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. [J Physiol Sci. 2006;56 Suppl:S2]

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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of AMPA receptors produces an immediate blockade of spine motility which is reversed as soon as the stimulus is withdrawn.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blockade of spine motility via NMDA receptors requires 30 min to develop and persists for hours after the stimulus is withdrawn.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. [J Physiol Sci. 2006;56 Suppl:S2]

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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. [J Physiol Sci. 2006;56 Suppl:S2]

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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. 2006;56 Suppl:S2]

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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 GAD67-GFP 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 whole-cell 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. [J Physiol Sci. 2006;56 Suppl:S3]

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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 high-field 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. [J Physiol Sci. 2006;56 Suppl:S3]

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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. c-Met+ 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. [J Physiol Sci. 2006;56 Suppl:S3]

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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. 2006;56 Suppl:S3]

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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. [J Physiol Sci. 2006;56 Suppl:S4]

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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 molecular-feature 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. 2006;56 Suppl:S4]

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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. [J Physiol Sci. 2006;56 Suppl:S4]

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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. [J Physiol Sci. 2006;56 Suppl:S6]

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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 circa-12 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 cyto- and 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. [J Physiol Sci. 2006;56 Suppl:S6]

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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:CLOCK-mediated transcriptional activation. On the other hand, we found Ser557-phosphorylated 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. [J Physiol Sci. 2006;56 Suppl:S6]

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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. [J Physiol Sci. 2006;56 Suppl:S7]

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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). [J Physiol Sci. 2006;56 Suppl:S7]

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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. [J Physiol Sci. 2006;56 Suppl:S8]

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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 down-regulate 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. [J Physiol Sci. 2006;56 Suppl:S8]

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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. 2006;56 Suppl:S8]

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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 negatively. [J Physiol Sci. 2006;56 Suppl:S9]

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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. [J Physiol Sci. 2006;56 Suppl:S9]

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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. [J Physiol Sci. 2006;56 Suppl:S9]

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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 NaPi-IIa 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. [J Physiol Sci. 2006;56 Suppl:S10]

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The brain astrocytes transport excess extracelluar K⁺ yielded by synaptic 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 (pH_i) change in a physiological range, K⁺ outflow and part of K⁺ influx may be finely controlled by pH_i. 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 astrocytes. [J Physiol Sci. 2006;56 Suppl:S10]

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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 re-localization 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. [J Physiol Sci. 2006;56 Suppl:S10]

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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. [J Physiol Sci. 2006;56 Suppl:S10]

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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 macrophage. [J Physiol Sci. 2006;56 Suppl:S11]

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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 (2005). 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). [J Physiol Sci. 2006;56 Suppl:S11]

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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 BCR-induced 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, DAG-dependent activation of TRPC3 plays an important role in BCR-mediated Ca²⁺ and MAPK signalling. [J Physiol Sci. 2006;56 Suppl:S11]

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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. [J Physiol Sci. 2006;56 Suppl:S12]

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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 (20-HETE), 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. [J Physiol Sci. 2006;56 Suppl:S12]

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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. [J Physiol Sci. 2006;56 Suppl:S12]

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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 week-old 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. [J Physiol Sci. 2006;56 Suppl:S13]

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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 estrogen-dependent 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 narrowed-down 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. 2006;56 Suppl:S13]

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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. [J Physiol Sci. 2006;56 Suppl:S13]

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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 self-excitation 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. 2006;56 Suppl:S14]

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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. [J Physiol Sci. 2006;56 Suppl:S14]

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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. [J Physiol Sci. 2006;56 Suppl:S14]

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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 large-scale network activities in the early development. [J Physiol Sci. 2006;56 Suppl:S15]

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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. [J Physiol Sci. 2006;56 Suppl:S15]

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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. 2006;56 Suppl:S15]

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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 (LN_vs). 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 LN_vs. 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. [J Physiol Sci. 2006;56 Suppl:S16]

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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). [J Physiol Sci. 2006;56 Suppl:S16]

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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. [J Physiol Sci. 2006;56 Suppl:S16]

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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 presented. [J Physiol Sci. 2006;56 Suppl:S16]

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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 metabolism. [J Physiol Sci. 2006;56 Suppl:S17]

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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. [J Physiol Sci. 2006;56 Suppl:S17]

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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 high-starch (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. 2006;56 Suppl:S17]

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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 differentiation. [J Physiol Sci. 2006;56 Suppl:S18]

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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. 2006;56 Suppl:S18]

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