Songs of passerine birds (like zebra finch) are learned motor behavior which used by males to attract females and to protect territories. Generally, bird songs are consist of several different song notes (elements), and these notes are produced in a fixed temporal order. Among the passerine birds, male Bengalese finches (BF) sing complex song which follows finite state syntax. The song control system of BF consists of a set of discrete nuclei including the HVC, RA and Nif. Previous lesion study (Hosino and Okanoya 2000) showed that Nif lesioned BFs sung simpler songs, with less phrases to phrases branching than that of prelesion birds. This finding suggests that Nif-HVC connection plays important role in generating song grammar in BFs. In this study, we perfused Nif with muscimol (GABA agonist) for 30 minutes via microdialysis probes as a perturbation on Nif-HVC system. Following a local administration of muscimol into Nif, song grammar of BF was modified. Some of chunks in their finite state grammar disappeared and pronounced elongation of introductorily notes' duration was observed in first 3 hours. In addition, we also recorded stuttering like repetitions of song notes. Nif is also known as one of auditory relay nucleus to HVC. Some of drug effects, therefore, are possibly caused by disruption of auditory feedback. Further detailed studies are needed to know function of Nif-HVC connection in relating to generate song grammar. [J Physiol Sci. 2006;56 Suppl:S98]

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To study the ontogenic change in stress susceptibility during postnatal brain development, we have previously examined the ontogenic changes of Fos expression in the paraventricular nucleus of the hypothalamus (PVN) after intraperitoneal (i.p.) injection of cinchophen. We have obtained evidence that Fos was induced by cinchophen in PVN after postnatal day (P)10. Such differential responses to stress have led us hypothesize that such stressful stimuli at various time point during postnatal development may differentially affect motor behavior in adult rat. To test this hypothesis, we applied an open field test and a rotarod test using rats that received i.p. injection of cinchophen on P5, P7, P10 and P15. There were no significant differences of body weight and muscle strength between them. In open field test, female rats that received cinchophen injection on P10 tended to stay longer in the center of the field at P30, suggesting that they showed anxiety to a novel environment. In rotarod test, there was no significant difference among all groups. These results indicate that postnatal stress by cinchophen may affect in part their motor behavior related to anxiety in adult rat. [J Physiol Sci. 2006;56 Suppl:S98]

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Secretion of HCO₃⁻ at the apical side of the epithelial cells of the choroid plexus is an essential step in the formation of cerebrospinal fluid (CSF). Anion conductance with a high degree of HCO₃⁻ permeability has been observed and suggested to be the major pathway for HCO₃⁻ transport across the apical membrane, but the molecular entity remains unknown. We identified the first molecular entity of apical choroid plexus HCO₃⁻ transport, a novel variant of NBC4 (NBC4g). Electrophysiological studies and pH-recovery assay showed that NBC4g has the electrogenic, DIDS-sensitive, and cAMP-dependent HCO₃⁻ transport activity. Furthermore, the contribution of NBC4g to choroid plexus HCO₃⁻ transport was demonstrated by RNAi-mediated knockdown of NBC4g using primary cultured cells; treatment with siRNA of NBC4g led to a similar degree of reduction in the transport activity as that observed by treatment with DIDS. Thus, our data strongly indicate that NBC4g is the long-sought transporter responsible for the HCO₃⁻ secretion from the choroid plexus into the ventricle thereby controlling the H⁺ buffering and pH of the CSF. [J Physiol Sci. 2006;56 Suppl:S98]

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Polychrolinated biphenyls (PCBs) and dioxins (PCDD, PCDF, coplaner-PCB) are the environmental chemicals that may affect the growth and homeostasis of many organs. WHO determined toxic index of dioxins as toxicity equivalent factor (TEF). However, we reported previously that the most toxic dioxin (TCDD) did not affect on thyroid hormone (TH) receptor (TR)-mediated transcription. On the other hand, a hydroxylated-PCB5005 whose TEF was almost negligible, strongly suppressed the transcription. In this study, we performed a reporter gene assay with several PCBs and dioxins, and observed opposite tendency from TEF: many PCBs with little TEF showed a greater effect than dioxins. Furthermore the magnitude of suppression by PCBs and PCDF in neuroblastoma derived cell line was greater than that in fibroblast derived cell line. To analyze further the effect of these chemicals in TR-TH response element (TRE) binding, electrophoretic mobility shift assay (EMSA) was performed. PCB congeners that suppressed TR-mediated transcription dissociated TR from TRE, indicating that PCB action is exerted through this mechanism.These results suggest that TEF of dioxins and PCBs do not always correctly indicate their toxicity. The suppression of TR-mediated transcription by PCBs and dioxins should be incorporated to construct a novel index of those chemicals. [J Physiol Sci. 2006;56 Suppl:S99]

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The anteroventral third ventricular region (AV3V), a pivotal area for autonomic functions, contains synaptic boutons with Glu and three subtypes of glutamate receptors (Glu-Rs). Although our previous data suggest involvements of NMDA- and metabotropic Rs in ADH release triggered by several stimuli, roles of AMPA/kinate (non NMDA)-Rs related to Na⁺ channels in hormone release and other functions have not been examined as yet. This study aimed to elucidate the issue through experiments in conscious rats with indwelling cerebral and vascular cannulae. Infusion sites in the brain were identified histologically after experiments. Topical AV3V infusion with a non NMDA-R agonist FWD augmented plasma ADH (pADH), glucose (Glc) and osmolality (Osm), and enhanced arterial pressure (AP) in a dose-dependent manner. All the responses of these variables were blocked by pre-administering NBQX, a selective non NMDA-R antagonist. When NBQX was applied to the AV3V structures such as the median preoptic nucleus, rises of pADH in response to systemic load of hypertonic saline or normo- or hypotensive bleeding were inhibited remarkably. The increases of AP, Osm or Na⁺ provoked by the osmotic load and the responses of plasma angiotensin II, Osm, Glc or AP caused by the bleeding were not affected significantly. These results suggest that AV3V non NMDA-Rs, as well as NMDA-Rs, may contribute to both the hyperosmotic and hypovolemic ADH secretion. [J Physiol Sci. 2006;56 Suppl:S99]

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Platelet-activating factor (PAF) is a highly potent stimulator of the secretion of cortisol, corticosterone, and aldosterone. We previously reported that PAF acts mainly through PAF receptor accompanied by the activation of protein kinase (PK) C. While, ACTH acts through ACTH receptor accompanied by the activation of PK A. In the present study, we studied the cross-talk in adrenal steroidogenesis among PAF, ACTH, and angiotensin II (ANG II). 1) The administration of 1nM PAF or 10pg/ml ACTH significantly stimulated cortisol secretion. The rate of secretion peaked 2.5-5 or 10-12.5min after infusion of PAF or ACTH. When concurrently applied 1nM PAF with 10pg/ml ACTH evoked cortisol secretion less than additional and peaked 5-10min. 2) Aldosterone secretion in response to ANG II significantly stimulated at 1nM and peaked 15-20min after the infusion of ANG II. The administration of 10nM PAF did not induce significant aldosterone secretion. The concurrent administration of 1nM ANG II with 10nM PAF significantly inhibited the secretion of aldosterone to ANG II. 3) Aldosterone secretion in response to ACTH significantly increased at 1ng/ml and peaked 15-20min after the infusion of ACTH. The concurrent administration of 1ng/ml ACTH with 10nM PAF significantly evoked aldosterone secretion almost additional. The rate of secretion peaked 0-2.5min after infusion of the mixture. These results implicate that a cross-talk between the PK A system and the PK C system regulates the cortisol and aldosterone secretion. [J Physiol Sci. 2006;56 Suppl:S99]

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Ghrelin is an acylated brain-gut hormone secreted primarily from stomach. The major active form of ghrelin is a 28-amino acid peptide with an n-octanoyl (C8) modification at Ser3 residue. There also exist other acylated forms of ghrelin, such as n-decanoyl (C10) ghrelin. The previous study demonstrated that intravenous administration of C8-ghrelin increases appetite and food consumption in rodents. To elucidate effects of acyl-modified ghrelins on appetite, we have therefore measured concentrations of acylated ghrelins in stomach and plasma of control and fasted mice. In control, the C8-ghrelin concentration in stomach was higher than the C10-ghrelin. In 48h-fasted mice, the C8-ghrelin significantly decreased whereas the C10-ghrelin significantly increased. Consequently, the C10-ghrelin was greater than the C8-ghrelin in plasma of 48h-fasted mice. Intraperitoneal (ip) injection and intracerebroventricular (icv) injection of C8- or C10-ghrelin significantly facilitated the food consumption 2h after the injection. Ip injection of either C8- or C10-ghrelin had a similar effect. On the other hand, 2h after icv injection of C8-ghrelin, the food consumption was greater than that 2h after icv injection of C10-ghrelin. These results suggest that the condition of energy metabolism influences the acyl-modification of ghrelin, and C10-ghrelin has a site-dependent activity for the stimulation of appetite in mice. [J Physiol Sci. 2006;56 Suppl:S100]

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Objective- A qualitative descriptive study was conducted to determine the religious practices and beliefs related to menarche and menstruation among middle aged women and adolescent girls in the district of Colombo within Buddhist, Hindu, Islamic and Roman Catholic religions.Method-Information was gathered from the middle aged women and the adolescent girls by focus group discussion and by a self-administered questionnaire respectively. Pertinent religious scripture were identified through key informants of each religion. Results-The religious taboos observed during menstruation included not participating in religious activities, avoidance of sex and cooking. Restricted religious activities were observed among middle aged women of Buddhist, Hindu and Islam religions. Buddhist adolescent girls have given up this practice. Avoidance of sexual intercourse during menstruation was observed in all four religions. Hindu's observed unique practices such as restriction of water during menarche and reduced household activities during menstruation. Hindu and Islam women follow the scriptures strictly whereas the Buddhist practices seem to be influenced by the Hindu culture. Except Roman Catholics, others observe restrictions during menarche although there are no statements in scriptures of the four religions studied.Conclusion-Unsafe practices are still continuing among women and adolescents despite statements in religious scriptures. [J Physiol Sci. 2006;56 Suppl:S100]

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Although it is known that drebrin is expressed in the rostral migratory stream (RMS), a neurogenesis region in adult rat brain, the isoform of drebrin is unknown. In this study, a drebrin A specific antibody (DAS2) and another specific antibody (M2F6) recognizing drebrin E and A were used to identify the drebrin isoform that is specifically expressed in migrating neurons. To investigate the immunocytochemical characteristics of drebrin-positive cells, we carried out double-labeling with M2F6 and an antibody of PSA-NCAM, GFAP, or Ki-67. In addition, we performed double labeling of staining of RMS with DAS2 and M2F6 to identify the drebrin isoform. We also analyzed adult rat brains whose unilateral olfactory bulb (OB) had been removed. The migrating cells in subventricular zone of RMS were strongly stained with M2F6, but not with DAS2. This indicates that drebrin E but not A is expressed in these cells. These packed, bi-polar cells expressed PSA-NCAM but not GFAP. Some of them had been undergoing proliferation because their nuclei expressed Ki-67. These findings suggest that M2F6-positive and DAS2-negative cells are migrating neuronal precursors. Unilateral olfactory bulbectomy significantly increased the total area of M2F6-positive and DAS2-negative cells in the ipsilateral RMS comparing with the contralateral side, which is consistent with the previous report about the increase of migrating neuronal precursors by the bulbectomy [J Physiol Sci. 2006;56 Suppl:S101]

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Oral opening of multicellular organisms is generally formed at the anterior end of the animal. Phylum Cnidaria is so far the only exception where oral opening is formed at the posterior end of the animal expressing Wnt-3a homologues (1), a typical gene which is expressed specifically at the posterior end. Why this opposite oral-aboral polarity relative to A-P polarity appeared only in this phylum remains unknown. Expression of Hox-1 homologues at the oral end has been proposed as evidence that oral end represents anterior end even in Cnidaria (2). Here we report that the aboral end of Hydra bears several similarities to the oral end of higher organisms. [J Physiol Sci. 2006;56 Suppl:S101]

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Purpose: We attempt to classify the retinal proteins displayed on two-dimensional (2-D) gels based on their time-dependent expression patterns, which we designate "proteomic trajectory ", along the postnatal developmental axis. Methods: Retinas of C57/B6 mice were collected at postnatal (P) days 1, 3, 5, 7, 9, 14, 21 and at the adult stage. After separating the proteins by 2-D gel electrophoresis, the gel images were analyzed by Progenesis workstation. Protein spots were quantified and normalized, and the proteomic trajectory along the developmental axis was obtained. The results were averaged from four independent experiments. The proteomic trajectories were clustered by self-organizing mapping (SOM) using GeneCluster2. For protein identification each spot was excised and subjected to in-gel digestion by trypsin, followed by peptide mass fingerprinting (PMF). PMF search and confirmation were performed by MASCOT. Results and Conclusions: We identified ca 400 proteins. These proteins can be clustered by SOM into four major types, each exhibiting characteristic proteomic trajectory: Juvenile-type, showing abundant expression in the early postnatal stages and declining along the maturation process; Transient-type, showing transient expression during the development; Adult-type, showing increased expression in the later stages of development toward maturation; and Constitutive-Type, being expressed relatively constant during the entire developmental stages. [J Physiol Sci. 2006;56 Suppl:S101]

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The genetic background and key gene for congenital scoliosis has not yet been clarified. Ishibashi rats (IS) have congenital malformations of the lumbar vertebrae leading to kyphoscoliosis similar to that seen in human. This study investigated characteristics and gene expression of IS to provide insights into human congenital scoliosis.To characterize skeletal malformations of lumbar vertebrae in IS, radiographic and staining studies were performed.Then the gene expression profile of Hox10 and 11 paralogues, whose critical roles in determination of phenotypes of lumbar and sacral vertebrae are well known, between IS and Wistar strain rats by Real Time-PCR was studied.Significant differences on skeletal structures between IS and Wistar were found: (1) transitional vertebrae; (2) anterior wedged vertebra; (3) union of anterior vertebrae; (4) an additional vertebra (7th lumbar vertebra). Especially, transitional vertebra was frequently observed.Staining studies of IS fetuses revealed the fusion of primary ossification centers in the lumbar vertebral column, which was not observed in cervical and thoracic vertebral column.Regarding gene expression of Hox10 and 11 paralogues, the expression of some of these paralogues had low level in lumbar/ sacral region of vertebral column compared with that of Wistar.Our results indicate that Hox10 and 11 paralogues play critical roles in generating vertebrae of IS phenotype. Further work is in progress to elucidate the expression profile of Hox10 and 11 paralogues in the axial skeleton of IS. [J Physiol Sci. 2006;56 Suppl:S102]

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Arousal from hibernation with a rapid increase of the energy metabolism suggests being exposed to a strong oxidation stress whenever the animal awakes, therefore being considered to have an innate anti-oxidation mechanism to prevent pathological troubles. To elucidate the state of the oxidation stress, endogenous anti-oxidants were quantified along the time course of hibernation. Very slow flow (3.5 μL/h) brain microdialysis enabled temperature independent sampling of the brain extracellular fluid (ECF) during hibernation, arousal and cenothermia in Syrian hamsters (Mesocricetus auratus). Brain tissue and dialysates were analyzed to provide the first profile of ECF changes in levels of ascorbic acid (AA), glutathione (GSH) and uric acid (UA) during hibernation and the transition to cenothermia. Brain tissue content of AA and GSH were unchanged between hibernation and cenothermia, however arousal was associated with substantial oxidation of AA from the brain ECF and plasma compartments. ECF-GSH increased during arousal. Brain tissue UA content was decreased 50% during hibernation. ECF-UA levels were unchanged in hibernation and cenothermia, however transiently increased 100% during arousal. The results suggest that arousal from hibernation is a suitable experimental model for examination of the mechanisms by which non-pathological tissue integrity is maintained in the face of the generation of free radicals during increasing metabolism, temperature and cerebral reperfusion. [J Physiol Sci. 2006;56 Suppl:S102]

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Body temperature (T_b) is different between male and female, e.g. daily change in T_b is fluctuated with menstruation cycle in female rats. We hypothesized that estrogen plays a crucial role in the sex difference in T_b. Methods (1) Daily change of T_b was measured after gonadectomy in male and female rats. After the measurement, silicon tubes containing 17-beta estradiol (E₂) crystalline, aimed to maintain blood estrogen constant, were subcutaneously placed in the rats. Then T_b measurement was repeated. (2) Thermoregulation during 2-h heat exposure at 34°C or cold exposure at 5°C was assessed in gonadectomized female rats, and the same protocol was conducted in those with E₂ tubes. Results (1) Compared with male rats, T_b rhythm in female gonadectomized rats became unstable, showing 2-4 h irregular oscillations. T_b rhythm remained unchanged in male gonadectomized rats. In female gonadectomized rats with E₂ tubes, T_b rhythm returned to the normal level. However, there was no influence of E₂ on T_b rhythm in the male rats. (2) Both in the heat and cold, gonadectomized female rats could not maintain their T_b as those with E₂ tubes. Histological analysis for the rat brain showed that Fos-immunoreactive cells in the hypothalamus were smaller in the rats without E₂ tubes. Conclusion These results show that estrogen is involved in the thermoregulation in female rats. Estrogen may modulate thermal sensitivity to the environment at the level of the hypothalamus [J Physiol Sci. 2006;56 Suppl:S102]

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An Epidermiologic study was carried out in the dry season on 250 commercial motor bike riders from five different parks. 69% of the bike riders were in the 31-40 and 41-50 age range while 31% were in 21-30, 51-60 and 61-70 age range. Half of the population studied were normotensive.Arterial hypertension was found in 25%of the examined workers (p<0.05), borderline hypertension was found in 26%of the workers (p<0.05). The severity of the hypertension increased with the age of the workers and the 31-40 age range had the highest incidence of hypertension accounting for 24(38%) of the total 63 frank cases of hypertension. The severity of the hypertension increased linearly with their duration of exposure to commercial motor bike riding(r=0.6, P<0.05). heart rate showed a progressive increase with age but a drop was observed in the 51-60 age range. The characteristics of the hypertension structure and it's interesting relation to age, number of years of commercial bike riding, heart rate and body weight is discussed. Of particular interest is the significant number of young adult bike riders found to be hypertensive. [J Physiol Sci. 2006;56 Suppl:S103]

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[Background] The frame pattern of substance for cell seeding can act not only on clustering of cells but also on cellular functions. However, the cellular kinetics depending on the frame pattern of habitat is not fully elucidated. We have established the technique for molding polymer resin with submicron accuracy, and have constructed many kinds of micro-frame patterns of substances. In this research, we explored micro-frame patterns suitable for neural network construction using PC12 cell line and evaluated the cellular functions on each micro-frame pattern. [Method] Micro-frame patterns were fabricated on the polymer resin of which the optical transparency was sufficient for the microscopic observation. [Results] One micro-domain was populated by 1-3 PC12 cells and a cellular network was formed by connecting one another through the open windows of the micro-domain. In addition, alterations in cellular growth and network formation occurred when the micro-domain structure was changed.[Conclusion] The results suggest that micro-frame patterns of substances play a critical role in cellular configuration. [J Physiol Sci. 2006;56 Suppl:S103]

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The pikas, Ochotona, living in cold zone or in high mountains prefer cold and are weak to heat. They were reported to be diurnal, most active at dawn and dusk, or active in day and night after the field observations. We have previously studied pikas' body temperature rhythm in their natural habitat using bio-telemetry devices and showed that the pikas are essentially diurnal and may vary their activity rhythm from diurnal in the relatively cool environment to a crepuscular (dawn and dusk) pattern in the relatively hot environment to avoid the heat during midday. In this study, we monitored body temperature in wild black-lipped pikas, Ochotona cruzoniae, in their natural habitat in Qinghai, China during mid-winter season. [J Physiol Sci. 2006;56 Suppl:S103]

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During brain ischemia, the excessive influx of Na⁺ is caused, resulted in the reversal of neuronal/astrocytic glutamate transporters; that is, glutamate and Na⁺ are co-transported to the extracellular space. Previous studies have revealed that this reversed uptake of glutamate occurs mainly via astrocytic GLT-1 and is the possibility cause of neuronal death. The present study aims at elucidating whether this reverse mode operation of GLT-1 has any functional meanings for astrocytes themselves. Analyses of the oxygen/glucose deprivation (OGD)-induced changes in the concentration of intracellular Na⁺ and Ca²⁺ have revealed that OGD produced Na⁺ overload, resulting in the reversal of Na⁺/Ca²⁺-exchanger (NCX). The reversed NCX then caused Ca²⁺ overload leading to the damage of astrocytes. When the cultures were treated with PACAP-38, a neuron-delivered peptide, to express GLT-1, the OGD-induced reversed GLT-1 released Na⁺ out of the cell, and significantly reduced the rise in intracellular Na⁺ and Ca²⁺ during OGD and the astrocytic cell damage. In contrast, however, OGD resulted in the co-transport of Na⁺ and glutamate out of astrocytes via reversed GLT-1, and the marked rise in the extracellular glutamate in neuron/astrocyte co-cultures produced excitotoxic neuronal death. These results suggested that ischemia-induced reverse mode operation of GLT-1 was toxic to neurons but beneficial to astrocytes by maintaining their Na⁺ gradient across cell membranes. [J Physiol Sci. 2006;56 Suppl:S104]

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Spontaneous oscillations in the intracellular concentration of calcium (Ca²⁺ oscillations) contribute to the regulation of gene expression. Here we investigated whether and how the dynamics of Ca²⁺ oscillations changed after sublethal preconditioning (PC) for PC-induced ischemic tolerance in neuron/astrocyte co-cultures. Ischemia was simulated by depriving co-cultures of both oxygen and glucose (OGD). The frequency of spontaneous Ca²⁺ oscillations decreased significantly between 4 and 8 h after the end of PC in both neurons and astrocytes. The reduction in oscillatory frequency caused by treatment with 2-APB, an inhibitor of IP3 receptors, resulted in the development of ischemic tolerance, in a suppression of the rise in the extracellular concentration of glutamate during OGD, and in a down-regulation of the expression of the glutamate transporter GLT-1. The expression of GLT-1 is known to be up-regulated by treatment with PACAP. Treatment with PACAP6-38, an inhibitor of PACAP receptors, decreased the oscillatory frequency and GLT-1 protein levels, and induced ischemic tolerance. In contrast, treatment with PACAP38 increased the oscillatory frequency, and antagonized both the PC-induced down-regulation of GLT-1 expression and ischemic tolerance. These results suggested that the sublethal PC insult suppressed the spontaneous Ca²⁺ oscillations regulating various gene expressions for the development of the PC-induced ischemic tolerance. [J Physiol Sci. 2006;56 Suppl:S104]

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We have recently found that an intraperitoneal (i.p.) injection of a synthetic double-stranded RNA, polyriboinosinic: polyribocytidylic acid (poly I:C), which mimics viral infection, induces interferon-α (IFN-α) and serotonin (5-HT) transporter (5-HTT) in the brain. To explore the functional significance of their expression, we determined extracellular concentrations of 5-HT and other monoamines such as noradrenaline (NA) and dopamine (DA) in the medial prefrontal cortex (mPFC) of freely moving rats using in vivo microdialysis method. Following an i.p. injection of poly I:C (3 mg/kg), NE levels in the mPFC transiently increased but returned to the basal level within 6 hrs after the injection. DA levels were not affected by poly I:C. On the other hand, 5-HT concentration in the mPFC decreased to 60-70% of the basal level until 8 hrs after poly I:C, while levels of a 5-HT metabolite, 5-hydroxyindole acetic acid, did not alter. The poly I:C-induced decrease in 5-HT was significantly attenuated by local perfusion with a selective 5-HT reuptake inhibitor (fluoxetine) in the mPFC. Microinjection of IFN-α into the mPFC also decreased 5-HT levels, which was again attenuated by perfusion with fluoxetine. It is considered that the poly I:C-induced 5-HTT, which is shown to be induced by IFN-α in astrocytes or endothelial cells, may scavenge extracellular 5-HT into the blood or cerebrospinal fluid, thereby decreasing 5-HT levels. We have reported that the decrease in 5-HT in the brain is closely related to the central mechanisms of fatigue. [J Physiol Sci. 2006;56 Suppl:S104]

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Diabetes mellitus (DM) frequently accompanies with contractile dysfunction and arrhythmia. L-type Ca²⁺ channel current (I_Ca(L)), transient outward current (I_to), delayed rectifier outward K⁺ current (I_K(delay)) and Na/Ca exchanger current (I_NCX) in ventricular myocytes were compared between control (LETO) and a type II DM model (OLETF) rats using a patch-clamp technique at 50 weeks of age to clarify electrophysiologic changes in diabetic heart. Blood pressure (BP) was measured at caudal artery by noninvasive tail-cuff method. After rats were anesthetized by sodium pentobarbital, electrocardiogram (ECG) was recorded by apex-base lead, and then hearts were excised and perfused with collagenase solution to isolate myocytes. Fibrosis of ventricles was histologically evaluated using Azan stain and connexin 43 protein (Cx43) expression was quantitated by western blot. Systolic and diastolic BPs were significantly elevated in OLETF rats. PQ interval and QRS duration were significantly prolonged and the cell sizes of myocytes were enlarged remarkably in OLETF rats. Current densities of (I_Ca(L), I_to, I_K(delay) and I_NCX were not changed in OLETF rats as compared with those in LETO rats. Although fibrosis was not seen in OLETF rat ventricles, Cx43 expression significantly decreased. We thought that the QRS duration was prolonged due to the delay in conduction of excitation in OLETF rat ventricles, which might be related with the decrease in Cx43 expression. [J Physiol Sci. 2006;56 Suppl:S105]

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Microglial cell has been demonstrated to be involved in various diseases, such as Alzheimer and Parkinson diseases, HIV encephalitis and multiple sclerosis. In spite of the facts that stress plays crucial roles in the progression of clinical diseases, the involvement of stress on the microglial activity remains to be elucidated. Based on finding that stress induced the elevation of proinflammatory cytokines, we hypothesized; (1) physical/emotional stress may have some effect on the microglial activation, (2) IL-18, a proinflammatory cytokine and demonstrated to be increased in stress from the adrenal gland, may participate in the microglial activation. We employed restraint combined with water immersion stress for 2 hours as acute stress. Immediately after release from stress, rats were sacrificed for experiments. Immunohistochemistry with OX-42 revealed that acute stress provoked morphological microglial activation in the thalamus, hypothalamus, hippocampus and central grey. Semi-quantitative real time PCR and immunohistochemistry showed that stress significantly induced IL-6 and iNOS from microglia. In addition, intraperitoneal IL-18 administration (5 μg/rat) caused robust microglial activation in the brain in a similar fashion observed in stress. Furthermore, in-vitro studies using microglia cell line (MG6-1) demonstrated that IL-18 administration (up to 500 ng/ml) significantly induced iNOS, IL-6, and IL-18 in a dose dependent manner. Thus, the present study suggests that stress may stimulate microglial cells to produce several pro-inflammatory cytokines and iNOS at least through stress-induced circulating IL-18. [J Physiol Sci. 2006;56 Suppl:S105]

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Dendritic spines are multiple functional units that receive most excitatory inputs in central nervous system. Modification of dendritic spine number is associated with several neurological diseases and synaptic plasticity. Spikar is a novel molecule which was isolated as a drebrin-binding protein using yeast two hybrid screening. In rat primary cultured hippocampal neurons, GFP-Spikar was localized primarily in nucleus and dendritic spines, and lesser amounts in soma, dendritic shafts, and axons. In this study, we investigated the role of Spikar in cultured hippocampal neurons during development. Hippocampal neurons were transfected with a Spikar-shRNA expression vector or an empty vector as a control at several developmental stages. The Spikar-shRNA expression vector caused 60-90% knock down (KD) of endogenous Spikar. In early stage of development, Spikar KD did not affect the density of dendritic protrusions. In contrast, at a stage of synapse formation, Spikar KD reduced spine density without changing filopodia density. In more mature stage when majority of dendritic protrusions are dendritic spines, Spikar KD reduced spine density as well. These results suggest that Spikar plays a role in the maintenance of dendritic spines without affecting the filopodia formation. [J Physiol Sci. 2006;56 Suppl:S105]

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Ca²⁺-regulated exocytosis is enhanced by the PGE₂/cAMP pathway in antral mucous cells of guinea pigs. The inhibition of the PGE₂/cAMP pathway by a PKA inhibitor (H-89) or aspirin (ASA) decreased the frequency of ACh-stimulated exocytotic events by 60%. Indomethacin (IDM), however, decreased the ACh-stimulated exocytotic events only by 30%. Moreover, IDM increased the ACh-stimulated exocytotic events by 50% in H-89-treated or ASA-treated cells. IDM inhibits the synthesis of PGG/H and 15R-HPETE, while ASA inhibits only PGG/H synthesis. Thus, IDM accumulates arachidonic acid (AA). AACOCF₃ or ACA (PLA₂ inhibitors), which inhibits AA synthesis, decreased the ACh-stimulated exocytotic events by 60%. IDM, however, did not increase the frequency in AACOCF₃-treated cells. AA increased the frequency of ACh-stimulated exocytotic events in AACOCF₃- or ASA-treated cells, similar to IDM in ASA-treated cells. Moreover, in the presence of AA, IDM did not further increase the ACh-stimulated exocytotic events in ASA-treated cells. The PGE₂ release from antral mucosa indicates that inhibition of PLA₂ by ACA decreases AA accumulation in unstimulated and ACh-stimulated antral mucosa. The dose-response study of AA and IDM demonstrated that the concentration of intracellular AA accumulated by IDM is less than 100 nM. In conclusion, IDM modulates ACh-stimulated exocytosis via AA accumulation in antral mucous cells. [J Physiol Sci. 2006;56 Suppl:S108]

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The effect of the immunosuppressant drug FK506 on microsomal Ca²⁺ release was investigated in rat pancreatic acinar cells. When FK506 (0.1-200 μM) was added to the microsomal vesicles at a steady state of ATP-dependent ⁴⁵Ca²⁺ uptake, FK506 caused a dose-dependent and a biphasic release of ⁴⁵Ca²⁺. Almost 10% of total ⁴⁵Ca²⁺ uptake was released at concentrations of FK506 up to 10 μM (K_m = 0.47 μM), and 60% of total ⁴⁵Ca²⁺ uptake was released at concentrations of FK506 over 10 μM (K_m = 55 μM). Preincubation of the vesicles with cyclic ADP-ribose (cADPR: 0.5 μM), which is known to modulate the ryanodine receptor, increased the FK506 (< 10 μM)-induced ⁴⁵Ca²⁺ release (V_max value of the release: 8.1% without cADPR vs. 14.4% with cADPR). Preincubation with 200 μg/ml of heparin, an inhibitor of inositol 1,4,5-trisphosphate (IP₃) receptor, resulted in significant inhibition of the FK506 (30 μM)-induced ⁴⁵Ca²⁺ release. Subsequent addition of IP₃ (5 μM) after FK506 (100 μM)-induced ⁴⁵Ca²⁺ release did not cause any release of ⁴⁵Ca²⁺. These results indicate that there are two different types of FK506-induced Ca²⁺ release mechanisms in the endoplasmic reticulum of rat pancreatic acinar cells: a high-affinity mechanism of Ca²⁺ release, which is activation of the ryanodine receptor, and a low-affinity mechanism of Ca²⁺ release, which is activation of the IP₃ receptor. [J Physiol Sci. 2006;56 Suppl:S108]

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The application of thapsigargin (TG) or cyclopiazonic acid (CPA) to Fura-2-loaded chromaffin cells in the perfused rat adrenal medulla abolished a transient [Ca²⁺]_c rise induced by muscarine in Ca²⁺-free medium due to the depletion of Ca²⁺ stores. Either TG or CPA induced a sustained increase of [Ca²⁺]_c in Ca²⁺-containing medium, indicating that store-operated Ca²⁺ entry (SOCE) mechanism exists in this cell type. The TG-induced [Ca²⁺]_c increase was inhibited completely with 2 mM Ni²⁺ but only by 18% with 100 μM D600, whereas maintained [Ca²⁺]_c increases during prolonged stimulations with muscarine (100 μM) and high-K⁺ (40 mM) were inhibited by 100% and 51% with Ni2+, by 53% and 80% with D600, respectively. In cells to which muscarine and Ni²⁺ had been co-applied, Ca²⁺ stores remained depleted to induce a sustained SOCE after the two agents were washed out. In isolated chromaffin cells, CPA induced a much smaller extent of elevation in [Ca²⁺]_c, compared with that induced in cells being in the adrenal medulla, which possibly suggests that the mechanism involved in SOCE may be fragile and was impaired in dissociation. TG or CPA applied alone to the adrenal medulla did not elicit a detectable amount of catecholamine secretion despite the elevation of [Ca²⁺]_c, nor promoted secretory responses to a significant extent when applied during stimulation with high-K⁺. These results suggest that SOCE in rat chromaffin cells may not produce a sufficient increase in [Ca²⁺]_c near the secretory vesicles to trigger exocytosis. [J Physiol Sci. 2006;56 Suppl:S108]

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Purpose: Myosin II is one of the typical motor proteins and is classified as non-regulated, phosphorylatable and Ca-binding myosins. Physarum myosin II belongs to Ca-binding one. Myosin II regulated by Ca-binding has not yet expressed as a recombinant protein. Here, we report the expression of heavy mero-myosin of physarum myosin II together with preliminary characterizations. Method: We used baculovirus expression system. Sf9 cells were infected with the virus constructs. Result: When baculovirus of heavy chain(HC) fragments was infected together with those of phosphorylated light chain (PLC) and Ca-binding light chain (CaLC), Sf9 cells produced soluble HMM, which were recovered in the supernatant together with PLC and CaLC. The HMM showed Mg-ATPase activity of 0.21 (s⁻¹head⁻¹), and actin-activated ATPase activity with Vmax=1.27 (s⁻¹head⁻¹), and Km=1.8μM. The movement of actin filaments on the HMM-coated glass surface was sensitive to Ca²⁺. We will show the effect of Ca²⁺ on the movement of HMM associated with various kinds of light chains. [J Physiol Sci. 2006;56 Suppl:S109]

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At fertilization of mammalian eggs, the repetitive Ca²⁺ releases from intracellular stores through inositol 1,4,5-trisphosphate (IP₃) receptors are thought to be induced by the soluble factor originated from the sperm. Although phospholipase C zeta (PLCζ) is the possible candidate of such 'sperm factor', there are no evidence to show the elevation of intracellular IP₃ concentration at fertilization. We measured the changes in IP₃ concentration during Ca²⁺ oscillations in mouse eggs, using a novel FRET-based IP₃ probe, fretino. Eggs expressing fretino showed a transient decrease in FRET signal in response to the microinjection of IP₃, but not to inositol 1,3,4,5-tetrakisphosphate (IP₄). The signal decayed exponentially with a time constant of 100-180 s. When Ca²⁺ oscillations were induced in the eggs by insemination, the signal of fretino showed no detectable changes to indicate the increase in IP₃ concentration. On the other hand, eggs expressing a large amount of PLCζ showed significant decrease in the FRET signal from fretino. Furthermore, the FRET signal oscillated with Ca²⁺ in such eggs, suggesting the enhancement of PLCζ activity by cytoplasmic Ca²⁺. The magnitudes of Ca²⁺-induced IP₃ production in the fertilized eggs and in the eggs expressing PLCζ or PLCδ1, were also compared. [J Physiol Sci. 2006;56 Suppl:S109]

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Mitochondrial morphology is regulated by balance of fission and fusion events. Certain dynamin family members such as dynamin-related protein 1 (Drp-1) are involved in the regulation of mitochondrial fission. Drp-1 specifically controls mitochondrial outer membrane fission. However, very little is known about the mechanism that initiates mitochondrial fission by Drp-1. In the present study, we detected Drp-1 was phosphorylated by CaMKI in vitro. In primary cultured hippocampal neurons, high K⁺ stimulation induced phosphorylated Drp-1 increment and Drp-1 transition from cytoplasm to mitochondria and mitochondrial fragmentation. The effect of high K⁺ was inhibited by KN93 (CaMK inhibitor). In vitro experiment, we found phosporylation promoted the Drp-1 complexes formation. Although overexpression of GFP-hFis1-C did not alter mitochondrial morphology, it inhibited high K⁺ induced mitochondrial fission in neurons. These results suggest that Drp-1 dynamics and mitochondrial morphology may be regulated by CaMKI-induced Drp-1 phosphorylation. [J Physiol Sci. 2006;56 Suppl:S109]

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We reported previously that two store-operated Ca²⁺ channels (SOCs) could be separated by using Zn²⁺ in the submadibular acinar cells. In short, after depleting the Ca²⁺ stores with thapsigargin, SOC signals during readmission of external Ca²⁺ were detected. The signal showed two phases; the initial large transient and subsequent sustained phase. External Zn²⁺ inhibited the former but not the latter. External Ni²⁺ or excess of outside K⁺ markedly reduced both. Based on this observation, we studied Na⁺ entry through SOC. Loading benzofrane isophthalate (SBFI)-AM for 2 h at 37^oC to the cells, internal [Na⁺]_i was monitored with digital imaging methods. Prior elimination of external Na⁺ (replaced with an impermeable cation, NMDG⁺) induced a substantial increase in Na⁺ entry by Na⁺ readmission, and it was strengthened by a simultaneous elimination of external Ca²⁺. When Ca²⁺ stores were actively depleted with thapsigargin under Ca²⁺- and Na⁺-free condition, the largest Na⁺ signals were counted by the Na⁺ readmission. In contrast to the pattern of Ca²⁺ entry, that of Na⁺ was monophasic and inhibited by external Zn²⁺, Ni²⁺ and Ca²⁺ as well. The finding that external Ca²⁺ reduced Na⁺ signals suggests that Ca²⁺ store depletion induces Na⁺ entry through SOCs in a competitive manner with Ca²⁺. Collectively, after depletion of Ca²⁺ stores, Na⁺ may enter into the cells through the divalent cation-sensitive Ca²⁺-entry pathway in mouse submandibular acinar cells. [J Physiol Sci. 2006;56 Suppl:S109]

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Although the HNF4 α protein is known to function as a dimer and Q268X heterozygotes carrying mutations in its allele cause MODY1, it is still unclear whether Q268X and wild type HNF4 α can dimerize, and what causes such a distinct phenotype. We visualized the practical and mutual interactions of HNF4 α and Q268X HNF4 α using Fluorescence Resonance Energy Transfer (FRET). A transition in cellular localization was seen in Q268X-HNF4 α complexes from the nucleoplasm to the nucleolus, where wild type HNF4α is normally localized in COS7 and CHO cells. Furthermore, FRET microscopy showed that Q268X-HNF4 α bound to wild type HNF4α and accumulated in the nucleolus. SHP, which is a repressor of HNF4 α, also bound to Q268X and translocated to the nucleolus. The cellular localization of a deletion mutant of HNF4 α showed that the site contributing to nucleolar accumulation is P333 to I338. Some proteins displayed an altered cellular function after localization to the nucleolus. According to these results, transfer to the nucleolus of the heterodimer Q268X-HNF4 α must affect the function of HNF4 α. Consequently, this study showed that Q268X-HNF4 α dimerizes with wild type HNF4 α and also binds with the repressor and changes its localization to the nucleolus. These effects, together with transcription function, may lead to the distinct phenotype of MODY. [J Physiol Sci. 2006;56 Suppl:S110]

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We tested the effects of exposure to a switched 1.5 Tesla magnetic field on transient increase in intracellular Ca²⁺ stimulated by neurotransmitters in bovine adrenal chromaffin cells. [Ca²⁺]i was increased transiently by addition of acetylcholine (ACh) in Ca²⁺-free medium and the ACh-induced increase was inhibited significantly by 2 hr-exposure to the magnetic field. The exposure caused not only to decrease the peak value but also to slow the decay phase of [Ca²⁺]i after peak. Delay of the decay phase was also caused by addition of KCN in the presence of ACh. The intracellular ATP content and oxygen consumption were influenced by the exposure in glucose-free medium. Measurement of mitochondrial membrane potential by using fluorescent probe, JC-1 (Molecular Probe), showed depolarization of the membrane in both cells exposed to antimycin and the magnetic field. The cellular content of F-actin stained with fluorescent probe (Alexa fluor 488 phalloidin) was also decreased by the exposure. These effects of magnetic field would be related to the eddy current. [J Physiol Sci. 2006;56 Suppl:S110]

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The final steps of exocytosis are consisted of three steps, docking, priming, and fusion, in which the priming step is maintained by ATP. We examined the effects of Cl⁻-free solution on the priming step.The isolated antral mucous cells were obtained by a collagenase treatment, and observed using video-microscopy. In Cl⁻-free solution, Cl⁻ was replaced with NO₃⁻.Acetylcholine (ACh 1 μM) increases the frequency of exocytosis ; an initial phase followed by a sustained phase. The Cl⁻-free solution enhanced the initial peak frequency of ACh-evoked exocytosis approximately 4 fold respectively.To examine effects of Cl⁻-free solution on the priming step, intracellular ATP was depleted by anoxia (aerated with N₂ 100%) or dinitrophenol (DNP 100 μM). Depletion of ATP eliminated the initial phase of ACh-evoked exocytotic events. After ATP depletion, Cl⁻-free solution did not evoke any initial phase.However, when cells were first perfused with Cl⁻-free solution, and then ATP was depleted, ACh induced an initial phase. Moreover, cells were first stimulated with ACh, which depelets the primed granules, and after a short interval (9min), cells were stimulated with ACh again. The initial phase was induced by the second ACh stimulation during perfusion with Cl⁻-free solution, while it was not during perfusion with the control solution.Based on the observation, Cl⁻-free solution increases number of the primed granules, which enhances ACh-evoked exocytotic events in antral mucous cells. [J Physiol Sci. 2006;56 Suppl:S110]

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Tight junction is the essential structure for salivary epithelial cells to keep polarity and to secrete fluid unidirectionally. Previously, we established a system for primary culture of parotid acinar cells. Acinar cells isolated from the rat parotid glands formed large colonies and attached to the basement of dishes at 24 h after the dispersion. After 2 days, most cells spread as a monolayer whereas a part of cells formed hemispherical lumps. Analysis with electron microscopy suggests that cells in the lumps retained original tight junctions and lumens. On the other hand, cells in monolayer also formed tight junctions. Immunofluorescence microscopy showed claudin-1 was observed in the lumps, while claudin-4 was detected at the tight junctions in monolayer. Most cells in the monolayer that had claudin-4-positive tight junctions retained secretory granules containing amylase. Therefore, the claudin-4-positive cells were probably derived from acinar cells, but not from ductal cells. Immunoblotting analysis showed that claudin-4 was expressed at 24 h and its expression increased time-dependently during the culture, although it was not detected just after the dispersion. These results suggest that the expression of claudins changed from isotype 1 to 4 while the morphology of the acinar cells changed to monolayer. Claudins possibly have a correlation with the formation and maintenance of culture configuration in parotid acinar cells. [J Physiol Sci. 2006;56 Suppl:S110]

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In this study, the effects of extremely low frequency (ELF) region on the osteoblast-like cells (MC3T3-E1) for the differentiation was examined. Sinusoidal (60Hz) magnetic fields were about 3 mT. Collagen protein contents of cultures were measured microscopically by using ImSpector system. Using insulin-like growth factor I (IGF-I), the difference between the effect by exposure was examined with these during effects for the collagen content of these cells. From these results, the effects of exposure and IGF-I treatment caused significant increase on collagen synthesis of osteoblasts. It is supposed that the effects of magnetic fields go through the intercellular signaling pathway. Therefore, we experiment by the use of some inhibitors which block the intercellular signal transduction and examine which route the exposure passes in order to influence on differentiation of osteoblasts. As a results, it is suggested that ELF magnetic fields stimulate collagen synthesis because of activation of p38 MAPK and induce the cell differentiation. These results indicate that the mechanisms of differentiation related to IGF in the osteoblasts were altered by the magnetic fields of extremely low frequency. [J Physiol Sci. 2006;56 Suppl:S111]

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Na,K-ATPase is a membrane-bound protein that maintains intracellular ionic concentrations, i.e., low Na+ and high K+, using the energy from hydrolysis of ATP. We have now observed a rapid recruitment of Na,K-ATPase to the cell surface in responce to extracellular low calcium concentration in the parathyroid cell that releases PTH in responce to low calcium. Using isotope-labelled ouabain that is a specific ligand for Na,K-ATPase, we observed that the recruitment occurs in a few minutes. Biotinylation of the cell-surface proteins revealed that a considerable amount of Na,K-ATPase is present in intracellular region and bound to a specific protein. In analysis of the protein-deficient mice, the molecular association is required for a novel mechanism of rapid recruitment of Na,K-ATPase. This rapid recruitment is essential for PTH release that is the first step of calcium regulation of the whole body. This indicates the importance of NA,K-ATPase in the calcium homeostasis. [J Physiol Sci. 2006;56 Suppl:S111]

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In guinea pig antral mucous cells, acetylcholine (ACh) induces a biphasic increase in the Ca²⁺-regulated exocytosis: an initial transient phase followed by a sustained one. We studied the effects of cGMP on ACh-stimulated exocytosis in guinea pig antral mucous cells using video microscopy. Cyclic GMP enhanced the frequency of ACh-stimulated exocytotic events, while cGMP alone induced no exocytotic events under the ACh-unstimulated condition. Cyclic GMP did not affect either Ca²⁺ mobilization or cAMP accumulation. cGMP shifted the Ca²⁺ dose-response curve upward with no shift to the lower-concentration, indicating that cGMP increases responsibility of the Ca²⁺-regulated exocytosis, but not the Ca²⁺ sensitivity. When cGMP was added after ATP depletion by dinitrophenol (DNP) or anoxia (N₂ bubbling), ACh evoked only a sustained phase in the exocytosis without any initial transient phase. In contrast, when cells were pretreated with cGMP before ATP depletion, ACh evoked the biphasic exocytotic events. These observations indicate that cGMP modulates ATP dependent priming of Ca²⁺-regulated exocytotic events. In conclusion, cGMP increases the number of primed granules via acceleration of the ATP-dependent priming step, which enhances the Ca²⁺-regulated exocytotic events stimulated by ACh. [J Physiol Sci. 2006;56 Suppl:S111]

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Acridine orange (AO), a weakly basic fluorescent dye, is permeable to plasma and vesicle membranes and preferentially remains in intracellular acidic regions. Using fluorescence microscopy, we observed dynamic changes in AO-loaded cultured mouse cells during illumination with blue light. Immediately after the start of illumination, the successive disruption of vesicle membrane was observed as a flash of fluorescence, and shortly after that, blebs were formed on the plasma membrane regardless of the occurrence of vesicle disruption. Vesicle disruption was almost completely inhibited when cells were treated with the vacuolar H⁺-ATPase inhibitor bafilomycin A1 followed by staining with AO, but not when bafilomycin A1 was treated after AO staining. Thus, the filling of AO in the vesicle, which is driven by vacuolar H⁺-ATPase, is initially required for vesicle disruption. In contrast, bafilomycin A1 did not prevent plasma membrane blebbing, indicating that the blebs are formed independently of the vesicle disruption. Both the vesicle disruption and the formation of plasma membrane blebs were partially inhibited by removal of oxygen from the cell environment and by singlet oxygen scavengers, sodium azide, ascorbic acid, and L-histidine, but not by the hydroxyl radical scavenger dimethyl thiourea. Thus, both phenomena are likely caused at least in part by the generation of singlet oxygen. These photosensitive features of plasma and vesicle membranes may be based on the use of the photodynamic effect, such as cancer therapy. [J Physiol Sci. 2006;56 Suppl:S111]

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Insulin stimulates glucose transport into adipocyte and muscle cells by inducing the translocation of the insulin responsive glucose transporter GLUT4 from intracellular storage compartments to the plasma membrane. GLUT4 translocation is a complicated process involving budding and fission at the storage compartment, trafficking to the plasma membrane, and fusion at the plasma membrane. Here, we have established a new method to visualize the movement of single GLUT4 molecule in living cells. 3T3L1 adipocyte expressing exofacial-myc-GLUT4-eCFP was labeled with Qdot-conjugated Myc antibody in the presence of insulin. Qdot-GLUT4 complex was then endocytosed by washing out of insulin. Observation was performed under video-rate confocal microscope equipped with high sensitivity EMCCD camera so that movement of GLUT4 molecule can be tracked for ∼10sec. Movement of GLUT4 molecules was obtained before or after the 2nd insulin stimulation for 5, 15, and 30 min. Analysis of the diffusion of GLUT4 molecules showed that movements can be classified in either 1) free diffusion, 2) confined diffusion, or 3) transport and all these movement existed both at basal state and after insulin stimulation. Overall movement tended to be in confined diffusion at the basal state, but fraction of transported GLUT4 increased by insulin stimulation. Diffusion coefficient of GLUT4 was higher after insulin stimulation than basal state. Together, insulin increases the mobility of GLUT4 and enhances its translocation to the plasma membrane. [J Physiol Sci. 2006;56 Suppl:S112]

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CA neurons are involved in a wide spectrum of physiological functions in the brain including sensory, motor, emotional, autonomic and endocrine regulation. Most CA neurons are localized in the brainstem and hypothalamic regions and typically make clusters of cells, among which the noradrenergic (A1, A2, A6) and dopaminergic (A9, A10, A12) neurons predominate. In order to explore functional roles of these neurons, we sought to collect CA neurons selectively using TH-GFP transgenic mice in which GFP expression was driven under TH-promoter. Fetal (E14.5, E16.5, or E18.5) brain was extracted, and neurons were dispersed after treating with trypsin, then GFP-positive cells were sorted out by flow-cytometry (FACS). RNA was extracted from the GFP-positive (TH) neurons, reverse-transcribed, and analyzed by PCR. [J Physiol Sci. 2006;56 Suppl:S112]

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Electrogenic Na⁺-HCO₃⁻ cotransporter (NBCe) plays an important role in mediating HCO₃⁻ efflux and influx across the basolateral membrane in various HCO₃⁻-transporting epithelia, including kidney proximal tubules and pancreatic ducts. Its activity has been generally assessed by monitoring intracellular pH, Na⁺ concentration, or membrane potential, but electrophysiological properties of the cotransporter at the native state still remain largely unknown. Using the whole-cell patch clamp technique, we have recently, for the first time, identified and characterized membrane currents attributable to the activity of a NBCe expressed in acutely dissociated acinar cells (BPA cells) from bovine parotid that secretes large volumes of a HCO₃⁻-rich fluid. Under voltage-clamp conditions, the currents were dependent upon extracellular Na⁺ and HCO₃⁻ and DIDS-sensitive. Further analysis of the currents indicated that the stoichiometry of the native cotransporter is most likely to be 2 HCO₃⁻ : 1 Na⁺. We could also demonstrate that BPA cells express transcripts of NBCe1-B (bNBCe1-B) and that recombinant bNBCe1-B currents in HEK293 cells shares common electrophysiological and pharmacological properties with those of the native currents. This study represents an initial attempt to provide electrophysiological characterization of a NBCe expressed in a native HCO₃⁻-secreting exocrine gland. [J Physiol Sci. 2006;56 Suppl:S112]

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The concentration-sensitive Na⁺ channel (Na_C; c:concentration) works as a Na⁺ sensor and it opens when [Na⁺]_o changes. The present study was carried out to clarify the function of the Na_C as one of the regulating factors in cell growth, using rat C6 glioma cells, since they have Na_C in quantity. The image analysis of Na⁺ dynamics, using a Na⁺ indicator (SBFI) and ARGUS-50 (Hamamatsu Photonics), revealed an elevation of [Na⁺]_i when [Na⁺]_o was raised from normal (140 mM) to 190 mM. This increase was augmented when Na⁺ efflux was suppressed by inhibitors of the Na⁺ pump (ouabain) or of the Na⁺/K⁺/Cl⁻ cotransporter (bumetanide). Osmolarity alteration was not important because addition of mannitol to the external solution did not introduce any changes in [Na⁺]_i. The expression of immediate early gene egr-1, measured by the real-time PCR method, was reduced when [Na⁺]_o was raised or when [Na⁺]_i was elevated by a Na⁺ ionophore, monensin (Cell Biol Int 29:261-268, 2005). These procedures suppressed the rate of cell proliferation. When the expression of Na_C was selectively inhibited by RNA interference (RNAi) techniques, both [Na⁺]_i and the growth rate of C6 cells were less affected by [Na⁺]_o changes, indicating that Na_C was involved in cell growth (by controlling gene expression through introduction of Na⁺ into the cell). It is concluded that Na⁺ ions enter C6 glioma cells mainly through Na_C, and Na⁺ ions regulate cell growth by controlling expression of proliferation-related genes. [J Physiol Sci. 2006;56 Suppl:S112]

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The vacuolar-type H⁺-ATPase (V-ATPase) is an electrogenic H⁺ pump that is distributed in living organisms. By carrying out uphill H⁺ transport, V-ATPases acidify lysozomes and energize intracellular membranes. In osteoclasts, the V-ATPases are enriched in the plasma membrane faced to the bone surface (ruffled membrane) and serve as a major acid-secretion pathway required for bone resorption. In this study, we attempted to identify the pump currents of osteoclasts electrophysiologically and investigated their dependence on the membrane potential and/or H⁺ gradient, which may change widely under different functional states. Outward H⁺ currents were increased by intracellular dialysis with ATP up to 10 mM in dose-dependent manner. The V-ATPase current was evaluated by blockers for the V-ATPase, bafilomycin A₁ and N,N'-dicyclohexylcarbodiimide, in the presence of 5 mM ATP. The V-ATPase currents were decreased by hyperpolarization, but were still outward at -80 mV under pH_o/pH_i of 7.3/5.5. The current amplitude was decreased by either intracellular alkalization or extracellular acidification, but did not show current reversal. Significant outward H⁺ currents were seen at 0 mV even under pH_o/pH_i of 5.5/7.3. The data showed that the V-ATPase-medaited currents depends on both voltage- and pH gradients across the plasma membrane. The pump, however, could secrete H⁺ upon exposure to strong acids as far as energy is supplied sufficiently. [J Physiol Sci. 2006;56 Suppl:S113]

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We measured intracellular free Mg²⁺ concentration ([Mg²⁺]_i) with fluorescent Mg²⁺ indicator furaptra (mag-fura-2) in Ca²⁺-free condition (0.1 mM EGTA) at 25°C. After the cells loaded with Mg²⁺ in 24 mM-Mg²⁺ solution for 3 h, reduction of [Mg²⁺]_o to 1 mM caused a decrease in [Mg²⁺]_i in the presence of extracellular Na⁺ (Na⁺-dependent Mg²⁺ efflux). To study the effects of K⁺ on Na⁺-dependent Mg²⁺ efflux, the initial rate of decrease in [Mg²⁺]_i (initial Δ[Mg²⁺]_i) /Δt) was compared at high extracellular [K⁺] (75 mM) and K⁺-free (replaced by N-methyl-D-glucamine) conditions. [Na⁺]_o was kept constant at 70 mM, and membrane potential was set at -13 mV with amphotericin-B-perforated patch clamp technique. With the K⁺-based pipette solution, the initial Δ[Mg²⁺]_i/Δt values obtained in the presence of 75 mM K⁺ and 0 mM K⁺ in the perfusate were not significantly different (79.0±6.0% and 65.6±5.0%, respectively, of the control values measured at 140 mM [Na⁺]_o without any modification of extracellular and intracellular K⁺ and Cl⁻). Intracellular perfusion with K⁺-free (Cs⁺-substituted) solution from the patch pipette in combination with removal of extracellular K⁺ did not significantly change the initial Δ[Mg²⁺]_i) /Δt (77.7±8.2% of the control). Finally, the initial Δ[Mg²⁺]_i/Δt was unchanged by extracellular and intracellular perfusion with K⁺-free and Cl⁻-free solutions (71.6±5.1% of the control). These results suggest that K⁺ and Cl⁻ are not involved in the Na⁺-dependent Mg²⁺ efflux. [J Physiol Sci. 2006;56 Suppl:S113]

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Excitotoxicity is associated with stroke, brain trauma and neurodegenerative disorders. Focal swellings along dendrites called varicosities are a hallmark of acute excitotoxic neuronal injury. We previously reported that cultured mouse cortical neurons express the volume-sensitive outwardly rectifying (VSOR) chloride channel, which is involved in volume regulation after osmotic swelling. Here we studied a role of the VSOR chloride channel in excitotoxic neuronal injury in cultured mouse cortical neurons. The blockade of the VSOR chloride channel activity by NPPB (40 μM), phloretin (100 μM) or IAA-94 (1 mM) during excitotoxic stimulation inhibited varicosity formation and necrotic neuronal death. On the other hand, a GABA_A receptor/chloride channel blocker, bicuculline (10 μM) or picrotoxin (100 μM), failed to inhibit neuronal necrosis induced by excitotoxicity. On-cell patch-clamp studies revealed robust VSOR chloride channel activity on varicosities during exposure to NMDA. These results suggest that the VSOR chloride channel is involved in aggravation of excitotoxicity by serving as the pathway for chloride influx, which induces varicosity formation and cell swelling leading to necrotic cell death. [J Physiol Sci. 2006;56 Suppl:S113]

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Around 70% of brain tissue is composed of glial cell, which regulates the homeostasis of various neurotransmitters, ions and water in the brain. However, little studies have been performed on the effects of CNS-acting drugs on glial function. We have examined the effect of various tricyclic antidepressant agents: amitriptyline, imipramine, nortriptyline, and desipramine, on a K⁺channel responsible for the glial K⁺-buffering action. The glial K⁺ -buffering channels are composed either of homomeric assembly of Kir4.1 or of heteromeric assembly of Kir4.1 and Kir5.1. In this study, Kir4.1 homomeric channels were exogeneously expressed in tsA201 cells and whole-cell currents were recorded using a patch-clamp technique. Application of each of the various tricyclic antidepressants immediately and reversibly caused a reduction of inward and outward currents through this channel. The inhibition was stronger as the membrane was more depolarized. Development of the current blockage was well fitted with a single exponential function. These results indicate that the block of Kir4.1 channels by these antidepressants was clearly in a voltage- and time-dependent fashion. Thus, various tricyclic antidepressants may act as inhibitors at the glial Kir4.1 channels. We conclude that the inhibition of the glial Kir4.1 channels by these drugs underlies the therapeutic effects and some of the side effects, particularly seizures in overdose. [J Physiol Sci. 2006;56 Suppl:S113]

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Cell shrinkage is a hallmark of apoptosis. We previously demonstrated that the apoptotic volume decrease, which represents an early-phase event of apoptosis, is induced by K⁺ and Cl⁻ efflux. On the other hand, it is known that osmotic cell shrinkage directly leads to apoptotic death in cells that lack the ability of volume regulation, called regulatory volume increase (RVI). In HeLa cells that can exhibit RVI, however, strong hypertonic stimulation failed to induce cell death. Since we have recently showed that the hypertonicity-induced non-selective cation channel (HICC) plays an important role in the RVI process in HeLa cells, we used flufenamate, a HICC blocker, to induce persistent cell shrinkage. Hypertonicity-induced cell death and activation of caspase-3 was enhanced by flufenamate in a concentration-dependent manner. The concentration dependency was in good accord with that for HICC current inhibition. These results suggest that HeLa cells are sensitized by inhibition of HICC to shrinkage-induced apoptosis. [J Physiol Sci. 2006;56 Suppl:S114]

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Iptakalim hydrochloride (IPT) is a novel ATP-sensitive potassium channel (K(ATP)) opener which has a different chemical structure from any other known K(ATP) opener, and produces vasodilution. In this study, we examined the effect of IPT on rat pancreatic beta-cell functions. In the perifusion experiment for islets, an application of IPT increased insulin secretion, tested with 5.5 mM glucose in the extracellular solution. Examined in isolated beta-cells loaded with fura-2, IPT elevated intracellular calcium concentration, which was restored by diazoxide. Under the patch-clamp whole-cell configuration, IPT induced depolarization in isolated beta-cells followed by action potential firing. The depolarization was associated with a decrease in membrane conductance resulting from a decrease in K(ATP) activity. Further, IPT applied into the bath solution inhibited K(ATP), recorded in the cell-attached mode. IPT applied to the intracellular surface of the membrane also inhibited K(ATP), recorded in the inside-out mode. These results indicate that IPT acts on pancreatic beta-cells as a K(ATP) blocker, which in turn causes electrical excitation of the cell and insulin secretion. [J Physiol Sci. 2006;56 Suppl:S114]

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We have recently demonstrated that maxi-anion channels constitute a major pathway for the regulated release of ATP. It is widely held that a voltage-dependent anion channel (VDAC) located in the plasmalemma that normally functions in the mitochondrial outer membrane is the most likely candidate protein of this channel. This hypothesis was based on the similarity of shared biophysical properties, such as the large unitary conductance and bell-shaped voltage dependency of the maxi-anion channel and mitochondrial VDAC. In the present study, we deleted each of the three genes encoding the VDAC isoforms individually and collectively. We have demonstrated that maxi-anion channel (around 400 pS) activity in VDAC-deficient mouse fibroblasts was unaltered. The channel activity was similar in VDAC1/VDAC3 double-deficient cells and in double-deficient cells with VDAC2 protein depleted by RNA interference. VDAC deletion slightly down-regulated, but never abolished, the swelling-induced ATP release. The lack of correlation between VDAC protein expression and maxi-anion channel activity strongly argues against the long held hypothesis of plasmalemmal VDAC being the maxi-anion channel. Details of the biophysical profile, such as the different potassium-to-chloride and glutamate-to-chloride selectivity and a different pattern of the voltage-dependent gating provide independent support for our conclusion. [J Physiol Sci. 2006;56 Suppl:S114]

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Chloride ions subserve many physiological functions, including regulation of cell volume, intracellular pH, fluid secretion, and stabilization of the resting membrane potential. Cl⁻ is absorbed from the gastrointestinal tract is mediated by Cl⁻/HCO₃ ⁻ exchanger. Recent studies have suggested that a major Cl⁻/HCO₃ ⁻ exchanger is SLC26A3. Since multiple isoforms of the Cl⁻/HCO₃⁻ exchanger are co-expressed in an intact colonic cell, complicating the functional analysis of an individual isoform, we generated an N-terminal hemagglutinin epitope-tagged human SLC26A3 construct and expressed transiently in CHO cells by using inducible gene expression systems. Using this system, we have previously characterized SLC26A3 by measuring of its activity with fluorescent pH-sensitive indicators, BCECF. To assess the validity of pH measurements, we measured the Cl⁻/HCO₃⁻ exchange activity by using chloride-sensitive dye, MQAE. We first measured Cl⁻/HCO₃⁻ exchange activity by MQAE and then measured its activity by using pH sensitive dye in the same cells. Cl⁻/HCO₃⁻ exchange rate measured by using MQAE was 10-20-fold greater than the rate measured by using BCECF. In addition, a carbonic anhydrase inhibitor acetazolamide partially inhibited Cl⁻/HCO₃⁻ exchange activity. These results suggest that even in the presence of a carbonic anhydrase, its reaction rate is not enough for intracellular pH measurements to assess the Cl⁻/HCO₃⁻ exchange activity in CHO cells. [J Physiol Sci. 2006;56 Suppl:S114]

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