EEG related to time dependent varying tasks are signals of non-stational processes, and ordinal information criteria for which stational processes are assumed can not be applied directly. In this paper, we propose an analysis method for EEG signals related to time dependent varying tasks using an information criterion which has been published by one of the authors. By using the method, time dependent varying information amount of the time dependent varying EEG can be evaluated related to a time dependent varying task. A task performed as an example was a color object image in which the color varies continuously depending on the time axis starting from a natural color to a strange color on the object. The total time of the variation was 20 seconds. The time dependent varying color image was displayed on a CRT display. The subjects were five graduate students. For measurements of EEG at observations of the image, an electrode cap with 19 electrodes has been mounted on the subjects. For each electrode, information amount analysis was performed on the time axis. The averaged result of 19 electrodes show that the information amount of alpha band decreases monotonously, and theta band increases monotonously with progress of time. It is supposed that the variation of the information amount in the example indicates activated interpretations related to the strange color on the object with progress of time. Though, EEG is taken up as an example in this paper, the method can be applied also for other signals observed from human body. [J Physiol Sci. 2007;57 Suppl:S71]

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We previously developed a method to localize a microelectrode tip with single-voxel accuracy using 4.7 T MRI. In that study, we validated the method's localization accuracy in vitro using a phantom (in-plane resolution: 50 µm) and in vivo using anesthetized monkeys (in-plane resolution: 150 µm). In the present study, we devised a custom-made non-magnetic mini-manipulator (Narishige) that allows for the easy implementation of the localization method for electrophysiological recording from behaving monkeys. With behaving monkeys, it is important to stably retain the microelectrode and so keep the tip in the same position when monkeys are anesthetized and transferred to the MRI scanner after the recording session. We implanted a microelectrode using our mini-manipulator to assess positional stability during the transition to the MRI, which could be performed on the either the day of the recording session or the next day. Across two successive days of implantation, the position of the microelectrode tip did not change on magnetic resonance images with an in-plane resolution of 150 µm (less than ± single voxel variation across days; 0.00 ± 0.94 voxel; equivalence test, P < 0.01). This stable maintenance of the microelectrode tip position across days enables electrophysiological recording in an awake state and MRI under anesthesia to be performed even on separate days. [J Physiol Sci. 2007;57 Suppl:S71]

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The natural environment is constantly changing. Humans and other animals should track these changes and alter their behavior to survive in such uncertainty. One choice which was correct a few seconds ago is not guaranteed to be correct now. The brain functions involved in the interactions between the environment and decision making, however, have not been well addressed. In this study, we aimed to see how monkeys extract information from the environment and adjust their behavior to current environment status, as well as neural correlates of the decision process. We used two Japanese macaques. Multiple electrodes were chronically implanted in prefrontal cortex. Their behaviors were recorded using a motion capture technique and video. In each trial, the monkeys were asked to take a food held in a researcher's hand. To complete the trial correctly, the monkeys should observe how researcher held the food and decide either the left or right hand to reach. Amount and type of food were varied to examine their adaptive behavior to the environmental context. We found that the monkeys adjusted their posture as a preparatory behavior. The behavior was systematic so that we could easily predict their future choice through the behavior by using linear regression model. The model could provide monkey's internal decision process virtually. There were various types of neuronal response found in PFC that were correlated to estimated decision processes. The results suggest PFC is involved in behavioral adjustment process under uncertain environment. [J Physiol Sci. 2007;57 Suppl:S71]

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Like human, one of primates, common marmoset shows complex and delicate social behavior which tentially makes this animal as a model for the study of neural system for the human social interaction and its disease.We have tested the social interaction of a female-male group of marmosets usually kept in individual cage and are attempting to translate the marmoset inner and outer emotional expression. In an objective manner by examining any relation betweentheir social context-dependent behavior and blood stress/ sex hormones levelover each behavior, we found reproducibly that each animal showed a traitof individual preference and the change of hormone level before and afterthe behavior test. [J Physiol Sci. 2007;57 Suppl:S72]

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The hypothalamic AMP kinase regulates feeding behavior in response to hormonal and nutrient signals. Decrease of AMPK activity in the paraventricular (PVH) and arcuate (ARH) hypothalamus plays an important role in anorexic effect of leptin. In the present study, we examined the long-term expression of constitutively-active AMP kinase (CA-AMPK) in the PVH in C57/BL6 mice in food intake and body weight, using lenti-viral vector with neuron specific-synapsin 1 promotor. CA-AMPK in the PVH gained body weight and increased food intake in mice: the increase in body weight was more than 10 g in CA-AMPK expressed mice 3 months after the infection, while the increase in the control mice was less than 4 g. Interestingly, CA-AMPK in the PVH increased the preference for high carbohydrate but not high fat diet, while the control mice preferred high fat diet rather than high carbohydrate diet. As shown in peripheral tissues by several reports, we found that CA-AMPK increased the expression of fatty acid oxidation-related genes such as PPAR alpha, FATP1, CPT1c, etc. in the PVH. Furthermore, direct administration of etomoxir, an inhibitor of CPT-1 and fatty acid oxidation in mitochondria, into the PVH in the CA-AMPK expressed mice, reversed the high preference for carbohydrate diet to the preference for high fat diet. Thus, these findings suggest that AMP kinase in the PVH regulates not only calorie intake but also the preference for carbohydrate and fat diets, by changing fatty acid metabolism in the hypothalamic nucleus. [J Physiol Sci. 2007;57 Suppl:S72]

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Cannabinoids exert their psychomotor actions through cannabinoid CB1 receptor in the brain. Genetic deletion of CB1 in mice causes various symptoms including change in locomotor activity, increased ring catalepsy, hippocampal plasticity, and impaired extinction of hippocampus-dependent learning. Although the cerebellar cortex contains the highest level of CB1 in the brain, cerebellum-related functional deficits have not been reported in CB1 knockout mice. To elucidate the roles of CB1 in cerebellar function, we subjected CB1 knockout mice to a delay version of classical eyeblink conditioning. We found that memory formation of delay eyeblink conditioning was severely impaired in CB1 knockout mice. In contrast, they exhibited normal performance in a trace version of eyeblink conditioning with 500-ms stimulus free interval intervened between the CS offset and the US onset, which is regarded as a hippocampus-dependent associative learning. We also found that intraperitoneal injection of the CB1 antagonist SR141716A (rimonabant) to wild-type mice caused severe impairment in acquisition but not extinction of the delay eyeblink conditioning. SR141716A treatment had no effect on the trace eyeblink conditioning with 500-ms or 750-ms trace interval. These results indicate that endogenous cannabioid signaling through CB1 is essential for cerebellum-dependent discrete motor learning especially for its acquisition phase. [J Physiol Sci. 2007;57 Suppl:S72]

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Tuberomamillary (TM) histamine (HA) neurons play an important role in the mechanisms of wakefulness (W). We have recently shown in the mouse that identified HA neurons are waking-specific and that they are all characterized by triphasic broad action potential. It is currently believed that HA neurons cease firing during sleep by an inhibitory mechanism mediated by GABAergic sleep-promoting neurons in the preoptic area (POA). In order to test this assumption, we have recorded single unit activity from the TM and POA in head-restrained, conscious mice and determined their unit activity profiles at the state transitions. Forty-two waking-specific neurons were recorded in the TM, while 7 slow-wave sleep (SWS)-specific and 8 SWS/paradoxical sleep (PS)-specific neurons were recorded in the POA. At the transition from W to SWS, TM HA neurons stopped firing clearly before the onset of EEG synchronization, the first sign of EEG sleep, with a mean (± S.E.M) latency of 1078.3 ± 72.7 ms, while both POA SWS-specific and SWS/PS-specific neurons began firing either at the onset or after the onset of EEG synchronization. At the transition from SWS to W, HA neurons displayed firing with a long delay (800.2 ± 62.7 ms) after the onset of EEG desynchronization, while the POA sleep-specific neurons stopped firing prior to the onset of EEG desynchronization. These results suggest that the neuronal activity of HA neurons during behavioral states is not controlled exclusively by POA sleep-promoting neurons. [J Physiol Sci. 2007;57 Suppl:S73]

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To clarify the role of brain histamine (HA) on the photic and non-photic entrainment of circadian behavioral rhythms, the formal properties in the histidine decarboxylase knock out mice (HDC-/-) of C57BL6CrSlc background were examined. Male adult mice were individually housed in a LD cycles. The spontaneous locomotor activity was continuously measured by thermal sensor. Then, animals were released into constant darkness (DD) for ca.4 weeks. Mean free-running period of spontaneous locomotor rhythm were in 23.88 h in HDC-/- mice (n=10) and 23.61h in control mice (n=10) (p<0.05). Phase responses to 60min ca.300lux light pulse were measured at different circadian phase. HDC-/- mice showed a PRC almost identical to that of the control. With an exception of smaller phase advance shifts at CT0 (HDC-/- 0.97h vs. +/+ 1.57 h, p<0.05). In order to examine whether the circadian behavioral rhythm in HDC-/- mice entrain to non-photic stimulus, after having shown stable free-running rhythm, they were transferred to a cage with a running wheel for 3h a day for 68 days in DD. Eight of ten control mice entrained to the schedule, and other two mice were showed a relative coordination during the schedule. Whereas, eight of ten HDC-/- mice showed entrainment to the schedule and other two mice showed a change of freer-running period. These results demonstrated that brain HA might affect the photic entrainment but not affect the non-photic entrainment in the mammalian circadian pacemaker. [J Physiol Sci. 2007;57 Suppl:S73]

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Circadian time-keeping system regulate behavior and physiological functions in most living organs. Recently, molecular links between the endogenous circadian clock and cell cycle regulation is reported. Biochemical changes, including the cancer an abnormal cell proliferation, induces dysfunction of clock in the suprachiasmatic nucleus a center of biological rhythm.Therefore, we examined an effect of carcinogenic drug on circadian-locomotor-activity rhythm. Diethylnitrosamine (DEN), which is widely used to cause hepatic carcinoma, was injected (i.p. 200 mg/kg) on rats kept under the light-dark (LD) cycle, constant dim light (DD) and constant light (LL). After the activity recording, livers of same specimens were histochemically analyzed using GST-P immunostaining, early detection marker of carcinogenicity, positive cells.After the DEN injection, decrease of activity mass on the next day was common in any of lighting conditions. Phase resetting under the LL and DD was observed without period length changes. The GST-P positive cell emerged most under the LD.It is known that locomotor activity rhythm and melatonin secretion couples well, and melatonin has a cancer chilling effect. The decrease of active mass under the LL suggested possibility to surpass restraint of melatonin secretion. [J Physiol Sci. 2007;57 Suppl:S73]

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To examine protein-DNA binding, a series of assays such as electrophoretic mobility shift assays (EMSA) have been developed. However, these are time consuming experiments, and contain several difficulties including the use of radioisotopes or specific antibodies. In addition, during gel electrophoresis, subtle binding may be dissociated. To overcome such difficulties, we developed a novel in vitro DNA-protein binding assay. This assay is based on a fluorescence-labeled DNAs and GST-fused proteins. After washing steps, immunological reaction was performed on the GST beads to detect binding activities that are measured by luminometer in solution in seconds. This technique is convenient because of its simple steps, in which radioisotopes are not required, and a small amount of garbage is produced. Thus, it may be useful for the screening of toxic compounds. By using this technique, we first confirmed the interaction of nuclear receptor (NR) with nuclear hormone response element (NRE). Moreover, we showed that GST-coactivator- or -corepressor-NR-NRE interactions. These results were compatible to those using EMSA or transient transfection-based reporter gene assays. Furthermore, we can apply this technique to study protein-protein or protein-RNA interactions.In summary, we developed a novel in vitro DNA-protein binding assay and this technique is useful to screen a large amount of DNA-protein interactions in a short time. [J Physiol Sci. 2007;57 Suppl:S74]

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Thyroid hormone (TH)plays an important role in the developing brain. Deficiency of TH during perinatal period induces abnormal brain development known as cretinism. The neurological mutant mouse, staggerer (sg), harbors a deletion within the gene encoding retinoic acid receptor-related orphan nuclear receptor (ROR) α. This mouse shows hypothyroid-mice like phenotype. We previously showed that an evident alteration of cerebellar neurotrophin mRNA levels in sg mice using semi-quantitative RT-PCR and in situ hybridization. Moreover we showed that the sg type ROR (RORsg) was not effective in augmenting TR action through various TREs, although wild type RORα augmented TR-mediated transcription. Since RORα and TR are coexpressed in many neurons including the Purkinje cell, RORα may be generally required for a full function of TR action. In the present study, we investigated the mechanism of crosstalk between TR and RORα. We showed that DNA-binding domain and ligand-binding domain are responsible to this augmentation using a series of TR-mutants and reporter gene assays. This augmentation may not due to direct binding of ROR to TR. On the other hand, it has been speculated that polychlorinated biphenyl (PCB) may be involved in the increase in number of cretinism. We previously showed that as low as 10⁻¹⁰ M of PCB, suppressed TR-mediated transcription. Low dose of PCB suppressed TR-ROR-augmented transcription. These results indicated that the augmentation of TR by ROR is possibly due to a novel mechanism and ROR did not rescue the suppression of TR-mediated transcription by PCB. [J Physiol Sci. 2007;57 Suppl:S74]

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Sphingosine 1-phosphate (S1P) is a lysophospholipid that exerts numerous biological activities, including promotion of angiogenic differentiation of vascular endothelial cells, both as a receptor ligand and as an intracellular second messenger. Because molecular mechanisms that underlie differentiation processes of pre-adipocytes to adipocytes (adipogenesis) remain incompletely understood, we explored the roles of sphingosine kinase-1 (SK-1), an enzyme that phosphorylates and converts sphingosine to S1P. RT-PCR assays revealed that the expression levels of SK-1 mRNA, but not those of SK-2, are upregulated during adipogenesis of mouse 3T3-L1 cells (4.0 folds at day 4 vs. pre-adipocytes, p < 0.005), concomitant with elevated intracellular S1P content (4.1 folds at day 5, HPLC analysis, p < 0.01). Conversely, cells transfected with siRNA specific to SK-1, which exhibit markedly reduced S1P content (61% vs. control), undergo lower degrees of adipogenesis, characterized by decreases in lipid droplet accumulation (Oil Red O staining, 51% vs. control) and in adipocyte marker gene expression (PPARγ and aP₂, RT-PCR). In vivo relevance of SK-1 in adipogenesis was explored in 13-weeks old ob/ob mice with obese phenotype, which exhibits markedly upregulated levels of SK-1 mRNA in both subcutaneous and epididymal adipose tissues (RT-PCR, p < 0.001 vs. wild type). These results suggest that the increased expression of SK-1, an S1P-producing enzyme, may promote adipogenesis, potentially providing a novel point of control in cardiovascular disorders associated with obesity. [J Physiol Sci. 2007;57 Suppl:S74]

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Although the anteroventral third ventricular region (AV3V), a pivotal forebrain area for autonomic functions, contains the receptors (-R) for GABA that might operate in association with those for excitatory amino acids, their roles in regulating ADH release and related phenomena have not been examined as yet. This study aimed to pursue the issue through experiments in conscious rats with indwelling cerebral and vascular cannulae. Infusion sites of drugs in the brain were identified through histological inspections. Administrations of a GABA(A) receptor antagonist bicuculline (BCC) into the AV3V caused prompt and remarkable augmentations in plasma ADH, osmolality, glucose, arterial pressure and heart rate, without affecting plasma electrolytes. When BCC was injected into the cerebral ventricle, relatively smaller or no responses were provoked in the variables. In contrast, AV3V injections of a GABA(B)-R antagonist phaclofen did not show any effects on those variables. The responses to AV3V applications of BCC were inhibited by preadministrations of a GABA(A)-R agonist muscimol, the treatments that affected none of the variables when injected alone. The applications of Glu-R antagonists MK-801 or NBQX were also potent to abolish the responses to AV3V BCC. These results suggest that in the AV3V GABA neurons may tonically operate to prevent ADH secretion and cardiovascular functions via the mechanisms related to glutamatergic neuronal activity. [J Physiol Sci. 2007;57 Suppl:S75]

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Platelet-activating factor (PAF) is a highly potent stimulator of the secretion of glucocorticoids. PAF acts mainly through PAF receptor accompanied by the activation of protein kinase (PK) C. While, ACTH and cholera toxin (CTX) acts through the activation of PKA. In the present study, we studied the cross-regulation of cortisol (F) secretion among PAF, ACTH, and CTX by perfused guinea pig adrenal gland. 1) The infusion of 1nM PAF or 10pg/ml ACTH for 5 min significantly stimulated F secretion. Concurrent application of PAF and ACTH evoked F secretion less than additional. The F response to repeated infusion of this mixture was reproducible. 2) The infusion of 20-2000 ng/ml CTX for 5 min dose-dependently augmented F secretion. The peak of F secretion to 20 ng/ml CTX reached a plateau about 40 to 60 min after start of infusion that persisted thereafter. The application of 10 nM PAF from 95 to 100 min after the infusion of CTX induced additional augmentation of F secretion above CTX-induced cortisol output. The application of 100 pg/ml ACTH had no effect. 3) When applied 10 nM PAF repeatedly from 95 to 100 min and from 140 to 145 min after the infusion of CTX, the completely same amounts of F were secreted above CTX-induced cortisol output. When applied 10 nM PAF 45 min before and 85 min after the infusion of CTX, almost same amounts of cortisol were secreted above basal and CTX-induced cortisol secretion.These results implicate that PAF not only induces cortisol secretion but also markedly augments Gs-activated cortisol secretion. [J Physiol Sci. 2007;57 Suppl:S75]

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The LC is the major source of NA innervation in the entire brain. In order to make clear the functions of the LC, we established a method to destroy selectively the NA neurons in the LC. Transgenic mice were used that expressed hIL2R driven by the DBH promoter. After injection of the immunotoxin (anti-hIL2R antibody conjugated with Psudomonas exotoxin) into the LC of a male transgenic mouse, the NA neurons were almost entirely ablated in the LC. After lipopolysaccharide (LPS) injection intraperitoneally (ip), the number of Fos-immunoreactive neurons increased dramatically in various brain regions in control animals. In animals whose LC had been destroyed, Fos expression was reduced markedly in the cortex, preoptic and hypothalamic areas as well as amygdaloid nuclei. LPS injection ip elicited marked increase in plasma ACTH in control animals, and this increase was not attenuated in the LC-ablated animals. In the elevated plus-maze, the ratio of the number of open arm entry to the total number of entry became increased in the LC-ablated animals in comparison with that in the controls.These results suggest differential roles of the LC in the neuroendocrine and behavioral responses. We are currently studying whether estrogen may modulate the effects of the LC using female mice with or without estradiol supplementation. [J Physiol Sci. 2007;57 Suppl:S75]

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Chinese herbs (CH) have been used as tradtional chinese medicine for deficiency of saliva secretion. However the mechanisms have not been clarified. This study was planned to investigate whether CH have direct effects on salivary gland or not. Submandibular gland was surgically isolated from Wistar male rat and vascularly perfused. The excretory duct was cannulated and the weight of secreted saliva was continuously recorded to calculate the fluid secretion. 20 kinds of CH were chosen, whose concentration in perfusate was prepared to be equal with the plasma concentration under clinical usage. At control, carbachol (CCh) was given at 0.2 μM. Saliva secretion formed an initial ephemeral peak at 30 s, and following gradual increase to a sustained level. By CH alone, no saliva secretion was induced by 19 kinds of CH chosen in the present experiments. However, during perfusion with 15 kinds out of 19 CH, overloading of CCh promoted the fluid secretion. The enhancement was classified to three patterns: 1) Overall sustained phase was slowly and continuously raised. 2) Sustained secretion rose continuously to reach maximum after 5-10 min, then sharply decreased to a lower sustained level than control. 3) The sustained secretion continuously increased to reach maximum after 5-10 min, then decreased slowly. Above three types of secretion were related respectively to the categories of Chinese herbs (yin-nourishing agent, qi-enhancing agent and blood activating agent). Present study introduced CH as a new tool to investigate mechanisms of fluid secretion by salivary glands. [J Physiol Sci. 2007;57 Suppl:S76]

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Orotic acid, a normal intermediate in pyrimidine synthesis, is converted to uridine 5'-triphosphate (UTP) that is utilized for the sythesis of RNA and for uridine diphosphosugars (UDP sugars) used in the glycosylation of basement membrane collagen. Therefore, as a precursor of uridine nucleotides, orotic acid incorporation is an important factor in glomerular and tubular basement membrane thickening, for example, observed in diabetes-induced renal hypertrophy. Orotic acid uptake is observed in normal liver and kidney, but its molecular mechanism is largely unknown. Because orotic acid is also known as a substrate of renal urate/anion exchanger, we investigated whether human renal urate transporter URAT1 mediates the transport of orotic acid using HEK293 cells expressing hURAT1 (HEK293-URAT1). Human URAT1 mediated a time- and dose-dependent uptake of orotic acid, with Km values of 5.2 μM. URAT1-mediated orotic acid transport was inhibited strongly by orotic acid and benzbromarone and moderately by uric acid, nicotinic acid, and probenecid. These results suggest that human URAT1 mediates the transport of orotic acid, and may function as one of its entrance pathway in the renal proximal tubular cells. [J Physiol Sci. 2007;57 Suppl:S76]

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Ca²⁺ is essential for the signal transduction pathways. We have previously demonstrated several Ca²⁺ mobilization mechanisms in odontoblasts such as; IP₃-induced Ca²⁺ release by PLC-coupled receptor activities, depolarization mediated Ca²⁺ mobilization and Ca²⁺ influx via store-operated Ca²⁺ channels. The [Ca²⁺]i levels are tightly regulated by the Ca²⁺ extrusion system including Na⁺/ Ca²⁺ exchangers (NCX) in the plasma membrane, however, no direct functional evidence has been obtained Na⁺/Ca²⁺ exchange pathway in odontoblasts. In this study, we investigated presence and functional characteristics of NCX in odontoblasts. NCX activities were measured with a fura-2 and patch-clamp technique. For Immunohistochemistry, cryostat sections were incubated with monoclonal antibodies against NCX, and labeled using fluorescent secondary antibodies. The reverse and forward exchange showed dependence on extracellular Ca²⁺ and Na⁺ concentrations, respectively. In addition, reverse exchange was blocked by an NCX inhibitor (KB-R7943) in a concentration dependent manner, while was not inhibited by SEA0400. NCX immunoreaction was observed on the odontoblast plasma membranes. These results indicate that odontoblasts express plasma membrane NCX3 which may operate Ca²⁺ extrusion after accelerated Ca²⁺ signals via store-operated Ca²⁺ channels during dentinogenesis. [J Physiol Sci. 2007;57 Suppl:S76]

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K-Cl cotransporters (KCCs) play significant roles in epithelial transport and cell volume regulation. Four KCC isoforms (KCC1-4) have been so far reported. We examined whether the KCC isoforms are expressed in the stomach. Western blot analysis showed that KCC3a and KCC4 were expressed in isolated gastric mucosa of rats and mice. Immunohistochemistry in the isolated gastric mucosa showed that KCC3a was expressed in the basolateral membrane and KCC4 was expressed in the apical membrane of the gastric parietal cells. KCC4 protein was found in human gastric tubulovesicles. Interestingly, KCC3a and KCC4 were abundantly expressed in the parietal cells located at luminal segment of the gland. Because the luminal segment parietal cells are much more active in HCl secretion than those of the basal segment, KCC3a and KCC4 may be involved in the mechanism of HCl secretion. In the LLC-PK1 cells stably expressing KCC3a (Tet-on system), ouabain-sensitive Na,K-ATPase activity was significantly higher than that of control cells. The expression level of Na,K-ATPase in the KCC3a-expressing cells was not significantly different from that in the control cells. EP level of Na,K-ATPase in the KCC3a-expressing cells was significantly higher than that in the control cells. KCC3a and Na,K-ATPase were present in the same lipid rafts. KCC3a may regulate gastric acid secretion cooperating with Na,K-ATPase in the basolateral membrane of parietal cells. [J Physiol Sci. 2007;57 Suppl:S77]

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Short-chain fatty acids (SCFAs) are 2-4 carbon monocarboxylates produced by bacterial fermentation in the large intestine, and known to induce a variety of physiological and pathophysiological effects on the intestine. In the previous study, we have reported that the SCFA receptor, GPR43, is expressed by the peptide YY-containing enteroendocrine cells and mucosal mast cells in the rat intestine. In the present study, we investigated the expression of the SCFA receptors, GPR43 and GPR41 in the human colon by RT-PCR, Western blotting, and immunohistochemistry, and the effects of SCFAs on transepithelial ion transport were measured by Ussing flux chamber technique. Segments of human colon were obtained with informed consent from the patients undergoing colectomy for carcinoma. Total RNA and proteins were extracted from the small sections in order to analyze the expression of SCFA receptors by RT-PCR and western blot, respectively. Moreover, mucosa-submucosal preparations were made by removing the muscle layer from the tissues, and short-circuit current were measured as an index of transepithelial ion transport. As a result, SCFA receptors were expressed in the mucosa, and propionate changed transepithelial ion transport in the human colon. [J Physiol Sci. 2007;57 Suppl:S77]

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Aquaporins (AQPs) are water channel proteins expressed in various organs and tissues. So far abnormal expression of AQPs has been reported in several cancer tissues, but the function of AQP in human cancer has been little known. Here, we compared the protein expression levels of AQP3, 4 and 5 between human gastric cancer tissue and its accompanying normal mucosa in the upper or middle part of the stomach. It was found that AQP5 was significantly upregulated in the intestinal type of gastric adenocarcinomas but not in diffuse type of adenocarcinomas. AQP5 was localized in the apical membrane of the cancer cells. Expression of AQP4 was down-regulated both in intestinal and diffuse types of adenocarcinomas. No significant expression of AQP3 was observed in the cancer and normal tissues. To clarify the function of AQP5 in gastric adenocarcinomas, we transiently expressed AQP5 in a poorly differentiated human gastric adenocarcinoma cell line (MKN45). Interestingly, AQP5-expressing cell increased the number of differentiated cells. The AQP5 expression increased an activity of alkaline phosphatase, a marker for the intestinal epithelial cell type of cancer cells and expression level of laminin, an epithelial cell marker. Upregulation of AQP5 may be involved in the tumor differentiation of human stomach. [J Physiol Sci. 2007;57 Suppl:S77]

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Transient receptor potential melastatine 8 (TRPM8), a member of TRP cation channel family, is activated by cooling and menthol. TRPM8 is expressed in a subgroup of sensory neurons in the trigeminal ganglia (TG) and the dorsal root ganglia (DRG) and in trigeminal nerve fibers of the tongue. If TRPM8 mediates cooling-induced cold sensation, TRPM8 should be expressed in cutaneous nerve terminals of sensory neurons. However, it has remained unresolved whether TRPM8 is expressed in nerve fibers of skin. Here we investigated the expression of TRPM8 protein in skin of nose, fingers of forelimb and hindlimb and ear of mice by immunohistochemical assay using self-made antibody against TRPM8. Tissues of skin of nose, fingers and ear were isolated. Cryostat sections (20 μm) were obtained and immunohistochemically stained. TRPM8 immunoreactivities were detected in nerve fibers of all of skins explored. These results indicate, for the first time, that TRPM8 protein is surely expressed in peripheral nerve endings at skin, implying that TRPM8 mediates cooling-induced cold sensation. [J Physiol Sci. 2007;57 Suppl:S78]

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TRPC6 is one of major TRP isoforms in vascular tissues and acts as a nonselective cation channel associated with vascular tone generation and remodeling. There is good evidence to suggest that activation and inactivation of TRP6 channel are effectively regulated via its phosphorylation by Ca²⁺-calmodulin dependent kinase II and protein kinase C, respectively. In this study, we show evidence that nitric oxide (NO)/cGMP/ PKG pathway may serve as another potent mechanism that would tonically inhibit TRPC6 channel activity.HEK293 cell expressing mouse TRPC6 protein responded to carhachol (100μM) to generate a non-selective cationic current with S-shaped double rectifying property (I_TRPC6). The magnitude of I_TRPC6 was greatly inhibited by pretreatment with a NO donor, S-nitrosoacetyl penicillamine (SNAP) (by 70% at 100μM) in a voltage-independent manner, but cell-surface expression of TRPC6 protein was not appreciably reduced. Similar extent of inhibition was produced by a membrane-permeable analogue of cGMP 8-bromo cGMP (8-Br-cGMP; 100μM). Both the inhibitory effects of SNAP and 8-Br-cGMP were nearly abolished by simultaneous administration of the PKG inhibitor KT5823 (10μM) or alanine substitution for one of PKG phophorylation motifs in murine TRPC6, T69. These results suggest that PKG-mediated phosphorylation is another powerful mechanism to control TRPC6 channel activity, which may in situ operate as a tonic negative feedback from adjacent endothelial cells which are producing NO continuously. [J Physiol Sci. 2007;57 Suppl:S78]

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The synthetic ciguatoxin, CTX3C has been shown to activate TTX-sensitive sodium channels (Na_v1.2, 1.4 and 1.5) by accelerating activation kinetics and shifts the activation curve to the hyperpolarizing direction¹. In this study, we examined the effects of CTX3C on one of TTX-resistant (TTX-R) sodium channels, Na_v1.8 Na⁺ channels. There are several evidences that Na_v1.8 is related to hyperexcitability observed in primary afferent neurons following nerve and tissue injury. Thus, Na_v1.8 has been considered to be the cause of hyperalgesia or neuropathic pain. On the other hand, ciguatoxin causes ciguatera, which is widespread fish poisoning and often presents neurotoxic symptoms, hyperalgesia or allodynia. We found 100 nM CTX3C is sufficient to modulate voltage-elicited currents of Na_v1.4 expressed in HEK293 cells at -100 mV, 10 times less dose are required than at -140 mV. However, no currents were observed without stimulus. Sustained currents of Na_v1.8, expressed in ND7-23 cells, were induced by application of 100 nM CTX3C without stimulus. The induced sustained currents were activated by depolarization to -80 mV, deactivated by hyperpolarization to -120 mV. These electrophysiological properties of Na_v1.8 well correlate with the sensory neurological symptoms associated with ciguatera fish poisoning. REFERENCE1. Yamaoka, K., M. Inoue, et al. (2004). Br J Pharmacol 142(5): 879-89. [J Physiol Sci. 2007;57 Suppl:S78]

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The ligands for most of the olfactory receptors (ORs) in mammals remain unknown, because it is difficult to express OR functionally in heterologous cells. It was recently demonstrated that coexpression with the receptor transporting proteins (RTPs) promotes OR functional expression in HEK293T cells. This finding enables us to repeatedly analyze more ORs function in heterologous cells. A murine olfactory receptor OR S6 was cloned from an isolated olfactory receptor neuron (ORN) that responded to octanedioic acid (cc8) and nonanedioic acid (cc9). In this study, we coexpressed RTP1, RTP2, Gα15-olf (Gα15 whose C-terminal seven amino acids are replaced with that of Gαolf) and an OR (OR-S6 or OR-S83) in HEK293 cells. Ligand specificities of ORs were analyzed by using Ca ²⁺ imaging. We found that HEK293 cells expressing OR-S6 respond to cc8 and cc9 similarly to the original ORN. Furthermore, OR-S83 ligand specificities in HEK293 cells were similar to that in original ORN. Next, we identified novel antagonists for OR-S6 in ligand candidates structurally related to cc8 or cc9. The relationship of structure and activity suggest that ligand length, functional group and interactions between receptor and medial parts of ligands involve in OR-S6 activation. [J Physiol Sci. 2007;57 Suppl:S79]

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We have previously shown that L-type Ca²⁺ channels provide a pathway for extracellular Ca²⁺ entry in cultured parathyroid cells from patients with secondary hyperparathyroidism. Because the cells previously examined were cultured in a standard DMEM, the ionized Ca²⁺ concentration (approximately 1.8 mM) is higher than that of our plasma (around 1.2 mM). We therefore asked whether the cells exhibit L-type Ca²⁺-channel activity when cultured in a medium containing Ca²⁺ of lower than 1.2 mM. The parathyroid cells were plated on a gelatin-coated thin cover glass and cultured in a medium containing Ca²⁺ of either 0.9, 1.2, or 1.8 mM. Two-days after starting cell culture, the cells, loaded with fluo-4 AM, were challenged to 150 mM K⁺ solution containing 1.5 mM Ca²⁺. Fluo-4 fluorescence was detected with a Nipkow-type confocal microscopy system. About 70% of the cells, cultured in 1.8 mM Ca²⁺, responded to the high K⁺ solution, exhibiting large fluo-4 Ca²⁺ transients. However, about 30% of the cells cultured in 0.9 mM Ca²⁺ responded to the high-K⁺ solution, showing the smaller amplitude of Ca²⁺ transients. The cells cultured in 1.2 mM Ca²⁺ appeared to show intermediate response. Thus, L-type Ca²⁺ channels may well be involved in the regulation of parathyroid hormone secretion under hypercalcemic and possibly normal conditions. [J Physiol Sci. 2007;57 Suppl:S79]

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Ethanol intake is well known as a predisposing factor for atrial fibrillation. While recent studies identified that abnormail automaticity in pulmonary vein triggers the onset of atrial arrhythmias. However, the underlying mechanism is unknown. To investigate whether low-voltage activated T-type Ca²⁺ channel remodeling is involved in the ethanol insult to myocytes, we performed the whole-cell patch-clamp studies targeted on the Ca_V3.1 and Ca_V3.2 T-type Ca²⁺ channels expressed in the HEK293 cells. Long-time exposure of ethanol (0%, 0.1%, 0.5% and 1%) to HEK293 cells did not modify current density of Ca_V3.1. Meanwhile, the current density of Ca_V3.2 was augmented by ethanol in a dose-dependent manner; 19.8% in 0.1% ethanol, 33.1% in 0.5% ethanol, and 35.5% in 1% ethanol, respectively. These results suggest that augmentation of Ca_V3.2 channel current caused by ethanol exposure may promote the pacemaker potentials, triggering abnormal automaticity in pulmonary vein and/or atrial myocytes. [J Physiol Sci. 2007;57 Suppl:S79]

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Atrial myocytes express α1 subunits of the L-type Ca²⁺ channel, Ca_V1.2 and Ca_V1.3. Ca_V1.3 activates and inactivates at voltages lower than those of CaV1.2 by approximately -15mV. We have previously reported that the replacement of the 2nd transmembrane domain of Ca_V1.2 by that of Ca_V1.3 (CD2) mimicked the negative shift of the steady-state inactivation. In order to elucidate the gating regulatory mechanism of Ca_V1.3, we expressed Ca_V1.2, Ca_V1.3, or Ca_V1.2/Ca_V1.3 chimera in BHK6 cells and examined their kinetics. The voltage-dependent inactivation (VDI) kinetics of Ca_V1.3 was much slower than that of Ca_V1.2. On the other hand, VDI was accelerated in CD2. Interestingly, substitution of Ile/Tyr (Ca_V1.2-type) for Val/His (Ca_V1.3-type) in IIS4 of CD2 slowed the inactivation kinetics. The replacement of C-terminal region of Ca_V1.2 by that of Ca_V1.3 (CTD) slowed the VDI kinetics. These results indicate that : 1) the insertion of Ca_V1.3-derived Val/His in IIS4 (voltage sensor) into Ca_V1.2 somehow accelerates the inactivation, but not in Ca_V1.3, 2) C-terminal region of Ca_V1.3 plays an important role in the slow inactivation kinetics of Ca_V1.3. It has been reported in Ca_V1.3(-/-) mice that action potential (AP) duration of the sinus nodal cells was shorter than that of wt. Our results suggest that Ca_V1.3 continues to be activated during APs, and thus contributes to maintain AP duration in atrial myocytes. [J Physiol Sci. 2007;57 Suppl:S79]

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Orexin-A and orexin-B, also called hypocretin-1 and hypocretin-2, act upon orexin receptor-1 and orexin receptor-2 in the brain, and participate in the regulation of feeding and sleep-wakefulness. Although orexin-containing neurons identified in the lateral hypothalamus project to the pedunculopontine tegmental nucleus (PPT), which is one of brain sites that control sleep-wakefulness, the action of orexins on PPT neurons is not known. Thus, we examined effects of orexins on PPT neurons using rat brain slice preparations and whole-cell patch clamp recording technique. Applications of orexin-A and orexin-B depolarized PPT neurons dose-dependently, and EC₅₀ values were greater for orexin-B than for orexin-A. The depolarization was associated with the increase in membrane resistance. When extracellular K⁺ concentration was increased and/or extracellular Na⁺ was replaced by N-methyl-D-glucamine (NMDG), the magnitude of the depolarization significantly decreased or disappeared. In addition, when extracellular K⁺ concentration was increased and intracellular Ca²⁺ was chelated by BAPTA contained in pipette solution, the magnitude of the depolarization decreased, but not disappeared. These results suggest that orexins affect to K⁺ channel and non-selective cation channel on PPT neurons via orexin receptor-1, and participate in the regulation of sleep-wakefulness via the excitatory effect on PPT neurons. [J Physiol Sci. 2007;57 Suppl:S80]

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Objective Ca²⁺ influx through voltage-dependent Ca²⁺ channels regulates vascular contraction and remodeling. However, the role of T-type Ca²⁺ channels (TCCs) has remained unknown in the ductus arteriosus (DA). Methods and Results 1) α1G, a major isoform of TCC in DA, was significantly up-regulated after birth. α1G was localized in the region of intimal thickening in rat perinatal DA. When the condition of culture media was changed from hypoxia (1% oxygen) to normoxia (21% oxygen), the expression of α1G mRNA was up-regulated by 1.5 fold in DA smooth muscle cells (SMCs), suggesting that the increase in oxygen tension is associated with the up-regulation of α1G mRNA in rat DA. 2) A highly selective TCC blocker, R(-)-efonidipine, significantly attenuated oxygen-induced vasoconstriction by 74% in rat DA at embryonic day 21 (p<0.01). The combination of a L-type Ca²⁺ channel blocker, nitrendipine, and R(-)-efonidipine induced further relaxation of DA, suggesting the additive effect of LCC and TCC. 3) DA SMC migration was increased in an extracellular Ca²⁺ concentration-dependent manner. DA SMC migration and proliferation were significantly decreased by stimulation of R(-)-efonidipine and by inhibition of α1G expression using α1G-specific siRNA. These data suggested that TCC promotes SMC migration and proliferation in rat DA. Conclusion TCC, which was up-regulated upon exposure to oxygen, regulated postnatal oxygen-induced DA closure through vasoconstriction and SMC migration and proliferation. [J Physiol Sci. 2007;57 Suppl:S80]

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Cardiomyocyte is known to hardly swell during the Na/K pump block, although it functions to maintain cell volume. Previously we analyzed the mechanism using a comprehensive mathematical model of cardiomyocyte, the Kyoto model, and proposed that plasma membrane Ca pump, reverse mode of Na/Ca exchanger (NCX) and window current of L-type Ca channel are the key factors responsible for the cardiac cell volume regulation. In the present study, we validated the hypothesis by conducting new experiments measuring [Na]i (monitored by SBFI), cell area and membrane potential (monitored by di-8-ANEPPS), with pharmacological interventions. When an NCX blocker 1 μM SEA0400 was applied to the cardiomyocyte after 60 min treatment with 40 μM ouabain, [Na]i started to increase, indicating the Na extrusion via reverse mode of NCX under the Na/K pump block condition. In addition, this drug caused a significant cell swelling, confirming the proposed mechanism that NCX in reverse mode prevents the cell swelling. When exposed to 1 μM isoproterenol, cardiomyocytes swelled after a delay, which was predicted to be triggered by L-type Ca channel window current. Additionally, application of an L-type Ca channel blocker 5 μM nifedipine eliminated the swelling almost completely. These experimental findings strongly supported the model prediction that the Ca transporters and channels take key roles in the cardiac cell volume regulation under the condition of Na/K pump block. [J Physiol Sci. 2007;57 Suppl:S80]

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We determined the roles of liver and splanchnic vascular bed in anaphylactic hypotension using in vivo and isolated perfused BALB/c mouse liver preparations. An intravenous injection of ovalbumin antigen into the Intact-sensitized mice caused a decrease in systemic arterial pressure (Psa) from 92±2 (SE) to 39±3 mmHg but caused only a slight increase in portal venous pressure (Ppv) from 6.4±0.1 cmH₂O to the peak of 9.9±0.5 cmH₂O at 3.5 min after antigen. The elimination of the splanchnic vascular beds by ligation of the celiac and mesenteric arteries, combined with total hepatectomy attenuated anaphylactic hypotension. The ligation of these arteries alone, but not partial hepatectomy (70%), also attenuated anaphylactic hypotension similarly. In contrast, sensitized mouse liver perfused portally at constant flow did not show anaphylactic venoconstriction, but showed substantial constriction in response to the anaphylaxis-associated substance of platelet-activating factor, indicating that venoconstriction observed in in vivo mice may be induced by mediators released from extrahepatic tissues. These results suggest that splanchnic vascular beds are involved in BALB/c mouse anaphylactic hypotension: They presumably act as sources of chemical mediators to cause the anaphylaxis-induced portal hypertension, which induced splanchnic congestion resulting in a decrease in circulating blood volume and thus systemic arterial hypotension. Mouse hepatic anaphylactic venoconstriction may be induced by factors outside the liver, but not by anaphylactic reaction within the liver. [J Physiol Sci. 2007;57 Suppl:S81]

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The effects of colorectal distension on heart rate (HR) and mean arterial blood pressure (MAP) were examined in anesthetized rats. A 2 cm balloon was inserted into the colorectum through the anus; the tip of the balloon was placed 6 cm proximally to the anal verge. The balloon was distended by inflating it with air using a syringe, and intra-balloon pressures of 40, 60 or 80 mmHg were applied for 20 s. HR and MAP were decreased by colorectal distension when intra-balloon pressures went up to 60 and 80 mmHg, but not 40 mmHg. The decreases in HR and MAP to colorectal distension were accompanied by decreases in the activities of the cardiac sympathetic efferent nerve and the renal sympathetic nerve which are indexes of vasoconstrictor. In acutely spinalized rats at the C2 level, the depressor response elicited by noxious colorectal distension was reversed to pressor response which was accompanied by an increase in the renal sympathetic nerve activity, while the response of HR was abolished. We concluded that noxious colorectal distension elicits 1) reflex decreases in HR and MAP via depression of sympathetic efferent nerve activity, and its reflex center requires the presence of brain; 2) reflex increase in MAP via excitation of sympathetic efferent nerve activity in spinalized rats. When central nervous system is intact, this spinally induced pressor response to noxious colorectal distension appears to be suppressed by inhibitory pathways from the brain. [J Physiol Sci. 2007;57 Suppl:S81]

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We have studied arterial blood supply to exercising skeletal muscle and venous return from the muscle in humans. Voluntary exercise were performed for 1 min with 30% of the maximal voluntary contraction in the supine [static or intermittent handgrip exercise]. Each one also experienced respiratory interventions (deep breath and forced inspiratory or expiratory apnea). The B-mode image and blood flow of brachial artery or vein were recorded with Doppler flowmetry. The distinct differences in blood flow response to exercise were found between the brachial artery and vein, although the overall response was the same each other. First, venous blood flow showed stronger respiratory modulation and less pulsatile modulation than arterial blood flow. Second, venous blood flow increased at the onset of exercise, whereas arterial blood flow often decreased. Third, venous blood flow increased at the contraction phase of intermittent exercise, whereas arterial blood flow decreased. On the contrary, venous blood flow decreased at the relaxation phase of intermittent exercise, whereas arterial blood flow increased. The respiratory interventions strongly influenced brachial venous blood flow than arterial blood flow. Particularly, a transient inspiratory movement caused a large increase in venous blood flow but not arterial blood flow. Thus these findings reveal that voluntary exercise and respiration movement cause the differential responses in brachial venous and arterial blood flow in humans. [J Physiol Sci. 2007;57 Suppl:S81]

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Background: Although sympathetic overactivity and vagal withdrawal aggravate heart diseases, the mechanism of the vagal withdrawal is not fully understood. We hypothesized that angiotensin II (ANGII) would attenuate the acetylcholine (ACh) release in the ventricle. Methods: We implanted a dialysis fiber into the left ventricular free wall and measured the ACh concentration in the dialysate during bilateral vagal stimulation (1 ms, 10 Hz, 10V) in anesthetized cats. Effects of intravenous ANGII at 10 μg/kg/h were examined (n=6). To examine the role of ANGII type 1 (AT1) receptors, an AT1 antagonist losartan was administered intravenously (10 mg/kg bolus, n=5) or locally through the dialysis probe (10 mM, n=6). Results: Vagal stimulation increased ACh from 0.85±0.08 to 10.7±2.4 nM (mean±SD). ANGII suppressed the ACh release to 7.5±1.4 (P<0.01). Local administration of losartan did not abolish the suppressive effect of ANGII on the stimulation-induced ACh release (from 8.0±2.0 to 5.8±2.4 nM, P<0.01) whereas the intravenous administration did (from 7.5±3.0 to 7.4±3.6 nM). Conclusion: ANGII suppressed the vagal stimulation-induced ACh release in the left ventricle. AT1 receptors at the postganglionic vagal nerve terminals were not involved in the site of this action. ANGII might act on the parasympathetic ganglia, thereby reducing the postganglionic vagal nerve activity and also contributing to the aggravation of heart diseases. [J Physiol Sci. 2007;57 Suppl:S81]

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Backgrounds: Recent studies have reported carotid (CA) lumen dilation as a risk factor for cardiovascular disease. However, the relationship of CA hemodynamic status and elastic properties to CA enlargement has not been investigated systematically in humans. Methods and results: We recorded CA pressure and axial flow velocity waveforms simultaneously using phase-locked echo tracking method and continuous Doppler method in 56 healthy volunteers (age: 20-72 years). Diastolic lumen diameter (DD) of the common carotid artery (CCA) significantly increased with age (DD = 0.024X age+5.29, r=0.439, P=0.0007). DD also significantly correlated with carotid flow volume (CAFV, r=0.754), CA distensibility (r=-0.45), body surface area (BSA, r=0.371), CA pulse pressure (PP, r=0.352), CA systolic blood pressure (SBP, r=0.333), CA augmentation index (r=0.320), brachial(BA) SBP (r=0.289), BA PP (r=0.266). According to multivariate regression analysis, age, CAFV, and BSA were independent determinants of DD (model 1; R²=0.688, p<0.0001). R² was similar even after excluding age (model 2; R² =0.686, p<0.0001). Independent determinants were CAFV, BSA, CA SBP, and heart rate (HR). With aging, CA SBP and HR increased significantly, but CAFV and BSA did not change. Conclusions: DD in the CCA was a multi-factorial parameter. Increased CAFV, BSA, CA SBP, and HR were key contributors to CA lumen dilation, but only CA SBP and HR seemed to be associated with age-related changes. Results of the present study should aid the interpretation of findings in large-scale prospective studies. [J Physiol Sci. 2007;57 Suppl:S82]

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Background: Heart rate (HR) is one of the determinant factors of cardiac performance. However, there is no direct evidence for the effect of heart rate on the actin-myosin interaction (AMI). Purpose: To test the effect of HR on AMI in hearts with X-ray diffraction (XRD) analysis using SPring-8. Methods: Seven isolated isovolumically contracting rat hearts were mounted so that the X-ray beam (15.0 keV) passed the subepicardial region. We recorded XRD images and LV pressure at HRs of 120 and 300bpm under 0 mmHg end-diastolic pressure. Between two different HRs, we compared 1) the amount and duration of AMI from the intensity ratio reduction of inner (1,0) and outer (1,1) XRD, and 2) myosin-filament lattice spacing (MLS) from the position of inner XRD. Results: Increasing HR from 120 to 300bpm significantly decreased the intensity ratio reduction at the end-systole (1.05±0.15 vs. 1.70±0.31 unitless, p<0.01) and shortened duration of AMI (128±12 vs. 91±18ms, p<0.01), suggesting a smaller amount of AMI, but did not change MLS (35.4±0.5 vs. 35.3±0.5nm, NS). Conclusion: Increasing HR reduces the AMI without causing incomplete relaxation, indicating intact intracellular Ca²⁺handling. These results may derive from shortening of Ca²⁺-myofilament interaction. [J Physiol Sci. 2007;57 Suppl:S82]

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We have reported that a thermosensitive-TRP channel, TRPV4 (activated by >35°C heat) is expressed in hippocampal neurons, and enhances the neural activities through its activation at body temperature. We also observed the TRPV4 mRNA expression in astrocytes. We hypothesized that TRPV4 might be an important ion channel to regulate the excitability of astrocytes. We prepared the cultured astrocytes, and examined the cellular distribution of TRPV4. TRPV4 mRNA and protein were observed in only subsets of the astrocytes, indicating that we can classify the characteristics of astrocytes dependings on the TRPV4 expression. We also examined whether functional-TRPV4 was expressed in the astrocytes by a Ca²⁺ imaging method with Fura2. The subsets of astrocytes responded to the all reported stimulus (heat, hypotonicity and 4αPDD, which is a TRPV4 specific agonist). Following the increased calcium level in only subsets of astrocytes, significant calcium oscillations were observed in among most of the astrocytes, indicating that communications among astrocytes were initiated through the TRPV4 activations. We hypothesized that the calcium oscillations among astrocytes might be a critical determinant to regulate the neural activities. Therefore, we examined how astrocytes communicate each other. Upon activation of TRPV4 by 4αPDD in astrocytes, we used a biosensor method, in which the HEK293 cells expressing various neurotransmitter receptors were used to detect the neurotransmitter release using a whole-cell patch-clamp method. [J Physiol Sci. 2007;57 Suppl:S82]

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Small molecule G-protein Arfs and its effector enzyme phospholipase D (PLD) are essential regulator for intracellular vesicular trafficking from endoplasmic reticulum to Golgi apparatus. We previously reported that intracellular application of brefeldin A, Exo1, and N-terminal peptide of Arf1, Arf1 inhibitors, depressed K⁺-current response induced by dopamine (DA) in Aplysia neurons. It is recently known that, Arf also play a role in regulation of recycling of several receptors at the cytoplasmic membrane in association with PLD. Therefore, we examined a possible involvement of the receptor endocytosis and recycling in regulation of the DA induced response. Intracellular application of α-synuclein, an inhibitor of PLD, significantly depressed the K⁺-current response to DA. Furthermore, intracellular application of dynamin inhibitory peptide, which inhibits the endocytosis of receptors, gradually augmented the DA induced K⁺-current response. In contrast, extracellular application of monensine, which inhibits the recycling of endocytosed receptors to the cytoplasmic membrane, gradually suppressed the DA induced K⁺-current response. These results suggest that Arf1 and PLD regulate the DA-induced K⁺-current response through recycling of the DA receptor to the cytoplasmic membrane. [J Physiol Sci. 2007;57 Suppl:S83]

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We previously showed the rat corticospinal (CS) synapses were formed all the gray matter at P7; but the synapses in the ventrolateral (VL) area were eliminated from P8 to P10 and CS synapses were distributed predominantly in the dorsomedial (DM) area. This was accompanied by CS axonal regression from the VL area, whereas axons were almost unchanged in the DM area. However, our subsequent study with anterograde labeling of CS axons showed that after this synapse elimination, the " second wave " of the CS innervation arrived at P12: CS terminals again increase in the ventral and also dorsal side. In this study, we investigated the development of CS synaptic responses directly by optical imaging with the voltage sensitive dye. The ventralmost part of posterior funiculus of the spinal cord slice was stimulated to activate CS tract axons and the response signal of the membrane potential change was recorded, which is referred to as optically detected EPSP (optEPSP). The optEPSP intensity in the VL area once peaked at P6–7, but later reduced at P10–P11. This suggests the early synapse formation and succeeding elimination in the VL area. However, the optEPSP intensity in the VL area increased again from P13 and continued to increase until P18. The optEPSP intensity in the DM area was fairly constant for P6–P11, and also increased from P13. Those findings indicate that the second wave of synapse formation surges both in the DM and VL area after elimination of synapses in the VL area formed by the first wave. [J Physiol Sci. 2007;57 Suppl:S83]

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Syntabulin, interacting with kinesin-1, transports syntaxin-1 and mitochondria in the axon to the presynaptic terminal. To examine functional significance of syntabulin in presynaptic neuron, synaptic transmission was examined at a cholinergic model synapse formed between rat sympathetic ganglion neurons in culture. Knock-down of syntabulin expression with targeted small interfering RNAs (siRNAs) significantly reduced the incidence of synaptic coupling, measured by recording excitatory postsynaptic potentials (EPSPs), on 10, 17 and 24 days in culture. This finding suggests that syntabulin plays an important role in the synapse development. Furthermore, in long-term cultured neurons, deficit of syntabulin in presynaptic neurons reduced the amplitude of EPSPs (57%) in comparison with that in control synapses transfeted with a scrambled RNA. In general, the synaptic transmission reduces with repetitive stimulations. Syntabulin knock-down accelerates the reduction of synaptic transmission with high frequency stimulation at 1 Hz, but not with low frequency stimulation at 0.3 or 0.1 Hz. In addition, the recovery of EPSP amplitude after depletion of synaptic vesicles in the presynaptic terminal with a train stimulation at 5 Hz for 4 min remarkably slows down in the syntabulin impaired synapse. These findings suggest that syntabulin functions in a mature synapse to maintain synaptic transmission. [J Physiol Sci. 2007;57 Suppl:S83]

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At calyces of Held of immature rats, Ca²⁺- and calmodulin (CaM)-dependent inactivation of presynaptic Ca²⁺ currents (I_pCa) is proposed to underlie synaptic depression. At postnatal day (P) 7-9, I_pCa evoked by a 1-ms depolarizing pulse showed a prominent paired-pulse inactivation of Ca²⁺ currents (PIC) at 0.1-2 s inter-stimulus intervals, and CaM inhibitors attenuated PIC and short-term synaptic depression as reported. However, at P13-15, PIC became very small and CaM inhibitors no longer affected PIC or synaptic depression. In contrast, at P13-15, a CaM inhibitor still attenuated I_pCa inactivation during a long pulse (2 s) depolarization, to a similar extent as at P7-9. Consistently, CaM immuno-reactivity at P15 calyces was indistinguishable from that at P7. These results suggest that the residual intra-terminal Ca²⁺ concentration following a brief depolarizing pulse or an action potential is insufficient for CaM to inactivate I_pCa at P13-15. Whole-cell dialysis of P13-15 calyces with a pipette solution containing no Ca²⁺ buffer had no effect on PIC, whereas loading 10-mM EGTA into P7-8 calyces blocked PIC. We conclude that spatial segregation of Ca²⁺ channels in the nerve terminal, rather than an increase in endogenous Ca²⁺ buffer proteins, may underlie the developmental decline of CaM-dependent I_pCa inactivation. [J Physiol Sci. 2007;57 Suppl:S83]

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Presynaptic ryanodine-sensitive Ca²⁺ store has been suggested to be involved in long-term potentiation (LTP) at the hippocampal mossy fiber synapses. However, the mode of activation of presynaptic ryanodine receptors remains mostly unknown. In this study, the conditions to activate presynaptic Ca²⁺ store at the mossy fiber synapse were examined using presynaptic Ca²⁺ measurements in acute hippocampal slice preparations. Presynaptic Ca²⁺ transients evoked by single shocks to the mossy fibers were not affected significantly by application of the ryanodine receptor blocker TMB-8 (100 μM). In contrast, presynaptic Ca²⁺ transients elicited by train stimuli (20 Hz, 10 times) were partially suppressed by TMB-8, although the responses to the first stimulus were almost unaffected. TMB-8 also suppressed the latter phase, but not the initial phase, of presynaptic Ca²⁺ transients elicited by tetanic stimulation (100 Hz, 100 times) of the mossy fibers. These findings suggested that the presynaptic ryanodine receptors release Ca²⁺ from intracellular stores in a use-dependent manner, and thereby amplify unique presynaptic long-term synaptic plasticity at the hippocampal mossy fiber synapses. [J Physiol Sci. 2007;57 Suppl:S84]

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The glial glutamate transporter GLT-1 has a crucial role in preventing the glutamate-excitotoxic processes. Neurosteroid dehydroepiandrosterone (DHEA) is also supposed to exert a potential neuroprotective effect against the glutamate-exitotoxicity, however, the relationship between GLT-1 and DHEA is unknown. Using a real-time optical recording technique, the present study aimed to examine the effects of DHEA on the activity of GLT-1 in the rat hippocampal dentate gurus slices stained with the voltage-sensitive dye RH155. We found that an application of DHEA to the slices increased rapidly and dose-dependently the synaptically induced glial depolarization (SIGD), an indicative of GLT-1 activity. As DHEA had little effect on presynaptic glutamate releases, the DHEA-induced SIGD increase should not be a secondary effect of DHEA on the glutamate releasing machinery. The DHEA-increased SIGD was largely reduced by the inhibitors for sigma-1 receptor and protein kinase C, but not by those for Dopamine-1receptor and protein kinase A, suggesting that DHEA prevents the glutamate-excitotoxicity by a sigma-1 receptor/PKC-mediated up-regulation of GLT-1 activity. [J Physiol Sci. 2007;57 Suppl:S84]

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AMPA-type glutamate receptor (AMPA-R) is recycling rapidly at the parallel fiber synapse in cerebellar Purkinje cell (PC). Blocking of constitutive exocytosis of AMPA-R by intracellular application of tetanus toxin (TeTx) reveals constitutive endocytosis of AMPA-R, which is independent of AMPA-R activity. Constitutive endocytosis of AMPA-R does not require elevation of intracellular Ca²⁺ concentration, and it is suppressed by concomitant inhibition of protein kinase C (PKC) and MAP kinase (ERK1/2). The maximum suppression level of PF-EPSC by TeTx is similar to that of botulinum toxin C1 (BoTx C). Thus, toxin resistant pool of AMPA-R is suggested to be a stable pool of the synaptic AMPA-R. LTD is induced even at the maximum reduction of PF-EPSC by TeTx, suggesting that LTD involves the process of destabilization of AMPA-R. Here, we examined the effects of actin-polymerization on constitutive and activity-dependent AMPAR trafficking. Actually, jasplakinolide, a blocker of actin-depolymerization, suppress both constitutive and activity dependent endocytosis of AMPA-R. Since toxin B inhibits also AMPA-R trafficking, mediation through Rho/Rac/Cdc42 is demonstrated in actin cytoskeleton regulation relating to AMPA-R trafficking. Immunocytochemical analysis shows that jasplakinolide suppress internalization of GluR2 in cultured PC. Thus, involvement of actin cytoskeleton is suggested in both constitutive and activity dependent AMPA-R trafficking at PF-synapse in cerebellar PC. [J Physiol Sci. 2007;57 Suppl:S84]

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Recent data indicate the existence of accessory proteins that directly regulate the activation status of heterotrimeric G-protein, which may serve additional regulatory points for cellular events. We asked if they were involved in an adaptation of myocardium to ischemia. A cDNA library was generated from rat hearts subjected to repetitive transient ischemia that was induced by inflation of an implanted balloon around the coronary artery. Utilizing a functional screen in yeast for G-protein activators, we isolated a novel G-protein activator (AGS8). AGS8 mRNA was induced in response to ventricular ischemia but not by tachycardia, hypertrophy or cardiac failure. Hypoxia induced AGS8 mRNA in isolated adult ventricular cardiomyocytes but not other cell types suggesting a myocyte specific adaptation mechanism involving remodeling of G-protein signaling pathways. Subsequent studies indicated that AGS8 interacts directly with Gβγ and this occurs in a manner that apparently does not alter the regulation of the effector PLC-β2 by Gβγ. These data indicate that AGS8 provides an alternative signal input to G-proteins independent of receptors. Such mechanisms may provide an important role in the adaptation of the myocardium to ischemia. [J Physiol Sci. 2007;57 Suppl:S85]

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The proximal tubule reabsorbs about 70% of the filtered Na⁺ load. The K⁺ channels in proximal tubule cells contribute to this process through the formation of the membrane potential, which serves as a driving force for electrogenic passive transport of Na⁺. In cultured human proximal tubule cells, an inwardly rectifying K⁺ channel with an inward conductance of 40 pS is the most frequently observed K⁺ channel under the control condition. We previously reported that nitric oxide (NO) possessed a dose-dependent biphasic effect on this K⁺ channel, i.e. a cGMP/PKG-dependent stimulatory effect at low doses and a nitrosylation-dependent suppressive effect at high doses. We also confirmed that interferon-γ (IFN) chronically suppressed the K⁺ channel activity by enhancing expression of inducible NO synthase (iNOS). In this study, we examined whether IFN would acutely affect the activity of the 40 pS K⁺ channel, using the patch-clamp technique. In cell-attached patches, IFN (20 ng/ml) added to the bath increased channel activity within 1-3 min. IFN had no effect on channel activity in inside-out patches. The acute stimulatory effect of IFN was not abolished by a NOS inhibitor, L-NAME (100 μM), an inhibitor of soluble guanylate cyclase, ODQ (10 μM), or a PKG inhibitor, KT5823 (1 μM). On the other hand, a tyrosine kinase inhibitor, genistein (100 μM), reduced the stimulatory effect of IFN. These results suggested that IFN acutely stimulated K⁺ channel activity through activation of tyrosine kinases, but not through the NO/cGMP/PKG pathway. [J Physiol Sci. 2007;57 Suppl:S85]

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S1P is a bioactive lysophospholipid that induces a variety of biological responses in diverse cell types. We previously showed that S1P₂ receptor activates Rho, which in turn induces inhibition of Rac and cell migration in S1P₂-expressing CHO cells. Phosphoinositide 3-kinases (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, leading to stimulation of cell migration and cell survival. Recently, it has been shown that Rho and Rho-kinase stimulates PTEN, which mediates inhibition of Akt and cell migration in CHO and B16 cells. These observations led us to investigate the role of PTEN in S1P₂-mediated inhibition of cell migration and Rac. IGF I stimulated Akt and chemotaxis in the PI3K inhibitor-sensitive manner. Interestingly, S1P partially inhibited IGF I-induced Akt activation and completely inhibited IGF I-induced chemotaxis and Rac activation in S1P₂ overexpressing CHO cells. Either the dominant negative mutant of PTEN, the PTEN-specific siRNA or the Rho-kinase inhibitor Y27632 prevented S1P-inhibition of Akt activity, indicating that Rho kinase-PTEN mediates inhibition of Akt. However, either of these treatments failed to abrogate S1P-inhibition of chemotaxis and Rac. These data indicate that S1P₂-mediated inhibition of either Rac or migration is not dependent on the tumor suppressor PTEN in these three cell types. [J Physiol Sci. 2007;57 Suppl:S85]

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Rho-kinase (ROK)-mediated Ca²⁺sensitization of vascular smooth muscle (VSM) plays a critical role in abnormal VSM contraction such as vasospasm. We previously found that sphingosylphosphorylcholine (SPC) induced ROK-mediated Ca²⁺sensitization of VSM through the activation of Fyn, and eicosapentaenoic acid (EPA) selectively inhibited the Ca²⁺sensitization by blocking the activation of Fyn. In this study, we examined the inhibitory mechanism of EPA on the SPC-induced Ca²⁺sensitization. SPC induced VSM contraction with no elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_i), and EPA inhibited the SPC-induced contraction without affecting the high-K-depolarization-induced contraction and [Ca²⁺]_i elevation. In cultured VSM cells, SPC induced the translocations of Fyn and ROK to plasma membrane, where they were colocalized with membrane raft markers, caveolin-1 and cholera toxin subunit B. EPA inhibited the SPC-induced translocation of Fyn and ROK without affecting the localization of caveolin-1, whereas β-cyclodextrin, which selectively deprived membrane cholesterol and thereby removed caveolin-1 from plasma membrane, inhibited the SPC-induced translocation of Fyn and ROK and contraction. Those findings suggest that membrane rafts plays a pivotal role in the Ca²⁺sensitization mediated by SPC/Fyn/ROK pathway, and EPA blocks this pathway without affecting membrane raft property. [J Physiol Sci. 2007;57 Suppl:S86]

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The effect of etxtracellular ATP on cell migration was investigated using 3T3-L1 cell line. In control preadipocytes, staining of actin filaments with phalloidin revealed a network of actin stress fibers running through the cytoplasm constructing a cellular frame as is typically observed in fibroblasts. After the cells were incubated with 10 μM ATP for 5 min at 37^oC, actin filament structures underwent a significant change in distribution mediated through P2Y receptors, forming a peripheral meshwork of broad, sheet-like structure (lamellipodia) and surface projections of thin, needle-like structures (filopodia), generally found at the plasma membrane of migrating cells. We then examined whether purinergic stimulation enhances the migration of the cells using chemotaxis assay chamber or chemokinesis assay filter system. Preadipocytes were found to migrate towards the source of ATP (chemotaxis) with the maximal migration of 80 μm within 60 min at 37^oC. Random cell migration (chemokinesis) was also observed with a smaller magnitude. In contrast, mature adipocytes tightly adhered to the extracellular matrix and appeared to be almost immobilized during the incubation. These results indicate that the extracellular ATP acts as a chemoattractant for preadipocytes, but not for mature adipocytes. [J Physiol Sci. 2007;57 Suppl:S86]

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