicate that the
cytoplasmic domain takes several conformational states when the channel is actively gating. We have also investigated the effect of open channel blocker, tetrabutylammonium, on the conformational changes in the
Myofibrillogenesis regulator 1 (MR-1) as a
causative gene for a hereditary
channelopathy; A study on a large
Japanese family of paroxysmal dystonic
choreoathetosis (PDC)
Kinoshita, Ryo1; Matsuo, Hirotaka1; Kamakura, Keiko2;
Nakayama, Akiyoshi1; Chiba, Toshinori1; Tokunaga,
Motohide1; Ishimine, Hisako1; Tsukada, Shingo1;
Kobayashi, Yasushi3; Fukuda, Jun1 (1Physiol. Natl. Defense
Med. Col., Tokorozawa, Japan; 23rd Int. Med. Natl. Defense Med.
Col., Tokorozawa, Japan; 3Anat. Natl. Defense Med. Col.,
Tokorozawa, Japan)
Paroxysmal dystonic choreoathetosis (PDC) is thought to be a hereditary
channelopathy mapped to chromosome 2q32-36. By means of linkage
analysis on a large Japanese family, we have narrowed the PDC locus
that contains 32 candidate genes. Here, we report that a heterozygous
mutation (A7V) in one of such genes, myofibrillogenesis regulator 1
(MR-1), is responsible for PDC in the Japanese family. This is consistent
with the finding in American PDC families. We further report that there
are several other polymorphisms in MR-1 in the Japanese PDC family.
To characterize MR-1, we generated specific antibodies against MR-1
and performed the immunohistochemical analysis in rat brain. The results of the MR-1 localization will be discussed. Similar to other channelopathies such as epilepsy and migraine, PDC is characterized by
involuntary movement attacks, and is presumed to be induced by abnormalities of ion channels. Although MR-1 may be associated with some
ion channels, its physiological functions remain unclear. Further characterization of MR-1 including its molecular function and relationship to
ion channels, may facilitate not only to understand pathophysiology of
PDC, but also to develop effective therapies for paroxysmal neurological
J. Physiol. Sci., Vol. 56, Suppl., 2006
Ionic channels & receptors
Dopamine induces slow
afterdepolarization in lateral amygdala
neurons in vitro.
Yamamoto, Ryo; Ueta, Yoshifumi; Kato, Nobuo
(Integrative Brain Sci. Med. Kyoto Univ. Kyoto, Japan)
Multimodal regulation of mitochondrial
activity and plasmalemmal Ca2+ entry by
noradrenaline and glucagon in rat brown
Higure, Yoko; Suzuki, Yuka; Hayashi, Mamie; Kuba,
Masako; Kuba, Kenji (Lab. Anat. & Physiol., Fac. Nutrition,
The amygdala plays significant roles in regulating emotional states and
behaviors. Certain aspects of emotion are well known to be affected by
the domaminergic projection system, of which targets includes the
amygdala. Indeed, a large number of in vivo studies have shown that activation of dopamine (DA) receptors in lateral amygdala (LA) neurons
alter emotional expression. For understanding DA-based modulation of
emotion in the LA, it would therefore be beneficial to study effects of
dopamine on intrinsic properties of the LA neurons, which remain largely unknown. In the present experiments, whole cell patch clamp recordings were carried out in rat brain slices to investigate DA effects on LA
neurons. Application of DA depolarized resting membrane potential
markedly, and induced slow afterdepolarization (sADP) in LA neurons.
This sADP is induced in a voltage-dependent manner, and lasts for more
than 5 seconds. D1, but not D2, receptor agonists induced the same type
of sADP. Previous reports have repeatedly suggested that sADP in general is triggered by the calcium influx. Consistently, calcium channel
blockers inhibited the present DA-induced sADP, but sodium channel
blockers did not. Also, application of flufenamic acid (FFA), a calcium
activated non-selective cation channel (CAN) blocker, inhibited the DAinduced sADP and canceled out the DA-induced depolarization as well.
These results suggest that DA induces sADP in LA neurons by activating
D1 receptors, and this sADP is attributable to activation of CANs.
In brown adipocytes, mitochondria and endoplasmic reticulum (ER) are
found to couple each other via Ca2+, regulating plasmalemmal Ca2+ entry
(Kuba et al., this meeting). To study how noradrenaline and glucagons
that cause thermogenesis regulate this coupling and how it regulates
plasmalemmal Ca2+ entry, intracellular Ca2+ concentration ([Ca2+]i) and
mitochondrial membrane potential were measured by fluorometry in
cultured rat brown adipocytes. Isoprotelenol and glucagon caused bi- or
triphasic rises in [Ca2+]i. The first phase was accompanied by mitochondrial membrane depolarization. The second phase was paralleled by mitochondrial membrane repolarization and blocked by Ca2+ free solution,
indicating plasmalemmal Ca2+ entry. The third phase was blocked by
Ca2+ free, EGTA solution, but not by thapsigargin, a blocker of Ca2+
pump at ER, and enhanced at pH 9, but not in a Na+-free solution, indicating activation of STOC. A blocker of phospholipase C, U73122, enhanced the second and third phases of β3-adrenergic and glucagon
responses, while it blocked Ca2+ release by α1-adrenoreceptor activation
from ER and subsequent activation of store-operated Ca2+ entry. Thus,
the activation of β3-receptor and glucagons receptor causes multimodal
plasmalemmal Ca2+ entry via changes in mitochondrial membrane
potential and depletion of Ca2+ in ER via mitochondrial ER coupling.
Bidirectional Ca2+ coupling between
endoplasmic reticulum and mitochondria
and multimodal regulation of
plasmalemmal Ca2+ entry in rat brown
Kuba, Masako; Higure, Yoko; Susaki, Hisashi; Kuba,
Kenji (Lab. Anat. & Physiol., Fac. Nutrition, Nagoya Univ. of Arts
and Sciences, Aichi, Japan)
We have studied how endoplasmic reticulum and mitochondria communicate each other via Ca2+ and whether mitochondrial activity affects
plasma Ca2+ entry. Intracellular Ca2+ concentration ([Ca2+]i) and mitochondrial membrane potential were measured by fluorometry in brown
adipocytes in culture. FCCP, a protonophore, caused bi- or triphasic rises
in [Ca2+]i. The first phase was accompanied by mitochondrial membrane
depolarization. The second phase, whose rising phase paralleled mitochondrial membrane repolarization, was blocked by a Ca2+ free solution,
indicating the activation of plasmalemmal Ca2+ entry. The third phase
was blocked by a Ca2+ free, EGTA solution, but not by thapsigargin, and
enhanced at pH 9, but not in a Na+-free solution, indicating the activation
of store-operated Ca2+ entry (STOC). A blocker of phospholipase C,
U73122, accelerated the decay of the first phase and enhanced the second phase. At a high [Ca2+]i under the effect of thapsigargin, FCCP produced a large rise in [Ca2+]i and subsequent reduction, or directly
reduced [Ca2+]i for ten to tens of minutes. These results suggest that mitochondrial Ca2+ release and/or depolarization activates plasmalemmal
Ca2+ entry different from STOC and Ca2+ release from ER, which leads
to STOC activation, while Ca2+ release from ER activates Ca2+ accumulation in, or release from, mitochondria.
Nagoya University of Arts and Sciences, Aichi, Japan)
Kinetics of the divalent cation gate of gapjunction channel in guinea-pig ventricular
Matsuda, Hiroyuki1; Oka, Chiaki1; Matsuoka, Satoshi2;
Noma, Akinori2 (1Kyoto University Group in Leading Project for
Biosimulation, Kyoto, Japan; 2Department of Physiology and
Biophysics, Kyoto University Graduate School of Medicine, Kyoto,
Myocardial gap junction channels (Gap) are indispensable to action potential propagation. The channel gate (chemical gating) is regulated by
intracellular cations such as Ca2+, Mg2+ and H+, but its dynamic gating
properties have been scarcely examined. In this study, we investigated
effects of Mg2+ on the Gap conductance in paired cells dissociated from
guinea-pig ventriculues.The two-electrode whole-cell patch-clamp technique was applied to one of the paired ventricular myocytes (cell1). The
current response to±5 -mV voltage pulses was recorded every 400 ms. In
order to apply Mg2+instantaneously, we perforated the membrane of the
other pair of myocytes (cell 2), using a sealed pulsed nitrogen laser in the
presence of a given concentration of Mg2+ in the bath solution. Under
these conditions, the recorded current flowed from the cell 1 mostly
through the Gap into the cell 2 whose interior was short circuited to the
ground.The Gap conductance decreased in response to various Mg2+
concentrations ([Mg2+]i) in a dose-dependent manner (Hill coefficient:
3.84 EC50: 0.603mM). The conductance decay was well fit by a single
exponential function. The 1/τ - [Mg2+]i relationship was almost linear
over the range of [Mg2+]i from 1 to 10 mM. Our results suggest that the
Gap gating is regulated by multiple bindings of divalent cations,
including one rate-limiting step.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Ionic channels & receptors
Molecular mechanisms of regulation of
receptor type-specific Gq signaling by
Yoshihiro1,3; Saitoh,
Itoh, Masayuki2; Kubo,
Osamu2 (1Dept Mol Physiol, NIPS,
Okazaki, Japan; 2Dept Bio-Sci, Nagahama Inst Bio-Sci Tech,
Nagahama, Japan; 3SORST, JST, Kawaguchi, Japan)
The regulator of G-protein signaling type 8 (RGS8) has a high affinity
for Gαi and only a low affinity for Gαq. We previously reported that
RGS8 decreased the amplitude of Gq-mediated response in a receptor
type-specific manner and that RGS8S, a splice variant of N-terminus
region, induced less inhibition. Although molecular mechanisms
underlying receptor type-specific attenuation by RGS8 still remains
unclear, recent evidences have raised a possibility that RGS may interact
with certain GPCRs. Here we show by co-immunoprecipitation
experiments that RGS8 directly binds to the third intracellular (i3) loop
of M1- and M3-muscarinic AChR but not of M2, and that binding of
RGS8S is weaker. We observed that a deletion of N-terminal 9 aa of
RGS8 or substitutions of Arg-8 and Arg-9 of RGS8 for Ala reduced
binding with M1i3, suggesting the importance of N-terminal region. To
examine whether or not the interaction between RGS8 and M1 may
occur in living cells, we performed BRET analysis. The results showed
that RGS8 actually interacts with M1 and that the interaction of RGS8S
is less clear. We next analyzed electrophysiologically the inhibitory
effects of RGS8 w.t. and R8A/R9A mutant on Gq-mediated responses
using Xenopus oocytes, and observed that the inhibitory effect of RGS8
was decreased by the mutations. These biochemical and
electrophysiological results show that RGS8 inhibits M1-muscarinic
AChR-mediated responses by a mechanism which involves direct
interaction between N-terminus of RGS8 and i3 loop of M1.
TRPM2 activation in rat pancreatic islets is
involved in insulin secretion
Togashi, Kazuya1,2; Tominaga, Makoto1,2 (1Sec. Cell. Sig.
Okazaki Inst. Integrat. Biosci, NINS, Okazaki, Japan; 2Dept.
Physiol. Sci, Sch. Life Sci, Grad. Univ. Advanced Studies, Okazaki,
There are six thermosensitive TRP channels in mammals, and there
might be other TRP channels sensitive to temperature stimuli. Recently,
we have demonstrated that TRPM2 can be activated by exposure to
warm temperatures (>35°C) apparently via direct heat-evoked channel
gating. β-NAD+- or ADP-ribose-evoked TRPM2 activity is robustly
potentiated at elevated temperatures. We have also reported that, even
though cyclic ADP-ribose (cADP-ribose) does not activate TRPM2 at
25°C, co-application of heat and intracellular cADP-ribose dramatically
potentiates TRPM2 activity. Here we show that in rodent pancreatic
islets, TRPM2 is co-expressed with insulin, and mild heating (around
body temperature) of these cells evokes increases in both cytosolic Ca2+
and insulin release which is KATP channel-independent and cAMPmediated. Heat-evoked response in pancreatic islets was significantly
diminished by applying the known TRPM2 inhibitors; nonsteroidal antiinflammatory drug flufenamic acid (FFA), anti-fungal reagent econazole
or 2-aminoethoxydiphenyl borate (2-APB), and by treatment with
TRPM2-specific siRNA. These results suggest that TRPM2 regulates
Ca2+ entry into pancreatic β-cells at body temperature depending on
production of cADPR-related molecules, thereby regulating insulin
Swelling-activated chloride currents in
rabbit articular chondrocytes: inhibition by
arachidonic acid
Expression of Na+/Ca2+ exchangers in
mouse osteoclasts and their functional role
during bone resorption
Isoya, Eiji1; Matsuura, Hiroshi2; Toyoda, Futoshi2;
Okumura, Noriaki1; Kubo, Mitsuhiko1; Imai, Shinji1;
Matsusue, Yoshitaka1 (1Dep. of Orthop, Shiga Univ. of Medical
Kajiya, Hiroshi1; Li, Jing-Ping1; Nakao, Akihiro1;
Okamoto, Fujio1; Iwamoto, Takahiro2; Okabe, Koji1
Science; 2Dep. of Physiol, Shiga Univ. of Medical Science)
Articular chondrocytes play an important role in the formation of the cartilage in synovial joints, which is closely influenced by mechanical or
osmotic stress. In the present study, whole-cell membrane currents were
recorded from isolated rabbit articular chondrocytes during exposure to
hyposmotic external solution, under conditions where Na+, Ca2+ and K+
channels and electrogenic transporters were minimized. Articular chondrocytes responded to a hyposmotic external solution (Na+ reduction to
about 70% of control) with an osmotic cell swelling, which was consistently accompanied by the activation of an outwardly-rectifying Cl– current (swelling-activated Cl– current, ICl,swell). ICl,swell was practically
time-independent at potentials negative to +30 mV but exhibited a gradual inactivation at more positive potentials. Bath application of arachidonic acid (AA) reversibly and concentration-dependently blocked
ICl,swell with an IC50 of 0.58 µM and Hill coefficient of 1.9. The maximal
effect (100% block) was obtained with 10 µM AA. Neither
cyclooxygenase inhibitor indomethacin (10 µM) nor lipoxygenase
inhibitor nordihydroguaretic acid (NDGA, 3 µM) significantly affected
the inhibitory action of AA. In addition, PGE2, LTB4 and LTD4 did not
have any appreciable effect on ICl,swell, suggesting that AA directly
affected ICl,swell. The present study thus confirms the presence of ICl,swell
which exhibits a high sensitivity to inhibition by AA in rabbit articular
(1Fukuoka Dental College, Fukuoka, Japan; 2Fukuoka Univ.,
Fukuoka, Fukuoka)
The plasma membrane Na+/Ca2+ exchangers (NCXs) are bi-directional
transporter that catalyzes the exchange of Na+ for Ca2+ depending on the
electrochemical gradients. Mammalian NCX forms a multigene family
comprising NCX 1, NCX 2 and NCX 3. However, the expression and
functional role of NCXs in mammalian osteoclasts are still unknown.
The aim of present study is to clarify the expression of NCX and their
functional role during bone resorption in mouse osteoclasts. We examined the expression of NCX using RT-PCR, immunocytochemical and
Western blotting methods. The activation of NCX during bone resorption were assessed by measurement of intracellular Ca2+ concentration
([Ca2+]i) using fura-2 and the effect of NCX inhibitors on pit formation
assay. Mouse osteoclasts were expressed NCX 1 and 3, not NCX 2 using
RT-PCR, Western blotting and immunocytochemical methods. There are
some isoforms in mouse osteoclasts; NCX 1.3 and 1.4 in NCX 1 and
NCX 3.5 in NCX 3. Under the measurement of [Ca2+]i, low or free extracellular sodium increased [Ca2+]i in osteoclasts. The [Na+]o free-induced [Ca2+]i i increase was inhibited by NCX inhibitors. The NCX
inhibitors also decreased in pit area resorbed by osteoclasts in dose dependent manner. These results suggest that NCXs are expressed in
mouse osteoclasts and act as Ca2+ regulation during bone resorption.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Neurons & synaptic functions
Effects of a Gq inhibitor, YM-254890, on
carbachol-induced contraction of bovine
ciliary muscle
Yasui, Fuminori1; Takai, Yoshiko2; Miyazu, Motoi1;
Takai, Akira1 (1Dept. Physiol., Asahikawa Med. Coll., Asahikawa,
Japan; 2Dept. Ophthalmol., Sch. Med. Nagoya Univ. Nagoya,
In the ciliary muscle, a smooth muscle under parasympathetic control,
contraction is initiated by stimulation of muscarinic receptors of
M3subtype. It is established that the initial phasic component of the contraction is triggered by Ca2+ release from intracellular stores mediated by
Gq-linked signalling pathway. The tonic component is also known to be
highly dependent on Ca2+, but Ca2+ is now provided by influx through
receptor-operated cation channels rather than by release from stores [Takai et al.(2005) J Physol 559, 899-922]. However, little is known about
the signalling mechanism involved in this Ca2+ influx. Here we have examined effects of YM-254890, a putative specific Gq inhibitor on contraction and [Ca2+]i elevation induced by carbachol (CCh). For
mechanical experiments ciliary muscle bundles dissected from bovine
eyes were vertically mounted in an organ bath continuously perfused
with normal saline, and isometric tension was recorded using a U-gauge
transducer. Bath application of 2 µM-CCh caused a contraction. Both
phasic and tonic components of this response were abolished by YM254890 (3-10 µM). Using a Fluo-4 fluorescence method, we observed
that CCh (10 µM) induced an elevation of the [Ca2+]i in dispersed bovine
ciliary muscle cells. Both initial phase and sustained phase of this
response were also abrogated by YM-254890 (3-10 µM). Gq appears to
be critically involved in Ca2+ mobilization in tonic as well as phasic
component of the contraction of bovine ciliary muscle.
Abnormal regulation of ENaC and SGK1 by
aldosterone in Dahl salt-sensitive rat
Aoi, Wataru; Niisato, Naomi; Sawabe, Yukinori;
Miyazaki, Hiroaki; Marunaka, Yoshinori (Dept. Mol. Cell
Physiol., Grad. Sch. Med. Sci., Kyoto Pref. Univ. Med., Kyoto,
Disturbance of renal Na+ reabsorption develops hypertension in Dahl
salt-sensitive (DS) rat. Aldosterone plays a critical role in controlling renal Na+ reabsorption by stimulating expression of epithelial Na+ channel
(ENaC) and also activate an ENaC-regulating protein kinase, serum and
glucocorticoid-regulated kinase 1 (SGK1). Therefore, we studied how
aldosterone regulates ENaC expression and SGK1 in DS rat. Aldosterone (1.5 mg/kg B.W.) was subcutaneously injected into adrenalectomized DS and Dahl salt-resistant (DR) rats kept with normal (0.3%
NaCl) diet and saline for 2 weeks after adrenalectomy. RNA and protein
were extracted from the kidney 6 hr after the aldosterone application. Aldosterone decreased mRNA expression of β- and γ-ENaC in DS rat
unlike DR rat, while aldosterone increased α-ENaC mRNA expression
in DS rat similar to DR rat. Further, we found that aldosterone did not
affect SGK1 expression in DS rat but elevated it in DR rat. These
observations indicate that ENaC and SGK1 are abnormally regulated by
aldosterone in DS rats, suggesting that these abnormal responses to
aldosterone would be one of factors causing salt-sensitive hypertension.
Supported by JSPS 17390057, 17590191, 17790154.
Neurons & synaptic functions
The activation of phosphatidylinositollinked D1-like dopamine receptor
profoundly suppresses the exaitatory
transmission in the developing
Noriyama, Yoshinobu1; Ogawa, Yoichi2; Yoshino,
Hiroki1; Yamashita, Masayuki2; Kishimoto, Toshifumi1
The disorder of dopamine (DA) system may be related to neurodevelopmental dysfunction. However, the action of DA on synaptic transmission
during development is largely unknown. We studied the effect of DA on
GABAergic and glutamatergic transmission in neonatal rat hippocampus
from the early period of synapse formation by whole-cell patch-clamp
recordings from CA1 pyramidal cells. DA (100 µM) profoundly
decreased the amplitude of GABAA receptor-mediated postsynaptic
currents (GABAA-PSCs) to 32% in the first postnatal week, when
GABA provides excitatory drive. DA also decreased the amplitude of
AMPA receptor-mediated excitatory postsynaptic currents (EPSCs) to
29% in the second postnatal week, when glutamate responses first
appear. The DA-induced inhibition declined after these periods and
became only partial after postnatal day 30. Further we identified the
receptor subtype involved in the DA-induced inhibition as
phosphatidylinositol (PI)-linked D1-like receptor, since SKF 83959, a
selective agonist for PI-linked D1-like receptor, clearly mimicked the
action of DA, and U-73122, an inhibitor of phospholipase C,
significantly reduced the DA-induced inhibition. DA did not change the
response to puff-applied GABA or kainic acid, nor miniature GABAAPSC or EPSC amplitudes. These results suggest that the activation of PIlinked D1-like receptor profoundly suppresses the excitatory
J. Physiol. Sci., Vol. 56, Suppl., 2006
Neurons & synaptic functions
Chronic nicotine treatments increases
cholinergic modulation of GABAergic
synaptic transmission in the mouse
α-Adrenoceptive dual modulation of
inhibitory GABAergic inputs to Purkinje
cells in the mouse cerebellum
Miura, Masami1,2; Ishii, Katsuyoshi2; Sumikawa,
Katumi2; Aosaki, Toshihiko1 (1Tokyo Metropolitan Institute of
(Neuronal Circuit Mechanisms Research Group, BSI, RIKEN, Wako,
Gerontology, Tokyo, Japan; 2Univ. California, Irvine, USA)
The striatum, an input stage of the basal ganglia, contributes to habit formation as well as motor functions. Recent studies suggest the involvement of the dorsal striatum in the advanced stages of drug addiction.
Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the striatum and can control the excitability of medium-sized
spiny (MS) neurons, which provide the striatal output. We investigated
the effect of chronic nicotine treatment on GABAergic synaptic transmission in mouse MS neurons. Twice-daily subcutaneous injections of
nicotine (1 mg/kg) for 10-15 days did not change the electrophysiological properties of MS neurons or of three types of interneurons. However,
intrastriatal stimulation evoked multiphasic IPSCs more frequently in
MS neurons of nicotine-treated mice than in those of PBS-treated mice.
Multiphasic IPSCs consisted of early and late components, both of
which were mediated by GABAA receptors. However, the GABAB receptor agonist SKF97541 suppressed the early but not the late IPSC,
suggesting heterogeneity of GABAergic inputs to MS neurons. Furthermore, generation of the late IPSC required the activation of nicotinic
acetylcholine receptors (nAChR) because dihydro-β-erythroidine, an
antagonist of nAChR, suppressed only the late IPSC. These results
suggest that chronic nicotine treatment enhances the cholinergic
modulation of GABAergic synaptic transmission in the striatum and that
the enhanced inhibitory inputs.
In the cerebellar cortex, previous reports indicated that noradrenaline
(NA) enhances inhibitory synaptic transmission via β-adrenoceptorpathways. However, the effects of α-adrenoceptor activation on
cerebellar inhibitory postsynaptic currents (IPSCs) have not yet been
fully understood. Therefore, we investigated the effects of the α1- or α2adrenoceptor agonist on IPSCs recorded from mouse Purkinje cells
(PCs). The selective α1-adrenoceptor agonist phenylephrine (PE)
increased both the frequency and amplitude of spontaneous IPSCs
(sIPSCs). PE also enhanced the amplitude of evoked IPSCs (eIPSCs)
and increased the frequency but not the amplitude of miniature IPSCs
(mIPSCs). Moreover, PE decreased the paired-pulse ratio of eIPSCs and
did not change GABA receptor sensitivity in PCs. Conversely, the
selective α2-adrenoceptor agonist clonidine significantly reduced both
the frequency and the amplitude of sIPSCs. Neither eIPSCs nor mIPSCs
were affected by clonidine. Furthermore, presynaptic cell-attached
recordings showed that spontaneous activity of GABAergic
interneurons was enhanced by PE, while reduced by clonidine. These
results suggest that NA enhances inhibitory neurotransmitter release via
α1-adrenoceptors, which are expressed in presynaptic terminals and
somatodendritic domains, whereas suppresses the excitability of
interneurons via α2-adrenoceptors, which are expressed in presynaptic
somatodendritic domains. Thus, cerebellar α-adrenoceptors play roles in
a presynaptic dual modulation of GABAergic inputs from interneurons
to PCs.
Muscarinic suppression of Golgi cells
excitability in the mammalian cochlear
Irie, Tomohiko; Fukui, Iwao; Ohmori, Harunori (Facult.
Med., Kyoto Univ., Kyoto, Japan)
Dorsal cochlear nucleus (DCN) is known to process complex sounds.
The principal cells are known to integrate inputs from auditory nerve fibers (ANFs) and parallel fibers. Axons of granule cells form parallel fibers and convey multimodal information. Granule cells cluster around
ventral cochlear nucleus and DCN, and have mutual synapses between
inhibitory interneurons; Golgi cells. Thus, Golgi cells may have some
modulatory effects on parallel fiber activities; however, little is known.
We studied the excitability of Golgi cells and interpreted the roles played
by Golgi cells in the neuronal activity of DCN. By depolarizing current
injection, Golgi cells fired repetitively and the firing frequency increased
with current injection. At higher current intensity (300 pA-400 pA),
steep firing adaptation was observed. By hyperpolarizing current
injection, a depolarizing voltage sag emerged due to h-current activation.
EPSCs evoked by ANFs stimulation were of multiple-peaks suggesting
inputs through polysynaptic pathway. Because some cholinergic
projections were expected, we tested cholinergic agonists: Carbachol
induced a membrane hyperpolarization, accompanied with a decrease in
the input resistance; muscarine evoked similar responses. These indicate
the activation of muscarinic receptors, which hyperpolarized Golgi cells
through the activation of GIRK.
Therefore, cholinergic innervation may contribute in modulation of
parallel fiber activity through inhibitory Golgi cells.
Hirono, Moritoshi; Matsunaga, Wataru; Obata, Kunihiko
Disruption of Clathrin-mediated
Endocytosis of Synaptic Vesicles by
Calpain-dependent Cleavages of
Amphiphysin I
Wu, Yumei; Tomizawa, Kazuhito; Wei, Fanyan; Omori,
Iori; Nishiki, Tei-ichi; Matsui, Hideki (Dept. Physiol. Grad.
Sch. Med. & Dent., Univ. Okayama, Okayama, Japan)
Amphiphysin I, a member of the BAR (Bin-Amphiphysin-Rvsp) protein
super family, plays a key role in clathrin-mediated endocytosis of synaptic vesicles. Amphiphysin I mediates invagination and fission of synaptic vesicles in cooperation with dynamin, which senses and facilitates
membrane curvature by its BAR domain. In vitro, when present at higher
concentrations, amphiphysin I can stabilize membrane curvature, generating lipid tubules, forming ring structure with dynamin, and increasing
dynamin GTPase activity. In the present study, we found that amphiphysin I was cleaved to three fragments by treatment with high KCl (80 mM)
in the mouse hippocampus slices. The cleavages were inhibited by pretreatment with calpain inhibitors. Calpain also cleaved amphihysin I to
three fragments in vitro. We identified the three cleavage sites by mass
spectrometry. Amphiphysin I was cleaved at the sites of 322, 349 and
386. Calpain-dependent cleavages of amphiphysin I can induce the liposome tubulation as the same as wild- type amphiphysin I, but it cannot
form the ring structure with dynamin I under electron microscope. Moreover, transferrin uptake was inhibited by overexpression of the truncated
form of amphiphysin I compared with that of wild-type amphiphysin I in
COS-7 cell. These results suggest that amphiphysin I may be a substrate
of calpain in presynaptic terminus and the cleavages are important for
the regulation of clathrin-mediated endocytosis of synaptic vesicles.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Neurons & synaptic functions
Protein kinase inhibitor staurosporine
reduces the number of readily releasable
synaptic vesicles at the calyx of Held
Kanda, Takeshi; Takahashi, Tomoyuki (Dept. Neurophysiol.,
Univ. Tokyo Grad. Sch. Med., Tokyo, Japan)
The efficacy of synaptic transmission is determined by the quantal parameters such as the number of readily releasable pool vesicles (N), release probability (p) and postsynaptic response to a single quantum of
transmitter (q). After massive vesicle exocytosis, N is replenished by
vesicle trafficking for maintaining synaptic efficacy. However the mechanism underlying vesicle dynamics remains unknown. As a first step, we
investigated whether staurosporine, a general kinase inhibitor, affects
quantal parameters at the calyx of Held synapse in the brainstem slices
of 12- to 15-day-old rats. Pretreatment of slices with 2 µM staurosporine
for 1h decreased the amplitude of evoked EPSCs and slowed their rise
time, but had no effect on their decay time. Staurosporine also reduced
the frequency of spontaneous miniature EPSCs without affecting their
amplitude (q) or kinetics. Estimation of quantal parameters, using the
tetanic stimulation protocol, revealed that staurosporine reduced Nq, but
not p. Staurosporine increased the magnitude of synaptic depression
during repetitive stimulation (1-100 Hz) supporting the depletion model
of synaptic depression. Staurosporin slowed recovery from depression
caused by 10-100 Hz-stimulation without affecting the fast phase of
recovery time course after 100 Hz-stimulation. We conclude that
staurosporine-sensitive protein kinases affect the size of readily
releasable pool and specifically accelerate a slow component of vesicle
mobilization thereby contributing to the replenishment of readily
releasable vesicles.
Positive modulation by proteinaseactivated receptor agonist peptides of
nociceptive transmission in the rat spinal
dorsal horn
Fujita, Tsugumi; Nakatsuka, Terumasa; Koga, Akiko;
Liu, Tao; Kumamoto, Eiichi (Dept. Physiol., Facult. Med., Saga
Univ., Saga, Japan)
G-protein coupled proteinase-activated receptors (PARs) have a unique
activation mechanism in that a proteolytically exposed N-terminal region acts as a tethered ligand. Four members of PARs such as PAR-1 and
PAR-2, which were identified by molecular cloning, can be activated by
synthetic peptides whose amino acid sequences correspond to the tethered ligands. Although PARs appear to be involved in nociceptive transmission in peripheral terminals of primary afferents, there is no report
about involvement of PARs in the transmission in the spinal dorsal horn.
In order to clarify a role of PARs in the nociceptive transmission, we examined how PAR-1 and PAR-2 agonist peptides affect glutamatergic
spontaneous excitatory postsynaptic currents (sEPSCs) in substantia gelatinosa (SG; lamina II of Rexed) neurons which are thought to play a
pivotal role in regulating nociceptive transmission to the CNS. We applied the blind whole-cell voltage-clamp technique to the SG neurons in
adult rat spinal cord slices. PAR-1 agonist peptide (SFLLRN-NH2; 1
µM) reversibly increased the frequency of sEPSC without a change in its
amplitude, while PAR-2 agonist peptide (SLIGKV-NH2; 1 µM) had no
effects on sEPSCs. Both peptides did not alter holding currents at -70
mV. These results indicate that PAR-1 but not PAR-2 agonist peptide
enhances the spontaneous release of L-glutamate from nerve terminals
in the SG. It is suggested that PAR-1s expressed in nerve terminals in the
SG may play an important role in producing nociception.
Melittin Enhances Inhibitory Synaptic
Transmission in Adult Rat Spinal Dorsal
Horn Neurons
Muscarinic responses in rat PAG neurons
Liu, Tao; Fujita, Tugumi; Koga, Akiko; Nakatsuka,
Terumasa; Kumamoto, Eiichi (Dept. Physiol., Facult. Med.,
of Intenal Medicine, Shiga University of Medical Science, Otsu,
Shiga, Japan; 2Institute of Physiology, University of Kiel, Kiel,
Germany; 3Department of Nursing, Shiga University of Medical
Science, Otsu, Shiga, Japan)
Saga Univ., Saga, Japan)
We have recently reported that a phospholipase A2 (PLA2) activator
melittin reversibly enhances glutamatergic excitatory synaptic transmission in substantia gelatinosa (SG; lamina II of Rexed) neurons of the rat
spinal cord. The SG neurons receive not only excitatory but also GABAand glycine-mediated inhibitory transmission. In order to know the effect of melittin on the spontaneous inhibitory transmission, we applied
the blind whole-cell patch-clamp technique to SG neurons in adult rat
spinal cord slices. Melittin (1 µM) superfused for 3 min gradually
increased the frequency and amplitude of spontaneous inhibitory
postsynaptic currents (sIPSCs) at a holding potential of 0 mV, which
were visible about 2 min after the beginning of its superfusion and
subsided within 8 min after washout. These facilitatory actions of
melittin were observed for GABAergic and glycinergic sIPSCs, which
were recorded in the presence of a glycine-receptor antagonist
strychnine (1 µM) and a GABAA receptor antagonist bicuculline (10
µM), respectively, and were reduced in extent by a PLA2 inhibitor 4bromophenacryl bromide (10 µM). A voltage-gated Na+ channel blocker
tetrodotoxin (TTX, 0.5 µM) had a tendency to inhibit the effect of
melittin on the GABAergic but not glycinergic sIPSC. It is concluded
that melittin enhances GABAergic and glycinergic inbibitory
transmission in a pre- and postsynaptic manner through the activation of
PLA2 in the SG; a part of the effect of melittin on GABAergic
transmission is due to its action on excitatory transmission.
Sanada, Mitsuru1; Alzheimer, Christian2; Maeda,
Kengo1; Yasuda, Hitoshi3 (1Division of Neurology, Department
The periaqueductal grey (PAG) of the brainstem is a central site for the
various physiological functions including cardiovascular control, defensive behavior and pain. Since the cholinergic modulation on PAG activity is still unknown, we used infrared-videomicroscopy in conjunction
with whole-cell recordings to elucidate the effects of acetylcholine on
PAG neurons in midbrain slices of juvenile rats. In current clamp mode,
40% of all PAG neurons examined showed depolarization of their membrane potential during the application of carbachol (CCh). On the other
hand, 20% of all PAG neurons examined were hyperpolarized by CCh.
The remaining PAG neurons (40%) were insensitive to CCh. Both the
depolarizing and the hyperpolarizing action of CCh were atropine-sensitive, indicating that they were mediated by muscarinic receptors. The depolarizing response to CCh was abrogated by M1 antagonist,
pirenzepine, while the hyperpolarizing response was abolished by M2
antagonist, gallamine. These results suggest that rat PAG neurons have
functional muscarinic receptors.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Neurons & synaptic functions
Facilitated NMDA-receptor activities in the
hippocampal CA1 area of mutant mice
lacking D-amino-acid oxidase
Watanabe, Masashi1; Maekawa, Masao2; Tsuda,
Masayuki2; Yamaguchi, Shigeki1; Hori, Yuuichi2 (1Dept. of
Anesthesiol., Dokkyo Univ. Sch. Med., Mibu, Japan; 2Dept. of
Physiol & Biol. Inf., Dokkyo Univ. Sch. Med., Mibu, Japan)
D-serine which is thought to facilitate the NMDA receptor, present in the
forebrain and co-localize with N-methyl-D-aspartate (NMDA) receptor.
We reported that the spatial learning in the water maze and tetanus induced hippocampal LTP in the slice preparations were facilitated in the
mutant mice lacking D-amino-acid oxidase, an enzyme which metabolizes D-serine. In the present report, we compared NMDA dependent
synaptic current and the effect of D-serine on NMDA dependent synaptic current in the hippocampal CA1 area between the wild-type mice and
mutant mice lacking D-amino-acid oxidase, to clarify whether NMDA
dependent synaptic current was enhanced in the mutant mice and whether the enhancement was D-serine dependent. Excitatory postsynaptic
currents (EPSCs) were recorded in CA1 pyramidal cells using whole cell
patch-clamp techniques by stimulating Schaffer collateral-commissural
fibers of the hippocampal slices. The ratio of NMDA receptor-mediated
EPSC amplitudes to non-NMDA receptor-mediated EPSC amplitudes
was significantly larger in the mutant mice than in wild-type mice. The
ratio of NMDA component to non-NMDA component of the wild-type
mice was significantly increased to the levels observed in the mutant
mice, when D-serine was added to perfusion medium. We suggest that
D-serine increased in the mutant mice brain facilitates NMDA-mediated
synaptic current.
Apparent lack of desensitization in the
AMPA-component of EPSC evoked in the
CA1 pyramidal neuron of hippocampal
slice of adult rat
Kimura, Shingo1; Kawasaki, satoshi1; Watanabe, Shuji1;
Fujita, Reiko2; Sasaki, Kazuhiko1 (1Dept. Physiol., Sch. Med.,
Iwate Med. Univ., Japan; 2Dept. Chem., Sch. Lib. Arts & Sci., Iwate
Med. Univ., Japan)
To study differences in nature between synaptic and extrasynaptic
AMPA-receptor in the adult brain, we recorded stimulus-evoked EPSC
and AMPA-induced current response from hippocampal pyramidal neuron of rat brain slice under whole-cell patch clamp. Application of either
NBQX or GYKI52466, AMPA antagonists, suppressed the both EPSC
and AMPA-induced current response. Current(I)-voltage(V) relationship of the EPSC and AMPA-response intersected the voltage axis at 0
mV showing linear I-V curve. The AMPA-induced response was markedly augmented as much as twice of the control in the presence of cyclothiazide (CTZ), an inhibitor of desensitization of AMPA-receptor,
whereas either EPSC or spontaneously evoked miniature EPSC was not
affected at all by the CTZ. Furthermore, neither the amplitude nor the
time course of the consecutive EPSCs evoked by repetitive stimulation
of Schafer collateral at 100 Hz was not affected significantly by the presence of CTZ, although cumulative current responses to repetitive application of AMPA, which exhibited occlusion, were greatly augmented by
CTZ in similar fashion to the single application of AMPA. The amplitude of the EPSC decreased in the presence of dihydrokainic acid
(DHK), an inhibitor of GLT-1 glutamate transporter, but addition of CTZ
to the DHK did not augment the EPSC amplitude. All these results suggest that synaptic AMPA receptor may lack the nature of desensitization
unlike the extrasynaptic AMPA-receptor.
Lidocaine transiently inhibits evoked
EPSPs in the rat hippocampal CA1 region
Tanaka, Eiichiro; Yamada, Aya; Higashi, Hideho
Facilitation of glutamatergic excitatory
synaptic transmission in hippocampal CA1
regions of traumatic brain injury model rats
(Deptartment of Physiology, Kurume University School of Medicine,
Kurume, Japan)
Hasuo, Hiroshi1; Cao, Ruifeng1; Ooba, Satomi1,2;
Akasu, Takashi1 (1Dept. Physiology, Kurume Univ. Sch. Med.,
Extracellular recordings were made from CA1 regions in the rat hippocampal slice tissues. Superfusion of slice preparations with lidocaine at
low concentrations (1 - 100 µM) induced a transient inhibition and
subsequent augmentation of the maximal slope of the field excitatory
postsynaptic potentials (fEPSPs). On the other hand, high concentrations
(300 - 1000 µM) of lidocaine only suppressed the maximal slope of the
fEPSPs. The amplitude of presynaptic volleys was simply suppressed by
lidocaine (1 - 1000 µM) in a dose-dependent manner. Pretreatment of
adenosine 1 (A1) receptor antagonist, DPCPX (1 µM) diminished the
transient inhibition of the fEPSPs. Intracellular recordings from CA1
neurons showed that lidocaine (3 - 100 µM) transiently suppressed the
amplitudes of the evoked fast EPSPs and, of the fast and late IPSPs.
Lidocaine at low concentrations (3 - 30 µM) also induced a DPCPXsensitive transient hyperpolarization in the CA1 and CA3 neurons. In the
presence of TEA (20 mM) and TTX (0.3 µM), lidocaine at the low
concentrations reduced the amplitude and duration of Ca2+ spikes
recorded from CA3 neurons. These results suggest that lidocaine at the
low concentrations (3 - 100 µM) transiently inhibites the evoked fEPSPs
via activation of A1 receptors at both pre- and post-synaptic sites in the
rat CA1 hippocampal neurons.
Kurume, Japan; 2Dept. Neurosurgery, Kurume Univ. Sch. Med.,
Kurume, Japan)
We investigated the effects of fluid percussion injury (FPI) on the
glutamatergic excitatory synaptic transmission of CA1 pyramidal neurons using conventional intracellular recording techniques. A moderate
impact (3.8-4.8 atm) was applied to the left hemisphere of the parietal
cerebral cortex by using a FPI device (HPD-1700, Dragonfly, Inc.). After a survival period of 7 days, horizontal brain slices containing hippocampus were cut. The slice preparations were devided into three groups
(control, FPI-ipsilateral, and FPI-contralateral). The control group includes the data from normal and sham-operated rats. There were no significant differences in resting and acting membrane properties among
three groups. However, the EPSPs evoked by stimulations of Schaffer
collaterals in the presence of bicuculline showed steeper input-output relationship (I-O) in the FPI-ipsilateral group. The number of spikes
evoked by EPSP in the presence of bicuculline was larger in FPI-ipsilateral group although there were no significant differences among three
groups in the absence of bicuculline. The paired-pulse facilitation ratio
in FPI groups was smaller than that of control group. Frequency of mEPSPs recorded in the presence of tetrodotoxin was higher than that of
control while the amplitude of mEPSPs was not different. These results
suggest that the post-traumatic facilitation of gultamatergic transmission
in pyramidal neurons of rat hippocampal CA1 is mediated by presynaptic origin.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Neurons & synaptic functions
Effects of propofol on tonic GABAergic
inhibition in rat neocortex layer V
pyramidal neuron
Neuronal glutamate transporters regulate
the activation of metabotropic glutamate
receptors in cerebellar Purkinje cells
Yam, Sumii1; Yamada, Junko2; Furukawa, Tomonori1;
Fukuda, Atsuo1,2 (1Dept. of Physiol. Hamamatsu Univ. Sch. of
Nikkuni, Osamu; Takayasu, Yukihiro; Iino, Masae;
Ozawa, Seiji (Dept. Neurophysiol., Gunma Univ. Grad. Sch.
Med., Hamamatsu, Japan; 2Grad. Sch. of Elec. Sci. and Tech,
Shizuoka Univ., Hamamatsu, Japan)
Med., Maebashi, Gunma, Japan)
GABAAreceptor mediated two inhibition models, phasic and tonic. Midazolam, benzodiazepine, and a widely used anesthetic, propofol potentiate GABAA receptor function. Bai et al. reported propofol, compared
with midazolam, had a lower potency but higher efficacy for increasing
the amplitude of the tonic current in the hippocampal CA1 pyramidal
neurons. We compared the effect of midazolam and propofol on the tonic
current in neocortex layer V pyramidal neuronsusing whole-cell patch
clam techniques from 2- to 3- week old rats. Propofol and midazolam
cause a concentration-dependent increase in the amplitude of the tonic
current. In the neocortex, midazolam increased tonic current more than
propofol. Picrotoxin blocked both midazolam and propofol effect on the
tonic current. In contrast, bicuculline blocked tonic current induced by
midazolam but not proporol. These suggest that these two drugs mediate
tonic current via different subtypes of GABAA receptors Referance
(1)Molecular Pharmacology 2001;59:814-824
Descending inhibitory synaptic inputs to
rat superior salivatory nucleus
Fujii, Akihito1; Mitoh, Yoshihiro2; Funahashi, Makoto3;
Kobashi, Motoi2; Takano-Yamamoto, Teruko1; Matsuo,
Ryuji2 (1Dept. Orthodontics and Dentofacial Orthopedics.,
Okayama Univ. Grad. Sch. Med. Dent., Okayama, Japan.; 2Dept.
Oral Physiol., Okayama Univ. Grad. Sch. Med. Dent., Okayama,
Japan.; 3Dept. Oral.Physiol., Hokkaido Univ. Grad. Sch. Dent.,
Hokkaido, Japan.)
We showed the excitatory (glutamate) and inhibitory (GABA and glycine) synaptic inputs to the superior salivatory (SS) neurons innervating
the submandibular salivary glands and tongue. However, the relationships between the higher and lower centers in the synaptic inputs have
not yet been examined. In the present study, we studied electrophysiologically the inhibitory synaptic inputs in brainstem slices obtained from
normal and decerebrate rats. The SS neurons were labeled by retrograde
axonal transport of a fluorescent dye. Whole-cell patch-clamp recordings were made from the labeled neurons. The currents were evoked by
agonists (GABA and Glycine) perfusion, and electrical stimulation near
the recording cell. After decerebration, agonists perfusion induced larger
currents, but their decay time constant were not altered as compared with
those of normals. This increase may result from receptor up-regulation
at postsynaptic membrane. By electrical stimulation, in 83% (n=34/41)
neurons, enhanced IPSCs were evoked, suggesting that the neurons have
the inhibitory inputs from both the higher and lower centers. In 17% neurons (n=7/41), no IPSCs were evoked, suggesting that the neurons have
only the descending inhibitory inputs from the higher centers. It is suggested that all SS neurons have the descending inhibitory synaptic inputs
from the higher centers.
Glutamate transporters are essential to remove synaptically released
glutamate in excitatory synapses. Glial glutamate transporters expressed
in Bergmann glia remove the majority of glutamate at excitatory synapses in cerebellar Purkinje cells (PCs) at early times after transmitter release. The neuronal glutamate transporter, the excitatory amino acid
transporter 4 (EAAT4), is concentrated at perisynaptic sites of PCs,
where metabotropic glutamate receptors (mGluRs) are located. To clarify the contribution of EAAT4 to the regulation of mGluR activation, we
recorded mGluR-mediated excitatory postsynaptic currents (mGluREPSCs) in cerebellar slices of mice deficient in EAAT4 and compared
them with those in wild-type (WT) mice. The amplitude of mGluR-EPSCs evoked by the stimulation of parallel fibers (PFs) was larger in
EAAT4-deficient mice than that in WT mice. However, the amplitudes
of PF-evoked mGluR-EPSCs in EAAT4-deficient and wild-type mice
were similar in the presence of the glutamate transporter antagonist DLthreo-β-benzyloxyaspartic acid (TBOA). mGluR-EPSCs evoked by the
stimulation of climbing fibers (CFs) were observed in EAAT4-deficient
mice but not in WT mice in the normal saline. When the function of
EAAT4 was inhibited by a pharmacological treatment, mGluR-EPSCs
were elicited by the stimulation of CFs even in WT mice. These results
indicate that EAAT4 plays a critical role in the regulation of the
activation of perisynaptic mGluRs at both PF and CF synapses in PCs.
Removal of extracellular divalent cations
induced a slowly synchronized rhythmic
activity in immature rat dorsal spinal cord
Yoshio, Harada (Dept. of Physiology, Nippon Medical School,
Tokyo 113-8602, Japan)
Immature rat spinal motoneurons showed a slowly synchronized rhythmic activity (sSRA) in divalent cation- free solutions. This sSRA was attributed to rhythmic oscillations of extracellular potassium ions in spinal
cord. So, possible occurrence of the same phenomena in the dorsal horn
at this same special situation, was investigated in this experiment. In the
isolated spinal cord preparation from newborn rats, extracellular neuronal activity was recorded from dorsal and ventral roots using suction
electrodes. After removal of extracellular divalent cations, sSRA could
be recorded from both the dorsal root (dorsal sSRA) and the ventral root
(ventral sSRA). The dorsal and ventral sSRA had a mirror image, but
ventral sSRA occurred slightly earlier. Both were recorded in rats soon
after birth, but not in older rats. Sectioning the border between the dorsal
and ventral horn eliminated ventral sSRA, but not dorsal sSRA. It is suggested that both dorsal and ventral sSRA depends on the activity of a
rhythm generator located in the dorsal horn. Several characteristic features of these two sSRA will be discussed.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Neurons & synaptic functions
Relationship between 5'-nucleotidase
activity and nicotine effects in rat
Sakai, Yasushi1,2; Hyuga, Masumi1; Homma, Ikuo2 (1Div.
Physiology, Sch.Nurs. & Reha. Sci. Showa Univ. Yokohama, Japan;
22nd Dept. Physiology, Sch. Med. Showa Univ. Tokyo, Japan)
It has been demonstrated that AMP is hydrolyzed to adenosine(ADO) in
the synaptic cleft by 5'-nucleotidase. This enzyme in the central nervous
system is known to participate in neuroprotective properties. However,
little is known about the relationship between 5'-nucleotidase activity
and nicotine effects. Nicotine significantly increased 5'-nucleotidase in
synaptosomes from hippocampus of adult rats. This activity was significantly inhibited by AMPCP which was a 5'-nucleotidase inhibitor. Nicotine significantly increased the hydrolysis of ATP, ADP, AMP and
ADO by analysis with HPLC. These hydrolysis of adenine nucleotides
were completely blocked in EGTA Ca2+-free buffer solution. We also examined the effect of ovariectomy (OVX) and estrogen replacement therapy (ER) on the activity of the enzyme that degrade adenine nucleotides
in female rats. The release of ATP, ADP, AMP and ADO from hippocampus slice preparations by nicotine application in the buffer solution
tended to decrease in the OVX group when compared to a control group.
ER reversed the inhibition of the release of these nucleotides observed in
OVX rats. The regulation of enzymes that hydrolyze these nucleotides in
the hippocampus is essential in the modulation of the processes of neuronprotective properties. Results suggest the presence of a strong relationship between 5'-nucleotidase activity and nicotine effects to the ADO
formation, and also to estrogen binding-sites.
NMDA receptor activity-dependent
oscillatory signal outputs from the
retrosplenial cortex triggered by a signal
input from the visual cortex
Yoshimura, Hiroshi1; Honjo, Makoto1; Mashiyama,
Yuichi1; Segami, Natsuki1; Sugai, Tokio2; Onoda,
Norihiko2 (1Dept. Oral and Maxillofaci. Surg., Kanazawa Med.
Univ., Uchinada-cho, Ishikawa, Japan; 2Dept. Physiol., Kanazawa
Med. Univ., Uchinada-cho, Ishikawa, Japan)
The retrosplenial cortex is located at a critical juncture between the visual cortex and hippocampal formation. Herein we show how signals
traveling from the visual cortex behave in local circuits of the retrosplenial cortex, using optical recording methods and application of caffeine
to rat brain slices. Electrical signals evoked in the primary visual cortex
penetrated into the deep layer of the retrosplenial granular a cortex (RSGa), and propagated further toward postsubiculum and upper layer. NonNMDA receptor-dependent initial traveling signal from the visual cortex
triggered NMDA receptor-dependent neural oscillation in the RSGa. Oscillatory signals originated from the local area in the deep layer of the
RSGa, and the signal spread back and forth toward the visual cortex and
postsubiculum, in addition to spreading toward the upper layer. From the
perspective of the RSGa, extrinsic signal inputs from the visual cortex
switched on neural oscillators in the RSGa that deliver NMDA receptordependent intrinsic signal outputs. Opening and strengthening of nonNMDA receptor-dependent input pathways from the visual cortex required NMDA receptor-dependent oscillatory neural activities. These
input and output relationships indicate that the retrosplenial cortex may
represent an important relay station between the visual cortex and hippocampal formation.
Differential effects of propofol on inhibitory
postsynaptic currents in CA1 pyramidal
cells and dentate gyrus granule cells of rat
hippocampal slices
Muscarinic inhibition on IPSC at SNr GABA
neurons is induced through the Gβγ
activation from Gq/11 linked with M3muscarinic ACh receptor.
Ishiguro, Masanori; Matsuyama, Kiyoji; Aoki, Mamoru
Miyazaki, Takefumi (Dept. of Physiol., Tokyo Med. Univ., Tokyo,
(Sapporo Med.Univ.Scl. of Med. , Sapporo, Japan)
We have recently reported that there exist regional differences of benzodiazepine effects in GABA-mediated inhibitory synaptic transmission in
vitro studies. In this study, we examined the effects of propofol, one of
the most popular intravenous anesthetic agents, on the inhibitory
postsynaptic currents (IPSCs) in CA1 pyramidal cells (CA1-PCs) and
dentate gyrus granule cells (DG-GCs) in rat hippocampal slices. The
monosynaptic IPSCs were evoked by electrical stimulation of GABAergic interneurons and recorded from CA1-PCs and DG-GCs by whole cell
patch-clamp technique. The effects of specific concentrations of propofol (0.1, 1, 10 and 100µM) on the IPSCs in CA1-PCs and DG-GCs were
examined at varying membrane potentials (20 mV steps, from -120 to
+40 mV). In the absence of propofol, at the clamped membrane potential
of-120mV and +40mV, IPSC amplitudes and decay time constants in
both CA1-PCs and DG-GCs were kept stable for at least 20 minutes.
When tested within this stable period, propofol was observed to increase
the amplitudes and prolonged the decay time constant of IPSC s in CA1PCs. However, propofol changed neither the amplitude nor decay time
constant of the IPSCs in DG-GCs. These results suggest that propofol
possesses differential effects on IPSCs in CA1-PCs and DG-GCs similar
to benzodiazepines. The mechanism for these differential effects could
be due to the different sensitivity to propofol of the GABAAreceptor
subtypes in the CA1-PCs and DG-GCs.
Although I have analyzed the mechanism of muscarinic inhibition on the
IPSC at substantia nigra pars reticulata GABA neurons of rat, the intracellular transduction mechanism is not clear yet. N-ethylmaleimide
(NEM), a membrane permeable inhibitor of PTX-sensitive G-proteins,
did not have any significant effect on the muscarinic inhibition at the
concentration of 100 µM. NEM itself significantly increased in the
amplitude of IPSC to 1.627 ± 0.166 of the control amplitude (n=5, mean
± S.E.M., p=0.019 by Student's t test paired). Muscarine (10 µM)
reduced the IPSC amplitude to 0.420 ± 0.104 under the control condition
and to 0.370 ± 0.050 of the increased IPSC in the solution with NEM,
respectively (p=0.738). ω-Agatoxin TK (ω-Aga TK), a selective P-type
Ca2+ channel blocker, exerted no inhibitory influence on the muscarinic
inhibition. The amplitude of IPSC was significantly attenuated to 0.443
± 0.139 (n=4, p=0.031) in the solution with ω-Aga TK (100 nM). The
muscarinic inhibition ratios were 0.430 ± 0.089 in control and 0.326 ±
0.077 of the decreased IPSC in the solution with ω-Aga TK, respectively
(p=0.386). These results mentioned above and those previously reported
suggest that Gβγ subunits dissociated from Gq/11 linked with M3receptors reduce the GABA release through the direct effect on the
release machinery, when M3-muscarinic ACh receptors at the
presynaptic terminal of a striato-nigral projection fiber is activated.
Hopefully, these observations contribute to the new approach of drug
therapy for the basal ganglia disturbance.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Neurons & synaptic functions
Visualization of synaptically released
glutamate by a novel opticalglutamate
Namiki, Shigeyuki; Sakamoto, Hirokazu; Iinuma, Sho;
Hirose, Kenzo (Dept. Cell Physiol, Nagoya Univ. Grad. Sch.
Rapid elevation of the synaptophysin
mRNA expression level in rat
somatosensory cortex induced by tactile
Med., Nagoya Japan)
Yokoyama, Osamu; Kumashiro, Mari; Iriki, Atsushi;
Ishibashi, Hidetoshi (Sec. Cogn. Neurobiol., Tokyo Med. Dent.
Glutamate is an essential excitatory neurotransmitter in the central nervous systems. For the understanding of mechanisms underlying synaptic
transmission, we developed a novel optical glutamate probe called
S403C-OG which consists of a recombinant glutamate binding domain
derived from GluR2 subunit of AMPA receptor and a fluorescent dye. To
visualize synaptically released glutamate, we immobilized S403C-OG
on the cell surface of the cultured hippocampal neurons and captured fluorescence images with CCD camera. With this maneuver, we successfully detected the glutamate release along active synapses in response to
electrical stimuli. The amount of glutamate release considerably varied
among locations within the same neuron, suggesting the spatial heterogeneity among the release sites. We also observed spontaneous and transient glutamate release events without electrical stimuli, which was
stochastic, variable in amplitude and spatially confined in small regions.
Neither application of tetrodotoxin nor removal of extracellular calcium
blocked the spontaneous glutamate release. The frequency of the release
increased upon application of high concentrations of sucrose which is
known to increase the frequency of miniature EPSC. Furthermore, we
succeeded in continuous monitoring of the changes in presynaptic activity induced by phorbol ester, indicating that our probe enable to directly
visualize the presynaptic activity. In summary, S403C-OG is useful to
address many fundamental issues related to glutamatergic synaptic
transmission in the central nervous system.
Synaptophysin is an integral membrane protein abundant in the synaptic
vesicle and is found in nerve terminals throughout the brain. Its function
has been implicated in various aspects of synaptic vesicle cycling such
as biogenesis of vesicles, the regulation of the SNARE complex formation, synaptic vesicle fusion with plasma membrane, endocytosis and recycling of synaptic vesicles. It was recently suggested that
synaptophysin is also involved in the modulation of activity-dependent
synapse formation under a competitive condition. In this study, we examined at the individual level whether tactile stimulation selectively influenced the synaptophysin mRNA expression level in the
somatosensory cortex of rats. Anesthetized rats were caressed on the
back by an experimenter' s palms for twenty minutes and the mRNA expression levels in the somatosensory cortex responsible for the back and
in the visual cortices five minutes afterwards were determined using
quantitative PCR methodology. The synaptophysin mRNA expression
level was selectively higher in the experimental group than in the control
group in the somatosensory cortex but not in the visual cortex. This result suggests that the mRNA expression level of synaptophysin induced
by neuronal activity is related to the regulation of synapse formation or
remodeling or both.
Effect of MEK inhibitor U-0126 on wind-up
action of dorsal horn nociceptive neurons
in rats
Kamo, Hiroshi1; Honda, Kuniya1; Kitagawa, junichi2;
Noguchi, Koichi3; Iwata, Koichi2 (1Dept. of Oral and
Maxillofacial Surgery, Sch. of Dent., Nihon Univ., Tokyo, Japan;
2Dept. of Physiology, Sch. of Dent., Nihon Univ., Tokyo, Japan;
3Dept. of Anatomy and Neuroscience, Hyogo College of
Med.,Hyogo, Japan)
It is well known that wind-up is the phenomenon that the second order
neurons increase in firing frequency following repetitive stimulation of
C-fibers. Recently, several lines of evidences suggest that the phosphorylation of the extracellular signal-regulated protein kinase (ERK) is involved in the hyperexcitability of the nociceptive neurons. However, it
is not known how the ERK is involved in the windup phenomena. The
present study was designed to evaluate the change in wind-up of DH nociceptive neurons i.t. administration of MEK inhibitor, U0126. Rats
were anesthetized with sodium pentobarbital (50 mg/kg, i.p.). When single neural activity was isolated, the left sciatic nerve was stimulated (0.5
Hz, 3 times higher than C-fiber threshold). After windup was observed,
U0126 (2.5 mM, 25 mM and 250 mM) was applied to the DH. The increased firing was significantly depressed following application of
U0126. DH nociceptive neurons were classified as WDR neurons. All
WDR neurons were located in laminae I-II in the DH. We could not observe any clear dose dependent effect of U0126 on DH nociceptive neurons. The mechanical and thermal responses were not affected by
U0126. The present findings suggest that the intracellular MAP kinase
cascade is involved in the central sensitization of the DH nociceptive
neurons without any effects on naturally evoked responses.
Univ., Tokyo, Japan)
Neurosteroid pregnenolone sulfate
enhances glutamatergic synaptic
transmission by facilitating presynaptic
calcium channels
Hige, Toshihide1; Hori, Tetsuya2; Fujiyoshi, Yoshinori1;
Takahashi, Tomoyuki2 (1Dept. Biophys., Grad. Sch. Sci., Kyoto
Univ., Kyoto, Japan; 2Dept. Neurophysiol., Univ. Tokyo Grad. Sch.
Med., Tokyo, Japan)
Pregnenolone sulfate (PREGS) is an abundant neurosteroid in the brain.
PREGS presynaptically facilitates glutamatergic synaptic transmission,
but underlying mechanism is not known. At the giant synapse, the calyx
of Held in the rat brainstem slices, PREGS potentiated nerve-evoked excitatory postsynaptic currents (EPSCs), and increased the frequency, but
not the amplitude, of spontaneous miniature EPSCs (mEPSCs). The EPSCs potentiations by PREGS and those by forskolin or phorbol ester did
not occlude with each other. In direct whole-cell recordings from presynaptic terminals PREGS accelerated activation kinetics of voltage-dependent Ca2+ channel currents. BAPTA (10 mM) loaded into the terminal
only partially attenuated this PREGS effect, suggesting that the main effect is independent of intracellular Ca2+. PREGS had no effect on presynaptic voltage-dependent K+ currents or resting conductance. Ca2+
imaging at the nerve terminal showed that PREGS increased Ca2+ influx
into the terminal at the resting membrane potential. Consistently the
PREGS-dependent increment of the mEPSC frequency was attenuated
by 300 µM Cd2+. The PREGS-induced Ca2+ current facilitation was
reversed by the PREGS scavenger cyclodextrin applied from outside,
but not from inside, of the nerve terminal. We conclude that PREGS, by
acting from outside of the nerve terminal, activates Ca2+ channels,
thereby increasing both evoked and spontaneous transmitter release at
the calyx of Held.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Neurons & synaptic functions
In vivo transduction of murine cerebellar
Purkinje cells by HIV-derived lentiviral
Depolarizing GABAergic mechanisms of
hippocampal seizure-like activity in posttetanic and low-Mg2+ conditions
Torashima, Takashi1,2,3; Yamada, Nobuaki2; Hirai,
Hirokazu2,3 (1Grad. Sch. Med. Sci. Kanazawa Univ., Kanazawa,
Fujiwara-Tsukamoto, Yoko1; Isomura, Yohikazu1,2;
Takada, Masahiko1 (1Dept. System Neurosci., Tokyo Met. Inst.
Japan; 2Advanced Sci. Res. Center Kanazawa Univ., Kanazawa,
Japan; 3PRESTO JST, Saitama, Japan)
Neurosci., Tokyo, Japan; 2Neural Circuit Theory, RIKEN BSI,
saitama, Japan)
Cerebellar Purkinje cells are key elements regulating motor learning and
motor coordination. Gene transfer into neurons, followed by the assessment of the effects on neural function, is an effective approach for examining gene function. However, this method has not been used fully in the
study of the cerebellum, because of the obstacle of delivering genes into
Purkinje cells. To overcome this, we used a human immunodeficiency
virus (HIV)-derived lentiviral vector and examined the transduction profile of the vector in the cerebellum. A lentiviral vector expressing GFP
was injected into the cerebellar cortex. Seven days after the injection,
GFP was predominantly expressed in Purkinje cells. GFP was also expressed, though less efficiently, in other cortical interneurons and Bergmann glias. In contrast to reported findings with other viral vectors, no
transduced cells were observed outside of the cerebellar cortex, even in
the deep cerebellar nuclei, pontine nuclei and inferior olivary complex,
which are synaptically linked with Purkinje cells or granule cells. Thus,
when HIV-derived lentiviral vectors were injected into the cerebellar
cortex, transduction was limited to the cells in the cerebellar cortex, with
the highest tropism for Purkinje cells. These results suggest that HIV-derived lentiviral vectors are useful for the study of gene function in
Purkinje cells as well as for application as a gene therapy tool for the
treatment of diseases that affect Purkinje cells.
GABA is known to be a major inhibitory neurotransmitter in mature
mammalian brains. However, the effect of GABA can be converted into
depolarizing or even excitatory when the postsynaptic Cl– concentration
becomes relatively high. We have recently shown that seizure-like afterdischarge induced by tetanic stimulation in normal ACSF (post-tetanic
afterdischarge) is mediated by GABAergic excitation in mature hippocampal CA1 pyramidal cells. Here we investigated the possible contribution of similar depolarizing/excitatory GABAergic input to seizure-like
afterdischarge induced in a low extracellular Mg2+ condition, as another
experimental seizure model (low-Mg2+ afterdischarge). Perfusion of
GABAA antagonists abolished low-Mg2+ afterdischarge in most cases.
Each oscillatory response during low-Mg2+ afterdischarge was dependent on Cl– conductance and contained an F–-insensitive depolarizing
component in the pyramidal cells. Perforated patch-clamp recordings revealed that GABA responses were indeed depolarizing during low-Mg2+
afterdischarge. Moreover, interneurons in the strata pyramidale and
oriens discharged in oscillatory cycles more actively than those in other
layers. These results suggest that the depolarizing GABAergic input may
facilitate oscillatory synchronization among hippocampal CA1 pyramidal cells during low-Mg2+ afterdischarge in a fashion similar to the expression of post-tetanic afterdischarge.
Transneuronal regulation of synapse
formation and plasticity by Cbln1
Hirai, Hirokazu1,2; Miyazaki, Taisuke3; Miura, Eriko3;
Watanabe, Masahiko3; Morgan, James I4; Yuzaki,
Michisuke5 (1Innovative Brain Sci. Advanced Sci. Res. Center
Kanazawa Univ., Kanazawa, Japan; 2PRESTO JST, Kawaguchi,
Japan; 3Dept. Anatomy Hokkaido Univ. Sch. Med.; 4St. Jude
Children's Res. Hosp., Memphis, TN, USA; 5Dept. Physiol. Keio
Univ. Sch. Med., Tokyo, Japan)
Cbln1 is the prototype for a family of four brain-specific proteins
(Cbln1-Cbln4) of unknown function that was first identified by virtue of
its harboring a naturally occurring 16 amino acid peptide, cerebellin.
Cbln1 is a cerebellum-specific protein and structurally related to the C1q
and Tumor Necrosis Factor families of proteins. We show here that
Cbln1 is secreted from cerebellar granule cells as a glycoprotein and is
essential for three processes in cerebellar Purkinje cells: the matching
and maintenance of pre- and post-synaptic elements at parallel fiberPurkinje cell synapses, the establishment of the proper pattern of climbing fiber-Purkinje cell innervation, and the induction of long-term depression at parallel fiber-Purkinje cell synapses. Interestingly, the
behavioral, physiological and anatomical phenotype of cbln1-null mice
precisely mimics loss-of-function mutations in the orphan glutamate receptor, GluR∆2, a gene selectively expressed in Purkinje neurons.
Therefore, Cbln1 secreted from presynaptic granule cells may be a
component of a previously undocumented trans-neuronal signaling
pathway that controls synaptic structure and plasticity.
Possible roles of synaptic vesicles in
plasticity of enhanced neurotransmission
via Ca2+-induced Ca2+ release at frog motor
nerve terminal.
Narita, Kazuhiko1; Himi, Naoyuki1; Ochi, Kazunori1;
Suzuki, Naoya2; Kuba, Kenji3 (1Kawasaki Med. Sch.,
Kurashiki, Japan; 2Sch. of Sci., Nagoya Univ., Nagoya, Japan;
Univ. Arts. Sci. Fac. Nutri, Nagoya, Japan)
Ca2+-induced Ca2+ release (CICR) takes place in response to Ca2+ entry
via the activation of type-3 ryanodine receptors (RyRs) after its use- and
Ca2+-dependent priming, and amplifies impulse-evoked transmitter release in frog motor nerve terminals. Since the activation of CICR occurs
in less than 1 msec after a nerve impulse, the site of Ca2+ release is close
to the high [Ca2+]i microdomain, where the machinery of the exocytosis
is activated. Then, the most possible Ca2+ stores, on which RyRs reside,
would be synaptic vesicles. We studied here the effects of loading Ca2+
chelator into synaptic vesicles on the priming and induction of CICR.
EGTA was loaded into synaptic vesicles by incubating preparations in a
Ca2+-free, EGTA (1mM) and Mg 2+ (1 or 10mM) solution for 20-30 min,
in which endocytosis still took place following high frequency stimulation of the nerve in low Ca2+ (0.15-0.5mM), high Mg2+ (6-10mM) solutions. After loading EGTA, tetanus-induced rises in end-plate potential
(EPP) amplitude and miniature EPP (MEPP) frequency, reflecting the
priming and activattion of CICR, became slower in onset and smaller in
amplitude and rate of rise. Results are in favor of the idea that synaptic
vesicles are involved in the priming and activation of CICR and so synaptic plasticity.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Neurons & synaptic functions
Blockade of L-type, but not T-type, calcium
channels enhanced LTD magnitude
induced with low frequency stimulation at
hippocampal CA 1 synapses
Layer-dependent effects of Homer1a/Vesl1S on long-term depression at
corticocortical synapses in the rat visual
Udagawa, Rie; Nakano, Makoto; Kato, Nobuo (Dept. of
Ueta, Yoshifumi1; Yamamoto, Ryo1; Sugiura, Shigeki2;
Inokuchi, Kaoru3; Kato, Nobuo1 (1Dept. Integrat. Brain Sci.,
Integrative Brain Science Kyoto Univ. Grad. Sch. Med., Kyoto,
To induce long-term depression (LTD) at hippocampal CA1 synapses,
[Ca2+]i must sufficiently rise in CA1 pyramidal cells. Each type of calcium entry is believed to have a distinctive role in LTD induction. The
present study focuses on roles of voltage gated calcium channels
(VGCCs) in LTD induction at Schaffer collateral-to-CA1 synapses in the
hippocampal slices. VGCCs are known to be involved in many important cellular processes including synaptic plasticity. In control experiments, the magnitude of LTD varied depending on stimulus frequencies.
Also, LTD evoked by any of the tested frequencies (0.5-2Hz) required
NMDA receptor activation. T-type VGCCs turned out to have no significant role in LTD induction, since T-VGCC blockade did not change normal LTD induction. When L-type VGCCs were blocked with
nimodipine, LTD magnitude was enhanced with low frequency (0.5-Hz)
stimulation and was reduced with high frequency (1- or 2-Hz) stimulation. We were particularly interested in the enhancement of LTD by LVGCC blockade in response to low frequency (0.5 Hz) stimulation,
since we had previously observed ryanodine receptor activation could
also enhance LTD in response to the same low frequency stimulation.
Application of calcium store depleter, thapsigargin, in addition to nimodipine canceled out the LTD enhancement. Hence, intracellular calcium
release seemed to play a part in nimodipine-induced LTD enhancement
that we observed in a low stimulus frequency range.
Grad. Sch. Med., Kyoto Univ., Kyoto, Japan; 2Med. Genet. Res.
Center, Nara Med. Univ.; 3Mitsubishi Kagaku Inst. Life Sci.)
In the central nervous system, synaptic efficiency is modifiable in activity-dependent manners. Homer1a/Vesl-1S, an activity-dependently inducible member of the scaffold protein family Homer/Vesl, has been
implicated in long-term up-regulation of synaptic efficiency (various
forms of LTP), as well as in short-term modification of AMPA receptors
(arguably, both up- and down-regulation). It is not clear, however,
whether Homer 1a takes part in induction of LTD (long-term depression). The present experiments examined roles of Homer 1a in inducing
LTD at a variety of corticocortical synapses in rat visual cortex slices by
using whole-cell patch clamp. Homer 1a was injected by diffusion from
patch pipettes. With or Without Homer 1a injected, LTD induction was
attempted by pairing 1 Hz stimulation with post-synaptic depolarization
for 10 min. Without Homer 1a, LTD was induced at synapses between
layer IV axons onto layer II/III pyramidal cells, those between layer II/
III axons onto layer V pyramidal cells and those between layer II/III axons onto layer VI pyramidal cells. However, at synapses between layer
IV axons onto layer VI pyramidal cells, LTD induction was failed. With
Homer 1a protein injected, on the other hand, LTD induction was reduced in magnitude at layer II/III-to-layer VI pyramidal cell synapses,
but not at the other synapses examined, suggesting a synapse-specific effect of Homer 1a.
Auditory fear conditioning increases cell
proliferation in the basomedial nucleus of
rat amygdala
Interaction of GluRδ2 and PICK1 implicated
in the induction of cerebelallar LTD
Mori, Takatoshi1,2; Moriya, Takahiro1; So, Kenji1;
Ozawa, Hiroki2; Shinohara, Kazuyuki1 (1Div. Neurobio. &
Sch. Sci. Kyoto Univ., Kyoto, Japan; 2CREST, JST, Kawaguchi,
behav., Dpt. Trans. Med. Sci., Nagasaki Univ. Grad. Sch. Biomed.
Sci., Nagasaki. Japan; 2Div. Neuropsy., Dpt. Trans. Med. Sci.,
Nagasaki Univ. Grad. Sch. Biomed. Sci., Nagasaki. Japan)
The glutamate receptor δ2 subunit (GluRδ2) is selectively expressed in
cerebellar Purkinje neurons (PNs) and is involved in the long-term
depression (LTD). However, little is known about the mechanism of its
action. Acute expression of the wild-type GluRδ2 in the GluRδ2deficient PN rescued the LTD induction, suggesting the direct role of
GluRδ2 in LTD. To identify the critical region of GluRδ2 necessary for
the LTD induction, we constructed and expressed various mutant
GluRδ2 proteins in the GluRδ2-deficient PNs. The mutant GluRδ2
possessing the membrane-proximal 21 amino acid residues (AAs) in Cterminal cytoplasmic region rescued the LTD induction, whereas the
mutant with membrane-proximal 13 AAs failed. In addition,
overexpression of the membrane-proximal 14-20 AAs fused to EGFP
suppressed the LTD induction in a wild type PN. These results suggest
that the membrane-proximal 14-20 AAs of GluRδ2 plays an essential
role in LTD. Then, we identified protein interacting with C kinase 1
(PICK1) as a molecule interacting with the membrane-proximal Cterminal region of GluRδ2 by yeast two-hybrid screening. PICK1 colocalized with GluRδ2 at spines of PNs, and immunoprecipitation assays
showed that GluRδ2 bound to PICK1 mainly through the membraneproximal 14-20 AAs. These results indicate that the membrane-proximal
14-20 AAs of GluRδ2 are essential for both LTD and interaction with
PICK1, and suggest that interaction between GluRδ2 and PICK1 might
be critical for the LTD induction.
It has been known that proliferating cells exist in the amygdala, but their
functions remain to be clarified. On the other hand, the amygdala is
known to play essential roles in the emotional learning. Therefore, we
examined whether the auditory fear conditioning affects the cell proliferation in the rat amygdala as well as other brain areas. Male SD rats
were used. On day 1, the rats of conditioned group received five pairings
of tone (20 sec) as the conditioned stimulus (CS) and foot shock (1 sec)
as the unconditioned stimulus (US) (CS/US group). The control group
rats received only CS (CS group). After the training, rats were injected
with BrdU. On day 2, rats were placed in a cage and exposed to the tone
and their freezing responses were evaluated. Thereafter, rats were perfused and their brains were removed. Serial 40 µm-thick coronal
sections of the brains were cut with a cryostat and BrdU
immunohistochemistry was performed. BrdU-labeled cells were
quantified in each brain area. There was no statistical difference in the
number of BrdU-labeled cells in subventricular zone, septal nucleus,
subgranular zone of dentate gyrus, or entire amygdala between two
groups. In the basomedial amygdala (BMA), BrdU-labeled cells were
more abounding in CS/US group. These results suggest that the cell
proliferation in the BMA may be involved in the formation of the
auditory fear conditioning.
Yawata, Satoshi1,2; Hirano, Tomoo1,2 (1Dept. Biophys, Grad.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Neurons & synaptic functions
Activity pattern-dependency of BDNF
release from mossy fiber terminals of
Suyama, Shigetomo; Ishizuka, Toru; Yawo, Hiromu
(Dept.Developmental Biology and Neurosciences, Tohoku Univ.
Grad.Sch. of Life Sciences, Sendai, Japan)
Brain-derived neurotrophic factor (BDNF), which was first identified as
a molecule regulating neuronal survival and differentiation, has a pivotal
role in the regulation of synaptic plasticity, especially long-term potentiation (LTP). Recent studies have shown that BDNF is transported anterogradely along axon and stored in the presynaptic terminals. The mossy
fiber (MF) terminals, which are axon terminals of the granule cells in the
dentate gyrus (DG) of the hippocampus, contain the highest concentration of BDNF in the CNS. These observations led us to hypothesize that
BDNF is released from MF terminals and that its release is dependent on
the activity pattern. We made a Sindbis virus vector containing a mRNA
coding the fusion protein constract of BDNF and Venus, one of green
fluorescent protein derivatives, and inoculated it stereotaxically to the
DG cells of mouse hippocampus (P14-21). After 2-3 days, MF boutons
accumulating BDNF-Venus were identified in the acute slice under confocal microscopy. The activity-dependent BDNF release was measured
as a reduction of the fluorescence intensity of the individual presynaptic
terminal. We found that the activity patterns as high frequency stimulation and theta-burst stimulation are more effective on the BNDF release
than low frequency stimulation. It is suggested that BDNF is released
from the MF terminals during induction of LTP.
Functional development of neural circuit
formation in the embryonic chick olfactory
pathway: Optical imaging of voltagesensitive dyes
Sato, Katsushige; Momose-Sato, Yoko (Department of
Physiology, Tokyo Medical and Dental University, Graduate School
and Faculty of Medicine, Tokyo, Japan)
To understand the functional organization of the central nervous system
(CNS), it is essential to know how sensory information is processed
within the CNS. We have been approaching this topic by following the
ontogenetic patterning of neural circuit formation related to the cranial
and spinal sensory inputs using multiple-site optical recording techniques with voltage-sensitive dyes. In this study, we surveyed developmental organization of neural networks related the olfactory nerve (N. I)
in the embryonic chick forebrain. Stimulation applied to the olfactory
nerve elicited excitatory postsynaptic potential (EPSP)-related optical
signals in the olfactory bulb from the 7-day old embryonic stage (E7).
The EPSP was mediated by glutamate, and NMDA- and non-NMDA-receptor components were identified. In more developed stages, in addition to the responses in the olfactory bulb, another response area was
discriminated within the cerebrum, which seemed to correspond to the
higher-ordered nucleus of the olfactory pathway. The results suggest that
the olfactory pathway is functionally generated at early stages of development when neural networks related to other visceral and general somatic sensory inputs are also in the process of developing.
Repeated LTD induction causes longlasting synaptic elimination,
requiringprotein synthesis
Voltage-sensitive dye imaging of
oscillatory activity in the embryonic chick
olfactory bulb
Tanaka, Tsunehiro; Egashira, Yoshihiro; Kamikubo, Yuji;
Shinoda, Yo; Tominaga-Yoshino, Keiko; Ogura, Akihiko
Momose-Sato, Yoko; Sato, Katsushige (Department of
Physiology, Tokyo Medical and Dental University, Graduate School
and Faculty of Medicine, Tokyo, Japan)
(Dept. Neuro-sci., Grad. Sch. Frontier Biosci, Univ. Osaka, Osaka,
Synaptic plasticity is assumed as the cellular basis of memory. Long-termplasticity that lasts for days/weeks has not been fully analyzed, mainlybecause of the lack of model system. We found previously that the
repeatedinduction of LTP in cultured hippocampal slice caused a longlastingsynaptic enhancement accompanied by the formation of new synapses, which wasseparate from LTP itself. We found recently that the repeated induction ofLTD by mGluR activation in the same specimen
caused a long-lasting decreasein synaptic strength accompanied by the
elimination of synapses. Thus wepropose that these repetition-dependent
synaptic changes can serve as themodel system for the analysis of longterm plasticity. Here we addfollowing findings that support this proposal. 1) The synapse eliminationis independent of the means of LTD induction, since LTD induced not only byDHPG (dihydroxyphenylglycine, a
class I mGluR agonist) but also by a lowdose of NMDA (N-methyl-Daspartate, an NMDAR agonist) or by DHO(dihydroouabain, a Na/K-ATPase inhibitor) led to the equivalent synapticelimination, when repeated
three times (as monitored by electrophysiologicaland morphological indices). 2) The elimination required protein synthesis,since the application of anisomycin (an inhibitor of mRNAs translation toproteins)
suppressed the development of synapse elimination.
In an accompanying study, using a multiple-site optical recording technique with a voltage-sensitive dye, we examined the developmental organization of the olfactory pathway in the embryonic chick forebrain,
and showed that functional synaptic transmission in the olfactory bulb
was expressed at around E7. It is known that odor stimuli elicit oscillatory events in the olfactory bulb in various species. We found that oscillatory activity was also generated in the chick olfactory bulb during
embryogenesis. At early stages of development (E7-E8), postsynaptic
response-related optical signals evoked by olfactory nerve stimulation
exhibited a simple monophasic waveform that lasted a few seconds. As
development proceeded, the pattern of the optical signal became complicated, and oscillatory activity was observed in a later phase of the
postsynaptic response. The oscillation was restricted to the olfactory
bulb, and this spatial pattern was different from that of the propagating
wave activity termed the depolarization wave. We examined spatio-temporal patterns of the oscillatory activity in different stages, and studied
its developmental dynamics.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Neurons & synaptic functions
Activity-dependent synapse elimination of
corticispinal tract in mouse slice co-culture
Neural differentiation of neural stem cells
from adult human brain
Ohno, Takae; Sakurai, Masaki (Dept. Physiol. Teikyo Univ.
Masuda, Tadashi1; Moriya, Takahiro1; Terazon,
Hideyuki1; Ono, Tomonori2; Toda, Keisuke2; Baba,
Hiroshi2; Shinohara, Kazuyuki1 (1Division of Neurobiology
Sch.Med., Tokyo, Japan)
We have succeeded in reconstructing the corticospinal (CS) synapses in
vitro by co-culturing the rat sensorimotor cortex and cervical spinal cord.
In this in vitro model of CS projection, CS synapses are formed diffusely
throughout the spinal gray matter but later the synapses in the ventral
side are eliminated, which is NMDA-dependent (Ohno et al, J Neurosci
2004) with a critical period of 6-11DIV (Ohno & Sakurai, Neuroscience
2005). In order to use the genetically-modified mice to study the underlying molecular mechanisms of this developmental plasticity, we tried to
reconstruct the same system using mice. Entire blocks of the brain and
spinal cord were taken from P0 C57 BL/6 mice. Coronal slices of the
sensorimotor cortex and axial slices of the cervical cord were sectioned
(350 µm), and forelimb areas in the cortex was dissected from each
section. By recording field EPSP (fEPSP) along 80 µm-interval lattice in
the spinal gray matter in response to the stimulation of cortical deep
layer, we evaluated spatial distribution of synapse formation
quantitatively. Field EPSPs were recorded diffusely throughout the
spinal gray matter at 6-9 DIV, then the amplitudes of fEPSPs in the
ventral side began to decrease at 10 DIV, and dominated in the dorsal
area at 14-15 DIV. CS axon terminals labeled with biocytin
anterogradely distributed diffusely throughout the spinal gray matter at
7-9 DIV but the axons terminals in the ventral area were eliminated until
14 DIV. This synapse elimination from the ventral side was blocked by
APV, indicating that this process is also NMDA-dependent.
and behavior, Dept. of Translational Med. Sci., Nagasaki University
Grad. Sch. of Biomedical Sci.; 2Dept. of Neurosurg., National
Nagasaki Medical Center)
Recent reports suggest that the adult human brain contains undifferentiated, multipotent precursor cells or neural stem cells (NSCs). In this
study, we tried to isolate the NSCs from the surgically dissected hippocampus of the adult human using "Neurosphere" methods and characterize their ability of the proliferation and the differentiation. When the
dispersed cells from the adult human hippocampus were cultured in defined medium containing LIF (10 ng/ml), EGF (20 ng/ml), FGF2 (20 ng/
ml) and B-27 (2%), the sphere-forming cells which diameter is approximately 200 µm were observed. These cells can be maintained and
expanded in this condition for at least 6 months. The population
doubling time was approximately 16 days. Immunocytochemical
analysis showed that most of cells under this growth condition expressed
neural stem cell marker protein, nestin. To induce differentiation, growth
factors were removed from the medium. Immunocytochemical analysis
showed that many cells expressed neuron marker protein, Tuj1 and
astroglial marker protein, GFAP in this differentiation condition. These
results suggest that these cells are multipotent NSCs. To improve the
efficiency of neuronal differentiation, we are now examining the effects
of various soluble factors in the differentiation medium on the neural and
astroglial differntiation of the isolated NSCs.
Expression of the ABC transporter ABCA2
in rat myelinating and non-myelinating
Schwann cells
Developmental Changes of GABAergic
Inhibitory Synapses in the Deep Cerebellar
Saito, Takashi1,2; Yamada, Katsuya1,3; Wang, Yan1,2;
Tanaka, Yukiko1; Ishikawa, Kazuo2; Inagaki, Nobuya1,4
Saitow, Fumihito; Murano, Mitsumasa; Suzuki, Hidenori
(1Dept. Physiol., Akita Univ. Sch. Med., Akita, Japan; 2Dept.
Otolaryngol., Akita Univ. Sch. Med., Akita, Japan; 3Dept. Physiol.,
Hirosaki Univ. Sch. Med., and CREST, Aomori, Japan; 4Dept.
Diabetes & Clinical Nutrition., Kyoto Univ. Grad. Sch. Med., and
CREST, Kyoto, Japan)
We have previously shown in rat brain that ABCA2, which belongs to
the A subclass of ATP-binding cassette (ABC) transporter superfamily,
is predominantly expressed in the cytoplasm of oligodendrocytes but not
in GFAP+ astrocytes, CD11b+ microglia, or NG2+ progenitors. In addition, onset of ABCA2 expression in oligodendrocytes coincides with the
appearance of myelin segments immunolabeled with myelin basic protein, implying a role of ABCA2 in transport of substances related to myelination processes. Consistently, expression of ABCA2 was detected in
S100β+ Schwann cells in human and rat peripheral nerve. Unexpectedly,
however, ABCA2 also was detected in S100β-weakly positive cells
containing number of densely packed, thin axons in peripheral nerve,
implying expression of ABCA2 in non-myelinating Schwann cells.
Indeed, multiple immunolabeling with ABCA2, S100β, GFAP, and a
zinc finger transcription factor Krox20, one of the most reliable makers
for myelinating Schwann cells, revealed that ABCA2 is expressed not
only in myelinating Schwann cells but also in non-myelinating Schwann
cells. As number of non-myelinating axons are thinly wrapped by single
non-myelinating Schwann cells, ABCA2 might contribute to transport
of lipid components commonly required for surrounding myelinating
and non-myelinating axons.
(Dept. Pharmacol., Nippon Med. Sch., Tokyo, Japan)
Activity of the deep cerebellar nuclei (DCN) takes an important role in
outputting processed information from the cerebellum. In this study, we
first investigated modulatory effects of serotonin (5-HT) receptor on
GABAergic synapses in the rat DCN using whole-cell recordings in the
cerebellar slices. Both of an endogenous agonist 5-HT and a 5-HT1 agonist 5-CT decreased the amplitude of stimulation-evoked IPSCs (eIPSC) in DCN neurons, and their effect was reversibly abolished by a 5HT1A and 1B antagonist, cyanopindolol. Further, a selective 5-HT1A
agonist 8-OH-DPAT had no effects of the amplitude of eIPSCs. Based on
these results, the activation of 5-HT1B receptor is suggested to be responsible for decreasing the amplitude of eIPSCs. In the developing
DCN neurons, we next examined developmental changes in both the kinetics of GABAergic postsynaptic currents and the modulatory effects of
5-HT on GABAergic synapses in the rat DCN neurons. At younger stage
(around P14), eIPSCs showed slower kinetics and were more susceptible
to the 5-HT-induced modulation than those at older stage (around P21).
These pre- and postsynaptic parameters showed time-matched changing
during development. These results suggest that the information flow
from the cerebellar cortex is finely controlled at younger developmental
stages, which is important to form the normal cerebellar function in the
J. Physiol. Sci., Vol. 56, Suppl., 2006
Neurons & synaptic functions
Reduction in metabotropic glutamate
receptor-mediated presynaptic inhibition
of GABA/glycine synapses on developing
rat LSO neurons
Nishimaki, Takuya; Nishimaki, Takuya; Jang, Il-Sung;
Nabekura, Junichi (Dep Dev Physiol, NIPS, Okazaki; CREST,
JST, Japan)
The lateral superior olive (LSO) is the first auditory center that processes
differences in the sound level between the two ears. Here we report the
developmental changes in metabotropic glutamate receptor (mGluR)mediated presynaptic inhibition of GABAergic/glycinergic synaptic
transmission onto developing rat LSO neurons using conventional
whole-cell patch clamp technique. In addition to a developmental switch
of MNTB-LSO afferents from GABAergic to glycinergic IPSCs during
development, immature MNTB-LSO synapses could release glutamate
with GABA/glycine. Bath application of DCG IV, a selective mGluR 2/
3 (group II) agonist, greatly reduced IPSC amplitude in neonatal (< P5)
with a significant change in the paired-pulse ratio, which was eliminated
in the presence of group II antagonist, suggesting that DCG IV acts presynaptic mGluR 2/3 leading to reduce the release probability of GABA
and/or glycine release from presynaptic nerve terminals. However, the
mGluR-mediated presynaptic inhibition was gradually reduced with
postnatal development, in which DCG IV had little effect on MNTBevoked IPSCs recorded from P16-18 LSO neurons. At P5 LSO neurons,
presynaptic mGluR could be activated by endogenous glutamate released from the ipsilateral anteroventral cochlear nucleus (AVCN) afferent, but not from MNTB terminals. Based on these results, the functional
roles of presynaptic mGluR in the development of LSO neurons will be
further investigated and discussed.
EGF receptor (ErbB1) stimulation downregulates synaptic inputs of neocortical
GABAergic neurons
Namba, Hisaaki1; Nagano, Tadasato1,2; Zheng,
Yingjun1; Takei, Nobuyuki1; Nawa, Hiroyuki1 (1Dept. of
Mol. Neurobiol., Brain Res. Inst., Niigata Univ., Niigata, Japan;
2Dept. of Cell. Neurophysiol., Grad. Sch. of Med. Sci., Kanazawa
Univ., Kanazawa, Japan)
Neurotrophins and cytokines are involved in neuronal differentiation,
synaptic development and plasticity. In neocortical culture, we reported
epidermal growth factor (EGF) family (ErbB1 ligands; EGF, TGF-alpha,
HB-EGF) down-regulates an AMPA receptor molecule, GluR1. Using
neocortical cultures and EGF-administered animals, we electrophysiologically evaluated the effects of the EGF family on synaptic development and plasticity in the GABAergic neurons. In neocortical culture,
subchronic treatment with TGF-alpha reduced the expression of GluR1immunoreactivity in glutamic acid decarboxylase (GAD) 67 immunopositive GABAergic neurons. Whole-cell patch-clamp recording from
morphologically identified putative GABAergic neurons revealed decreases both in AMPA currents and amplitudes of mEPSCs by TGF-alpha. Subcutaneous administration of EGF for 14 days in neonatal mice
also decreased protein levels of AMPA and NMDA receptors in the frontal cortex. Immunohistochemical study revealed that the decrease in
GluR1 levels was relatively specific for the parvalbumin-positive
GABAergic neurons. Miniature analyses in cortical slices show that the
amplitudes of mEPSCs in the GABAergic neurons decreased significantly, whereas no alteration was observed in the pyramidal neurons.
Thus, activation of ErbB1 receptors during cortical development negatively regulates synaptic inputs and plasticity in the GABAergic neurons.
Single-cell-based analysis of neural
differentiation of the NSCs derived from
embryonic mouse striatum
Influences of neural activity on motility and
dendritic development of cortical
GABAergic neurons
Noda, Mitsuru1; Terazono, Hideyuki1; Moriya, Takahiro1;
Yamaguchi, Masahiro3; Yasuda, Kenji2; Nagata, Izumi4;
Shinohara, Kazuyuki1 (1Div. Neurobiol. & behav., Nagasaki
Maruyama, Atsushi1,2; Yanagawa, Yuchio3; Obata,
Kunihiko1; Tsumoto, Tadaharu1,2 (1RIKEN. BSI; 2SORST.
Univ. Grad. Sch. Biomed. Sci., Nagasaki, Japan; 2Dpt. Life Sci.,
Grad. Sch. Arts & Sci., Univ. Tokyo, Tokyo, Japan; 3Dpt. Physiol.,
Grad. Sch. Med., Univ. Tokyo, Tokyo Japan; 4Dpt. Neurosurg.
Radiol. Nagasaki Univ. Grad. Sch. Biomed. Sci., Nagasaki, Japan)
Neural stem cells (NSCs) are defined as self-renewing, multipotent progenitor cells that give rise to neurons, astrocytes and oligodendrocytes.Using single-cell-based on-chip cell-cultivation system, we
analyzed the process of neural differentiation from the NSC and examined the effects of BDNF, which is known to enhance neuronal differentiation.The NSCs were obtained from the striatum of E12.5 nestinpromoter GFP transgenic mice and an individual cell was placed into 32
pairs of agar microchambers connected by microchannels.The NSCs
were differentiated by media containing FBS, retinoic acid and forskolin
in the presence or absence of BDNF and cell adhesion, neurite outgrowth
were examined by recording a series of phase-contrast images and immunocytochemistry for neural marker, TuJ1.We observed that the NSCs
attached the microchamber and a part of cells exhibited neuron-like morphology and extended some neurites along microchannels.BDNF increased the rate of neurite outgrowth.It, however, failed to affect cell
adhesion.Thus, using this system, we could address the process of neural
differentiation from the NSCs in a single-cell-based level and could
demonstrate that BDNF had the ability to promote the neural differentiation.
Japan Science and Technology Agency; 3Grad. Sch. Med. Univ.
During development of cerebral cortex the two major types of neurons,
pyramidal and GABAergic neurons, migrate to their destination through
different routes and extend their dendrites at different timing. Questions
arise whether neuronal activity gives any influence on these processes of
neuronal development, and if so, whether its influence differs between
the two types of neurons. To address these questions, we used neuron
culture preparations of the visual cortex of GAD67-GFP knock-in mice,
in which GABAergic neurons can be identified by GFP. With time-lapse
imaging analysis we observed effects of drugs, which block or enhance
neural activity, on the motility and dendritic development of GABAergic
and pyramidal neurons. Until 7 days in vitro (DIV) GABAergic neurons
moved very quickly, although the movement was not smooth, while pyramidal neurons did not show such high motility. After their motility diminished, GABAergic neurons started to develop their dendrites. An
application of tetrodotoxin (TTX) increased the motility of GABAergic
neurons and extended the period when such high motility is maintained.
The application of TTX and antagonists for ionotropic glutamate receptors until 7 DIV retarded the dendritic development of GABAergic neurons, while did not significantly affect that of pyramidal neurons. These
results suggest that GABAergic neurons are more susceptible to neuronal activity than pyramidal neurons and activity may be a factor to stop
migration of GABAergic neurons and to start development of their dendrites.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Neurons & synaptic functions
Promoting action of endogenous and
exogenous epidermal growth factor-ErbB1
signals on the development of midbrain
dopaminergic neurons
Zheng, Yingjun1; Iwakura, Yuriko1; Namba, Hisaaki1;
Takei, Nobuyuki1; Sano, Hiromi2; Kobayashi, Kazuto2;
Nawa, Hiroyuki1 (1Dept. of Mol. Neurobiol., Brain Res. Inst.,
Niigata Univ., Japan; 2Dept. of Mol. Genetics, Inst. of Biomed. Sci.,
Fukushima Med. Univ. Sch. of Med., Japan)
Epidermal growth factor (EGF) binds to ErbB1 receptor and exerts a
neurotrophic activity on midbrain dopaminergic neurons. Here, using
EGF administrated animals and midbrain cultures, we investigated endogenous and exogenous ErbB1 activity on the development of dopaminergic neurons. Immunostaining of tyrosine hydroxylase (TH) revealed
that the chronic administration of ErbB1 inhibitors (PD153035,
ZD1839) to rat neonates prevented dopaminergic neurons from axonal
fiber outgrowth and striatal innervation. Further, protein levels of TH
and dopamine transporter decreased in the striatum but did not change in
the frontal cortex. In midbrain culture, EGF had promoting activities in
cell-survival and dopamine uptake. To monitor the development of intrinsic excitability of the dopaminergic neurons we prepared midbrain
cultures from TH-EGFP transgenic mice. Whole-cell current-clamp recording from EGFP positive dopaminergic cells revealed that chronic
treatment with EGF increased the number of action potentials induced
by current injections. Further, daily administrations of EGF in vivo increased AMPA-mediated synaptic responses in the dopaminergic neurons. These findings suggest that ErbB1 ligands such as EGF have a
neurotrophic activity for the development of midbrain dopaminergic
Kamiyama, Tsutomu; Sakurai, Masaki (Dept. Physiol,
Teikyo Univ. Sch. Med.,Tokyo, Japan)
In the previous study, we showed that the corticospinal (CS) synapses
and terminals are distributed in the whole spinal cord at P7 and then
eliminated from the ventrolateral side until P10. In this study, we further
investigated the development of CS synapses subsequent to the elimination electrophysiologically and morphologically. Field EPSPs of CS
synapses were evoked by stimulation of the medullary pyramid and recorded from the lower cervical cord (C7). For anterograde labeling of the
CS terminals at C7, biotin dextran amine (BDA) was injected to the sensorimotor cortex. The spinal cord were fixed and sectioned several days
after the injection. At the beginning of morphological study we determined the area of sensorimotor cortex projecting to C7. The amplitude
of the largest negative peak of the field EPSPs continuously increased
until 4 or 5 postnatal week. On the other hand, the spatial pattern of the
distribution of the potentials was nearly constant after P10. The terminal
distribution was analyzed by counting the number of axons within ventrolateral and dorsomedial area. After a relatively stable period of P10
and P11 following the elimination, the number of the terminals increased
again in the ventrolateral area, which was the case with the dorsomedial
area. The continuous amplitude increase of largest negative field EPSPs
may be an electrophysiological counterpart of CS terminals re-innervation. Thus development of the CS innervation seems to consist of at least
two steps.
Presynaptic TRPV1 and nACh receptors
mediate robust synaptic facilitation in area
postrema neurons of the rat
Kawa, Kazuyoshi (Dept. Neurophysiol.,Graduate Sch. Med.,
Tohoku Univ. Sendai, Japan)
Inhibitory synaptic transmission and its modulation in neurons of the
area postrema (AP), one of autonomic nuclei in the medulla, were studied using whole-cell patch-electrodes applied to slices from newborn
rats. When external saline containing 20 mM KCl was applied from a "Y
tube" to AP neurons, which were whole-cell clamped at -10 mV, massive
inhibitory postsynaptic currents (IPSCs) were induced. Most of the
evoked IPSCs were blocked by bicuculline, indicating GABAergic identity, while the remaining minority of synaptic currents was sensitive to
strychnine. When nicotine (5-100 µM) or capsaicin (0.1-1 µM) was
applied to AP neurons, robust appearance of IPSCs with GABAergic
identity was induced. After blocking action potential generation in the
slice with tetrodotoxin (1 µM), nicotine or capsaicin could still induce
GABAergic IPSCs. The nicotine-induced presynaptic facilitation was
significantly inhibited by mecamylamine, and it was slightly inhibited
by dihydro-β-erythroidine and negligibly inhibited by α-bungarotoxin.
Interestingly, responses to capsaicin of the synaptic facilitation showed
marked desensitization; even after five minutes of rigorous washout, the
magnitude of synaptic facilitation by capsaicin was 10-30% of that
evoked by the first application. It is concluded that nicotinic receptors,
as well as capsaicin receptors (presumably TRPV1), are expressed at
GABAergic presynaptic terminals in area postrema neurons and play a
distinctive role in controlling excitability of these neurons for their
proper function in the autonomic system.
Corticospinal re-innervation following
elimination of early formed synapses
during development
Hydrophilic changed [Ala4Lys5]-des-acyl
ghrelin(1-10) fails to elicit cardiovascular
responses in the rat nucleus tractus
Tsubota, Yuji; Owada-Makabe, Kyoko; Yukawa,
Kazunori; Maeda, Masanobu (Dep. Phys. Wakayama Med.
Univ. School Med. , Wakayama, Japan)
The neuronal mechanisms underlying the cardiovascular activities of
des-acylated ghrelin (DAG) and ghrelin remain unclear. Not only ghrelin
but also DAG that is endocrinically inactive form of ghrelin without
binding efficacy to growth-hormone secretagogue receptor type 1a (GHSR-1a) on the pituitary grand exerts cardiovascular actions when microinjected into the rat nucleus tractus solitarius (NTS). These responses
may be attributable to a receptor(s) other than GHSR-1a. DAG lacks hydrophobic octanoylation at Ser3 from ghrelin but retains hydrophobicity
at Phe4Leu5. We studied the cardiovascular effects in the rat NTS of a
synthetic peptide with a hydrophobic-to-hydrophilic substitution at
Phe4Leu5 to Ala4Lys5 of DAG(1-10). The intra-NTS microinjection of 80
or 200 pmol/100 nl of the synthetic peptide produced no changes in the
rat mean arterial pressure and heart rate. In addition, pretreatment with
200 pmol of the synthetic peptide had no antagonistic effect on the cardiovascular response induced by 80 pmol of DAG or native ghrelin. The
synthetic peptide was incapable of evoking hypotensive and bradycardic
responses in the NTS. Our results suggest that hydrophobicity at amino
acid position 4 to 5 of DAG may be essential to bind a new receptor and
to evoke the cardiovascular responses in the NTS.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Neurons & synaptic functions
Spontaneous activity of locus coeruleus
neurons is reduced in spontaneously
hypertensive rat (SHR) , AD/HD model rat.
Kidani, Yuri; Ishimatsu, Masaru; Akasu, Takashi (Dept. of
Physiol, Kurume Univ. Sch. of Med., Kurume, Japan)
The main problem of attention deficit/hyperactivity disorder (AD/HD),
one of developmental disorders of children, is difficulty in control or restrain of behavior. An imbalance of dopaminergic and noradrenergic
transmission is proposed for neural mechanisms of AD/HD. On adult
rats, we have already reported that methylphenidate, the most common
therapeutic agent for AD/HD produced a hyperpolarizing response associated with decrease in a membrane resistance by activation of the inward rectifier K + channels via α2-adrenoceptor of locus coeruleus (LC)
neurons where the most major source of noradrenergic tones in CNS.
The spontaneously hypertensive rat (SHR) are often used as a model of
AD/HD. Behavioral studies established that SHR displayed
hyperactivity, impulsivity, poor stability of performance and poorly
sustained attention, when compared with their normotensive WistarKyoto (WKY) control rats. In the present study, the tonic activity of LC
neurons of juvenile SHR was examined by using a whole-cell patch
clamp technique and compared with WKY. As a result, the resting
membrane potential of LC neurons in SHR (-47.7±0.39 mV) was
significantly decreased when compared with WKY (-51.4±0.63 mV).
Nevertheless, the frequency of spontaneous action potentials of SHR
(0.64±0.24 Hz) was significantly lower than that of WKY (2.14±0.53
Hz). These results suggest that noradrenergic spontaneous activity on
LC neurons in SHR is reduced compare with WKY.
Distinct presynaptic mechanisms underlie
firing frequency-dependent modulation of
synaptic transmission in the solitary
Yamamoto, Kiyofumi; Yamada, Chiaki; Imura, Taiko;
Shigetomi, Eiji; Kato, Fusao (Lab. Neurophysiol., Dept.
Neurosci., Jikei Univ. Sch. Med., Tokyo, Japan)
The afferent fibers in the vagus nerve transmit visceral information encoded as varying firing frequency to the second-order neurons in the nucleus of the solitary tract (NTS) and dorsal motor nucleus of the vagus
(DMX). The purpose of this study was to examine how the firing frequency affects transmission efficiency at these synapses. EPSCs evoked
by the solitary tract (TS) stimulation were recorded from DMX and NTS
neurons in the thick brainstem slices of young rats. TS stimulation at various frequencies (0.1 - 20 Hz) revealed distinct frequency-dependent responses in the EPSC amplitude among different types of neurons
recorded. When stimulated at 20 Hz, NTS neurons and low-pass type
DMX neurons exhibited marked amplitude reduction (<30% of the first
EPSC) within 10 pulses, whereas high-fidelity (hi-fi) type DMX neurons
presented only modest attenuation (>40%). These neurons exhibited distinct short-term plasticity as revealed by paired-pulse ratio (PPR) evaluation. Surprisingly, unlike the NTS neurons, PPRs in the low-pass DMX
neurons were not significantly affected by changes in [Ca2+]o, suggesting
distinct mechanisms for their short-term depression. These results indicate that the transmission efficiency between the visceral afferents and
second-order neurons depends largely on the firing frequency mostly
through distinct target cell-dependent presynaptic mechanisms, which
might result in differential activation of distinct components in the solitary complex network.
Norepinephrine content was increased in
locus coeruleus and medial prefrontal
cortex of spontaneously hypertensive rat
(SHR), AD/HD model rat
Ikeura, Sawako; Kidani, Yuri; Ishimatsu, Masaru;
Akasu, Takashi (Div. Integ. Auton. Func., Dept. Physiol., Kurume
Univ. Sch. Med., Kurume, Japan)
It has been reported that the role of norepinephrine (NE) and dopamine
(DA) in the brain is closely related to the etiology of attention-deficit/hyperactivity disorder (AD/HD). In this report, by using the HPLC method,
the amounts of NE and DA in the locus ceruleus (LC), the medial prefrontal cortex (mPFC), and the striatum (Str) were measured and compared spontaneously hypertensive rats (SHR), an AD/HD model rat, and
Wister-Kyoto rats (WKY) as the contrast rat. The results showed that the
amount of NE were significantly larger than those in the mPFC and Str
of WKY. On the other hand, the amount of DA has proven to be large in
Str, and very small in the LC and the mPFC. This is also the case with
SHR; the amount of NE and DA in LC, mPFC, and Str shows the same
pattern. However, in the LC, the amount of NE in SHR was increased
significantly more than that in WKY. The same tendency can be seen in
mPFC, however, in Str, the amount of NE did not show any increase,
compared with that in WKY. Furthermore, the amount of DA in the LC
was increased significantly in SHR, compared with that in WKY, whereas any significant difference could not be recognized in mPFC and Str.
These results suggest that the increase of NE and DA in the LC and
mPFC of SHR is related to the characteristic behavior in AD/HD.
Expression of exogenous protein in the
primary afferent neurons for the visceral
sensation by in-vivo gene transfer
Shigetomi, Eiji; Yamada, Chiaki; Kato, Fusao (Lab
Neurophysiol, Jikei Univ Sch Med, Tokyo, Japan)
In order to understand the mechanism how the visceral information is
transmitted to the brain, it is indispensable to identify the roles played by
already-identified molecules in the synaptic transmission between the
vagal afferent fibers and the second-order neurons in the nucleus tractus
solitarii (NTS). The molecules underlying the regulation of transmitter
release at this synapse are synthesized in the cell bodies located in the
nodose ganglion (NG). Here we challenged to establish an optimized
method for efficient in-vivo gene transfer into the NG neurons and evaluated expression of the gene product in the NG and its centrally projecting axons. In young Wistar rats, electrical pulses optimized for
electroporation were delivered to the NG immediately following injection of pCAGGS-EGFP plasmid vector (5 µg/µl; through courtesy of
Drs. J. Miyazaki and K. Nakajima). Two days after delivery, the NG was
dissected out and fixed in 4% paraforaldehyde. In some cases, acute
brainstem slices including the NTS were prepared and examined with a
confocal microscopy a few weeks later. A large portion of somata in the
NG and a large number of fibers projecting from NG expressed EGFP.
EGFP fluorescence was also detected in the solitary tract and
presynaptic termini in the NTS in the brainstem slices. This technique
might be applicable to analyzing specific molecule function in the
transmitter release in the central termini of the visceral afferent nerves.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Sensory functions
uptake to mitochondria accompanied
by depolarization of mitochondrial
membrane potential occured when [Ca2+]i
increased to a critical level at frog motor
nerve terminal
Effect of noise on contrast detection
sensitivity in human visual perception
Sasaki, Hitoshi1; Todorokihara, Masayoshi2; Ishida,
Takuya1; Miyachi, Junichiro1; Matsuura, Sumie1;
Kitamura, Tahei3; Aoki, Ryozo1 (1Dept. Physiol. & Biosignal.,
Nagoya Univ., Nagoya, Japan)
Osaka Univ. Grad. Sch. Med., Suita, Japan; 2Dept. Phys. & Elec.,
Osaka Pref. Univ. Grad. Sch. Eng. , Sakai, Japan; 3Dept. Elec. Eng.
& Elec., Col. Industri. Tech., Amagasaki, Japan)
To investigate the Ca2+ clearance mechanisms in presynaptic terminals,
we measured Ca2+ dynamics during nerve stimulation in frog neuromuscular junctions with a low affinity Ca2+ dye, Oregon Green 488
BAPTA 6F (Kd=13µM, pH=7.2). During 100Hz tetanus for 4 sec in a
normal Ringer's solution (1.8mM Ca2+, 0mM Mg2+), [Ca2+]i increased in
two phases, firstly steep rising (4 µM/sec for 0.2 sec) and secondly slow
increasing (0.8 µM/sec). Whenmitochondria were inhibited by rotenone
and oligomycin, slow phase disappeared and [Ca2+]i increased monotony
with rapid rate. Membrane voltage imaging of mitochondria with TMRE
suggested that Ca2+ uptake to mitochondria accompanied by
depolarization of mitochondrial membrane potential. When tetanus
frequency was reduced to 20Hz, [Ca2+]i dynamics did not affected by
rotenone and oligomycin, however, CCCP increased [Ca2+]i during
tetanus. The effect of rotenone and oligomycin appeared when [Ca2+]i
increased more than 0.6 µM during 40Hz tetanus. These results suggest
that Ca2+ uptake to mitochondria started when [Ca2+]i increased more
than 0.6 µM. CCCP had larger effect on [Ca2+]i increase during 40 Hz
tetanus than rotenone and oligomycin. These results suggest that CCCP
sensitive some Ca2+ clearance mechanisms other than mitochondrial
Ca2+ uptake contributed largely when [Ca2+]i was lower than some µM.
Recently it has been reported that background noise can improve detection sensitivity of sensory stimuli not only in animals, but also in humans. The present study was designed to examine how human visual
perception may be modified by superposition of noise. Twenty-two undergraduate students with normal or corrected to normal vision participated in this study. In a dim chamber, participants observed a signals of
a small light spot (white LED) with its intensity modulated by sine
waves, usually at a frequency of 1Hz. Random flickering light modulated by white noise was superposed on the signal. Contrast detection
threshold was measured with or without noise, using a psychophysical
method (the adjusting method). The threshold first decreased then increased as increase in the noise intensity, with the minimum value at
around just above the noise-threshold. Thus the increase in the contrast
detection sensitivity was found not only at subthreshold, but also suprathreshold intensity of noise. These findings were independent on the signal frequency (1-15Hz). A further experiment replicated these findings
using the up-and-down methods to measure the threshold in five participants (3 male and 2 female, aged 20-25 yr). We concluded from these
results that a certain amount of noise, even supra-threshold, can improve
contrast detection sensitivity in human visual perception.
Suzuki, Naoya; Iibohsi, Masahide (Dept. Phys. Sch. Sci.
Simulation analysis of the high-pass
filtering of the rod network in the retina
Kamiyama, Yoshimi (Inf. Sci. & Tech., Aichi Prefect. Univ.,
Sensory functions
It is known that the rod network in the retina behaves as a high-pass filter
to electrical signals. In the turtle and toad retinae, it was found that the
time to peak of the response was shorter in rods further away from a slit
of light. In the tiger salamander retina, it was shown that the voltage responses to square current injection became more transient as they travel
through the rod network. In previous studies, the high-pass filtering behavior has been attributed to an inductance element, a hyperpolarizationactivated current, or a K conductance activated by Ca. However, biophysical mechanism underlying the high-pass filter is not fully understood. The objective of this study is to analyze the functional roles of
individual ionic currents in the temporal filtering properties of rods
through computer simulations. A model of the rod photoreceptor network was developed. The model incorporates much of the known parameters in rod photoreceptors, i.e., the phototransduction cascade in the
outer segment, membrane ionic currents (ICa, IKv, IK(Ca), Ih, ICl(Ca)), intracellular calcium system and electrical junctions between rods.In simulation, the temporal filtering properties of the rod was analyzed. The
simulated result shows that single rod itself behaves as a high-pass filter.
The mechanism underlying the high-pass filter was examined by changing model parameters. The result suggests that IK(Ca), ICl(Ca) and Ih are responsible for the high-pass filtering. The model also well reproduced the
experimental observation that the shortening of the time to peak as the
signal propagates laterally.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Sensory functions
Efficiency of suprachoroidal-transretinal
stimulation on retinal neurons nearby the
Takashi2; Tano,
Kanda, Hiroyuki2,4; Fujikado,
Yasuo3; Sawai, Hajime1 (1Dept. Physiol.,
Grad. Sch. Med., Osaka Univ., Suita, Japan; 2Dept. Applied Visual
Sci., Grad. Sch. Med., Osaka Univ., Suita, Japan; 3Dept.
Ophthalmol., Grad. Sch. Med., Osaka Univ., Suita, Japan; 4Nidek
Co., Ltd. Gamagori, Japan)
As a method of retinal prosthesis we newly developed suprachoroidaltransretinal stimulation (STS), in which electrical current passed between scleral electrode and vitreous electrode (Kanda et al. '04). Previously we reported the spatial properties of STS (Kanda et al., PSJ
meeting '05), however the recorded neurons with the receptive fields
close to the scleral electrode were few. Here, we enlarged the sampling
especially within 2° from the scleral electrode and investigated whether
STS can activate neurons in the localized retinal area.
105 unit activities of relay cells in the lateral geniculate nucleus were
recorded from 12 adult cats. The response probability to STS of various
current intensities between 50-500 µA was examined for each unit, and
its relationship with the distance from the electrode was analyzed. It was
confirmed that the response probability depended on the distance from
the electrode. As the intensity decreased, the units with high response
probability limited near the electrode. For example, at 100 µA of STS,
the units with the probability over 80% were within 2.5° from the
electrode. The median of the probability of the units within 1° was the
highest among those with further distance when STS of 150 µA or 200
µA was applied. These results suggested that the retinal activation by
STS can be localized near the electrode with adequate current intensity.
Neuroprotective effect of transcorneal
electrical stimulation on optic nerve injury
Hata, Yoshio1; Miyake, Ken-ichiro1,2; Inoue, Yoshitsugu2
(1Div Integrative Biosci. Tottori Univ Grad Sch Med Sci, Yonago,
Japan; 2Div Ophthal and Vis Sci, Fac Med, Tottori Univ, Yonago,
Traumatic injury to the optic nerve often causes a rapid loss of vision
within several hours and leads retinal ganglion cells to cell death with
slower time course. The previous experiments demonstrated the neuroprotective effect of electrical optic nerve stimulation on the retinal cell
death over several days or weeks. However, it is not clear whether the
electrical stimulation effectively prevents the acute impairment of visual
function as observed in the traumatic optic neuropathy.
We examined whether the transcorneal electrical stimulation could
improve visual function after the optic nerve crush. A screw electrode
was secured on the skull over the visual cortex to record visually evoked
potential (VEP) in adult rats. VEP elicited by flash stimuli was recorded
before and after the optic nerve crush, and after the transcorneal
stimulation to estimate the degree of damage and the effect of
stimulation in individual animal. Calibrated optic nerve crush reduced
the amplitude of VEP significantly in all animals (30% of pre-crush
value on average). Transcorneal stimulation given through a bipolar
electrode fitted to a contact lens (intensity 500µA, duration 50µsec,
20Hz, 6hours) significantly enhances VEP amplitude (270% of the postcrush value on average) and the enhancement was preserved for one
week in most cases. These results suggest that the transcorneal
stimulation has a protective effect against acute impairment of visual
function by optic nerve crush.
Expression pattern of Nav1.1 in the rat
Kaneko, Yuko; Watanabe, Shu-Ichi (Dept. Physiol., Saitama
Med. Sch., Saitama, Japan)
Retinal ganglion cells and subsets of retinal amacrine cells generate
TTX-sensitive action potentials evoked by light stimulus. It has been reported that voltage-dependent sodium channel (Nav) α subunits, Nav1.1,
Nav1.2, Nav1.3, and Nav1.6, are expressed in the retinal ganglion cells
(Fjell J et al., Mol Brain Res 50:197-204, 1997; Boiko T et al., J Neurosci
23:2306-2313, 2003). However, subtypes of Nav expressed in retinal
amacrine cells have not been identified. To examine the specific Nav
subtypes expressed in the retinal amacrine cells, we applied in situ
hybridization on the rat retina with the RNA probes that recognize
Nav1.1, Nav1.2, Nav1.3, and Nav1.6. We found that Nav1.2, Nav1.3, and
Nav1.6 were localized in the ganglion cell layer (GCL). Interestingly,
Nav1.1 was expressed not only in GCL, but also in the inner nuclear layer
(INL). Cell bodies of Nav1.1 positive cells in INL were located on the
border between INL and the inner plexiform layer. It is probable that
these cells are subsets of amacrine cells. Difference in expression pattern
of Nav might reflex functional difference of action potentials between
amacrine and ganglion cells.
Electrophysiological Study of Rat
Collicular Responses to SuprachoroidalTransretinal Electrical Stimulation:
Evaluation of Pulse Parameters for
Artificial Retina
Kuroda, Masako1; Miyoshi, Tomomitsu1; Kanda,
Hiroyuki1,2,4; Fujikado, Takashi2; Tano, Yasuo3; Sawai,
Hajime1 (1Dept. Physiol. Osaka Univ.Grad.Sch. Med., Suita,
Japan; 2Dept. Applied Vis. Sci. Osaka Univ. Grad. Sch. Med., Suita,
Japan; 3Dept. Opthalmol. Osaka Univ. Grad. Sch. Med., Suita,
Japan; 4NIDEK CO., LTD., Gamagori, Japan)
We recently developed an electrical stimulation method for retinal prosthesis named Suprachoroidal-Transretinal Stimulation (STS, Kanda et
al., IOVS 2004). It was shown that a monophasic pulse of STS focally
applied between an anode on the sclera and an intravitreal cathode (inward STS) evokes well-localized field potentials in the superior colliculus (SC) of both normal and retinal dystrophic rats. When the outward
STS was delivered by reversing the stimulus polarity, the threshold was
much higher than that of the inward STS.
To avoid the electrochemical retinal damage due to charge imbalances,
a biphasic pulse must be used in the STS-based artificial retina. Thus, we
examined the SC response to biphasic STS comparing with that to the
monophasic STS. In normal rats, Electrically-Evoked Potentials (EEP)
to the single biphasic or monophasic STS were recorded from the SC
contralateral to the stimulated eye. In response to the biphasic STS the
late oscillatory components of EEP were greatly suppressed, although
the early component of EEP was smaller in amplitude than that to the
monophasic inward STS. Thus, biphasic STS may be beneficial for
generating artificial perception with high temporal resolution, as well as
being harmless.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Sensory functions
Dynamic change in orientation tuning of
neurons in the cat lateral geniculate
Naito, Tomoyuki1; Sadakane,
Masahiro2; Osaki, Hironobu3;
Sato, Hiromichi1 (1Grad.
Sch. Med., Osaka Univ,, Toyonaka, Japan; 2Grad. Sch. Front.
Biosci., Osaka Univ., Suita, Japan; 3Med. Sch., Osaka Univ., Suita,
Spatial-frequency dependent surround
suppression in early visual pathway
Osaki, Hironobu1; Naito, Tomoyuki2; Sadakane,
Osamu2; Okamoto, Masahiro3; Sato, Hiromichi2 (1Med.
Sch., Osaka Univ., Osaka, Suita, Japan; 2Grad. Sch. Med., Osaka
Univ., Toyonaka, Japan; 3Grad. Sch. Front. Biosci., Osaka Univ.,
Suita, Japan)
It is commonly believed that orientation selectivity first emerges in the
primary visual cortex (V1). In the present study, first, we examined the
orientation selectivity of LGN neurons using optimal and non-optimal
(in terms of stimulus size and spatial frequency (SF)) grating stimuli in
anesthetized cats. We found that although only about 10% LGN neurons
showed significantly orientation-biased response to the grating with optimal size and SF, about 90% of LGN neurons exhibited significant orientation selectivity to grating with diameters larger than its classical
receptive field (CRF) and SFs higher than the optimal for CRF response.
Then, stimulus-size tuning curves were made for responses to stimulation with preferred orientation tested with large stimulus and with orthogonal orientation. These two stimulus-size tuning curves exhibited
profile similar to each other under the optimal SF condition. However,
high SF grating caused stronger surround suppression for response to the
orthogonally oriented stimulus than that to the optimally orientated stimulus. These results suggest that there is orientation tuned surround suppression that is effective around the CRF boundary and its optimal SF is
tuned to higher than that of the CRF responses. We should further address how the orientation selectivity of LGN contributes to that in V1.
Neurons in the primary visual cortex (V1) change their responses depending on stimulus parameters such as orientation, spatial frequency
(SF), size and so on. In this study, we investigated how stimulus size effects on SF tuning property of neurons in V1 and lateral geniculate nucleus (LGN) of cats. First, we found that V1 neurons increased
sensitivity to high SF stimuli when small gratings were used and that SF
selectivity of V1 neurons was sharpened when large gratings were used,
according to the shifts of peak and high cut-off of SF tuning curves. Second, we measured area summation tuning curves under several SF grating conditions, and found that a higher SF stimulus caused a reduction
of the receptive field size and an increase of the surround suppression.
The same tendency was observed in LGN, which is the main source of
excitatory input to V1. This implies that the relationship between SF and
area summation properties observed in V1 has its origin in LGN and is
modified in the intracortical network. These results suggest how neural
circuit in early visual pathway changes its way of information processing
and how it reduces redundancy in various visual environments; for small
visual objects, neurons increase SF sensitivity to get fine resolution, on
the other hand, for large ones, they sharpen SF selectivity to reduce redundancy.
Temporal-frequency dependent surround
suppression in early visual pathway
Sadakane, Osamu1; Naito, Tomoyuki1; Osaki,
Hironobu2; Okamoto, Masahiro3; Sato, Hiromichi1
(1Grad. Sch. Med., Osaka Univ., Toyonaka, Japan; 2Med., Sch.,
Osaka Univ., Suita, Japan; 3Grad. Sch. Front. Biosci., Osaka Univ.,
Suita, Japan)
Neuronal responses of the primary visual cortex (V1) exhibit stimulussize tuning property, and they are suppressed by stimulation with grating
patches larger than their classical receptive field (CRF). Recently, it has
been revealed that size-tuning property of V1 neurons is dependent on
stimulus parameters, such as luminance contrast and spatial frequency.
In this study, we examined the effect of temporal frequency (TF) of sinusoidal grating stimulus on size-tuning curves of cat V1 neurons. Our
results showed that when the TF was higher than optimal, the strength of
surround suppression became weak and CRF-size became larger, suggesting that V1 neurons summate visual information from wider region
under high TF condition. We also tested the effect of changing stimulussize on TF tuning curve. Corresponding to above results, large grating
made peak and high cut-off of TF-tuning curve higher than those for
small grating. Then we examined neuronal responses of the lateral geniculate nucleus (LGN), and obtained basically similar results to those of
V1 neurons. These results suggest that neurons in early visual pathway
change their spatial integration property according to TF of stimulus, in
such a way that neurons integrate wide visual field for fast moving stimulus, whereas localized field for slow stimulus.
Surround suppression sharpens
orientation tuning in cat primary visual
Okamoto, Masahiro1; Naito, Tomoyuki2; Sadakane,
Osamu2; Sato, Hiromichi2 (1Grad. Sch. Front. Biosci., Osaka
Univ., Suita, Japan; 2Grad. Sch. Med., Osaka Univ., Toyonaka,
It is known that orientation tuning of neurons in the primary visual cortex (V1) becomes sharper as the size of stimulus increases beyond the
classical receptive field (CRF) (Orban et al., 1979; Chen et al., 2005;
Xing et al., 2005). We investigated relationships between the strength of
the orientation selectivity and that of the surround suppression in 74 V1
neurons recorded from anesthetized and paralyzed cats. The orientation
selectivity became stronger as an increment of surround suppression due
to an enlargement of stimulus that covered the CRF and the receptive
field surround (SRF). However, the preferred orientation of the neurons
was independent of stimulus sizes and did not vary according to a change
in stimulus size. Also, both the surround suppression and the orientation
selectivity of responses to the stimulus larger than the CRF were significantly stronger in layers II/III than in layers V/VI. We compared our results with predictions of a simple iceberg model where a large stimulus
that covered the CRF and SRF equally and linearly suppressed responses
to all stimulus orientations. The model, however, overestimated the
sharpening of the orientation tuning by large stimulus. These results suggest that surround suppression in V1 exhibits similar to but less-selective
orientation tuning than the response to CRF stimulation. Such an effect
of surround suppression can be a reason why the stronger orientation selectivity is observed for large stimulus.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Sensory functions
All-trans retinal acts as a photosensitizer in
frog rod photoreceptors.
Masutomi, Kosuke1; Koutalos, Yiannis2; Nakatani, Kei1
(1Grad. Sch. Life Sci., Univ. Tsukuba, Tsukuba, Japan; 2Dept.
Ophthal., Med. Univ. S.C., Charleston, S.C., USA)
It has been suggested that oxidation of rod outer segment (ROS) membrane lipids of photoreceptor cells may be involved in light-induced retinal degeneration. All-trans retinal is a potent photosensitizer and its role
in mediating photodamage, mainly lipid oxidation by producing singlet
oxygen, has been suspected for over two decades as shown in studies using ROS membranes or liposomes. Here we demonstrate that all-trans
retinal may actually act as a photosensitizer and produce photooxidation
in living cells. Exogenous all-trans retinal caused lipid oxidation under
UV light (365nm) in bleached bovine ROS membranes in proportion to
either the duration of UV radiation or the concentration of retinal. Similar results were obtained from dark-adapted ROS membranes with retinal released from rhodopsin after irradiation with long wavelength light
(530nm). Finally, consistent with previous studies which have implicated that accumulated all-trans retinal may cause light-induced oxidation
in photoreceptor cells, intact ellipsoid cells, which are able to quantitatively convert all-trans retinal to all-trans retinol, showed significantly
less oxidation compare to those without ellipsoid when exposed to UV
light after releasing all-trans retinal with 530nm light. UV did not have
much effect when there was no 530nm light treatment. These results indicate that all-trans retinal, which is released from rhodopsin by 530nm
light, is photosensitizing component in living cells and causes membrane
oxidation in living cells.
Early change of ocular dominance by brief
monocular deprivation in the
pharmacologically inhibited visual cortex
of kittens.
Morishima, Yu1; Sakamoto, Hroshi2; Akasaki,
Takahumi1; Hata, Yoshio1 (1Div Integrative Bioci, Tottori Univ
Grad Sch Med Sci, Yonago, Japan; 2Div Neurobiol, Sch Life Sci, Fac
Med, Tottori Univ, Yonago, Japan)
Monocular deprivation (MD) during an early postnatal period causes a
loss of visual cortical response to the deprived eye (Ocular dominance
(OD) shift). When MD is combined with inactivation of the visual cortex
by muscimol infusion, cortical neurons lose their response to the open
eye (reverse OD shift). Although these two forms of OD plasticity differ
in direction, it is largely unknown whether their other characteristics are
also different. Such information would be useful to clarify mechanisms
underlying these two plasticity. The normal OD shift proceeds rapidly
and the physiological effect of MD saturates after 48 hours in kittens.
Therefore, we examined how rapidly the reverse OD shift proceeds and
whether the shift could be induced in adults, to compare the dynamics
and age-dependency of two forms of OD plasticity. We have inhibited
the visual cortical neurons by infusing muscimol in four-week old kittens and adults. After 3-6 days of MD, OD of cortical neurons was evaluated by single-unit recording. The reverse OD shift was observed after
6-day MD as reported previously. After 3-day MD, however, the reverse
OD shift was not observed and the OD distribution remained similar to
that of normal animals. In adults, OD distribution in the inhibited cortex
remained unchanged even after 7-day MD. These results suggest that the
reverse OD shift might reflect a mechanism of developmental plasticity
that has a slower time course than the normal OD shift.
Effects of mouth guard wearing on
dynamic visual acuity
Hasegawa, Tatsuhisa1,4; Imai, Yuichiro2; Ishizashi,
Hiromichi3; Yamamoto, Kazuhiko2; Kirita, Tadaaki2;
Hisa, Yasuo4; Yamashita, Masayuki1; Wada, Yoshiro1
(1Dept. Physiology I, Nara Med. Univ., Nara, Japan; 2Dept.Oral and
Maxillofacial Surgery, Nara Med. Univ., Nara, Japan; 3Dept. Health
Science, Nara Med. Univ., Nara, Japan; 4Dept. Otolaryngology,
Kyoto Pref. Univ. Med., Kyoto, Japan)
To determine whether mouth guard (MG) wearing improves performance capacity of athletes, we examined the effects of MG wearing on
dynamic visual acuity (DVA). Subjects were asked to read and answer 3
numerals presented sequentially at random order. The numerals moved
left to right at 90 deg/sec on a CRT monitor under two conditions of head
motion: 1) stationary state, and 2) voluntary rightward rotation. In each
condition, thirty trials with and without MG were carried out and numbers of correct answers (full score; 3 x 30 = 90) were evaluated as DVA
score. We measured DVA improvement indexes (= DVA scores with MG
minus those without MG) and occlusal forces. In the condition 1, the correlation-coefficient between DVA improvement indexes and occlusal
forces was negative (r=-0.74) for nine subjects (20-32 years), whose
DVA improvement indexes were >0 in the condition 2. On the contrary,
the correlation-coefficient was positive (r=0.76) in the condition 2.
These results suggested that the effects of MG wearing on DVA strongly
depended on the condition of head motion and the occlusal force. As we
recorded simultaneously electromyographic activities of masseter and
sternocleidmastoideus, eye movements and head angular velocities,
these data will be described to discuss the effects of MG wearing on
Functional mapping of macaque primary
visual cortex with implanted multielectrode array
Miyakawa, Naohisa1; Blake, David2; Merzenich,
Michael2; Tanifuji, Manabu1 (1Integrative Neural Systems,
RIKEN BSI, Wako, Japan; 2Coleman Lab., KeckCenter for
Integrative Neurosci., UCSF, San Francisco, USA)
Functional mapping of macaque primary visual cortex with implanted
multi-electrode array Naohisa Miyakawa1, David Blake2, Michael
Merzenich2, Manabu Tanifuji1 1 Integrative Neural Systems, RIKEN
BSI, 2 Coleman Lab., KeckCenter for Integrative Neurosci., UCSFIt has
been known that in primates, different levels of visual information are
processed at different areas within the cortical visual streams. Much remain unknown on how each level of information is processed within
each area, but some evidence show possibility of information represented in a distributed manner within the local network of respective cortical
areas (Tsunoda et al. 2001). To understand the function of this local network activity, we have developed a chronically implantable multi-electrode array that is laid out in high-density grid configuration with 350
µm spacing. We implanted the array to the primary visual cortex of
macaque monkey, and detected multi-unit activity (MUA) from all
electrodes for as long as 3 month. We reconstructed a two-dimension
functional response map by visualizing the neuronal activity evoked
with grating stimuli of different orientations. The map showed
significant stability over the 3-month period, indicating that our array is
capable of monitoring cortical network activity with minimum damage
to the cortical tissue over this period.We will show our preliminary result
of multi-electrode array recording of infero-temporal (IT) cortex
neurons evoked by natural image stimuli.
J. Physiol. Sci., Vol. 56, Suppl., 2006
Sensory functions
Spatiotemporal dynamics of surround
suppression in cat V1: Stimulus-duration
and orientation-contrast
Stress responses to the heelsticks in
human infants were attenuated by their
own mother's milk odor
Shimegi, Satoshi; Kida, Hiroyuki; Ishikawa, Ayako;
Sato, Hiromichi (Grad. Sch. Med, Osaka University, Toyonaka,
Nishitani, Shota1; Takase, Ryuta1; Miyamura,
Tsunetake3; Tagawa, Masato2; Sumi, Muneichiro2;
Moriuchi, Hiroyuki2; Shinohara, Kazuyuki1 (1Div.
In the primary visual cortex (V1), a neuronal response to stimulation of
the classical receptive field (CRF) is suppressively modulated by the
stimulus presented at the receptive field surround (SRF). Using stationary flashes of sinusoidal grating as stimuli, we examined the dependency
of effect of SRF stimulation on the orientation-contrast (OC) between
CRF and SRF stimuli (Exp.1) and that on the presentation duration (50
ms vs. 50