Renal organic anion transporters play an important role in the handling of a number of endogenous and exogenous anionic substances in the kidney. In this study, we investigated prostaglandin E₂ (PGE₂) transport properties and intrarenal localization of mouse organic anion transporter 3 (mOat3). When expressed in Xenopus oocytes, mOat3 mediated the time- and concentration-dependent transport of PGE₂ (Km: 1.48 μM). PGE₂ transport mediated by mOat3 was trans-stimulated by intracellular glutarate injected into the oocytes. PGE₂ efflux via mOat3 was also trans-stimulated by extracellular glutarate. Thus, mOat3 was shown to mediate the bidirectional transport of PGE₂, partly coupled to the dicarboxylate exchange mechanism. Immunohistochemical study revealed that mOat3 protein was localized at the basolateral membrane of renal proximal and distal tubules. Furthermore, diffuse expression of mOat3, including expression in the basolateral membrane in macula densa (MD) cells, was observed. These results indicate that mOat3 plays an important role as basolateral transport pathway of PGE₂ in the distal nephron including MD cells that may constitute one of the indispensable steps for renin release and regulation of the tubuloglomerular feedback (TGF) mechanism. [J Physiol Sci. 2007;57 Suppl:S217]

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Albumin has a thiol group, called HMA (reduced form) when it is a free state. In contrast, albumin in which the thiol group is oxidized is called HNA (oxidized form). The major HNA is mixed disulfide with cystine (HNA-1), while the other is more oxidized state (HNA-2). We have studied the albumin thiol oxidation and the serum protein carbonyl formation in patients with chronic kidney disease (CKD). In CKD, the stage is classified by estimating the value for creatinine clearance using the Cockcroft-Gault formula (stage 1 to 5). In this study, 25 patients with CKD were divided into four groups (group A = stages 1 and 2; group B = stage 3a; group C = stage 3b; group D = stages 4 and 5). While the stage was advanced, mean value for HMA fraction was progressively decreased from 79.0 (group A) to 65.3% (group D) and that for HNA fraction was increased from 21.0 (group A) to 34.7% (group D). Serum protein carbonyl content was also progressively increased from 0.73 (group A) to 1.09 nM/mg (group D). In relationship between the thiol oxidation and the carbonyl formation, value for carbonyl content was not correlated with that for HNA-1 (R = 0.35, P = 0.09) but significantly with that for HNA-2 (R = 0.58, P <0.005). These results suggest that the measurements for albumin thiol oxidation and serum protein carbonylation in patients might be a useful biomarker in advancing stages of CKD, such as progressing renal dysfunction. [J Physiol Sci. 2007;57 Suppl:S217]

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In the present study, a month recording of renal sympathetic nerve activity (RSNA) was carried out to study the potential role of arterial baroreceptor in long-term regulation of RSNA and sodium excretion during sodium loading in freely moving rats. Wistar rats, which were previously sino-aortic denervated, were instrumented chronically with electrodes for the measurements of RSNA and electrocardiogram. At least 7 days after the implantation of the electrodes, the animals were housed in individual metabolic cages and then RSNA, heart rate and sodium balance were measured over three weeks. Animals were allowed to drink 3 different concentration of sodium chloride solutions; that was 0, 50, 308 mEq/NaCl. The rats were exposed to either 3 days sodium loading (308 mEq/NaCl) or unloading (0 mEq/NaCl) following 3 days control (50 mEq/NaCl) period. RSNA remained unchanged during both 308 mEq/NaCl and 0 mEq/NaCl loading. Heart rate also did not change significantly relative to the control levels throughout the period of both 308 mEq/NaCl and 0 mEq/NaCl loadings. Sodium excretion increased and decreased significantly (p<0.05) over the 3 days loading and unloading of sodium, respectively. These results suggest that baroreflex control of RSNA seems to play a minor role in regulating sodium excretion during prolonged sodium loading/unloading in rats. [J Physiol Sci. 2007;57 Suppl:S218]

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The kidney macula densa (MD) control the tubuloglomerular feedback (TGF) system for regulation of the total body fluid volume. The MD cells express cyclooxygenase-2 (COX-2) and COX-2-derived prostaglandins stimulate the renin release from the granular cells of afferent artelioles, especially during volume depletion. However, the subcellular signaling mechanism of COX-2 gene expression remains unclear. For this purpose, newly established mouse macula densa cells (NE-MD) were used to directly determine whether COX-2 gene expression was regulated by either low [NaCl] concentration or intracellular cAMP/Ca²⁺. In the present study, COX-2 gene expression was evaluated by real-time quantitative PCR. Real-time PCR showed a significant increase in COX-2 gene expression in the samples with low [Cl⁻] solution and culture media plus furosemide, an inhibitor of Na⁺-K⁺-2Cl⁻ cotransporter, compared to controls. However, no increase was observed in the samples with a low [Na⁺] solution. Low Cl⁻-induced increase in COX-2 expression was completely reversed in the presence of either BAPTA-AM, a membrane-permeable Ca²⁺ chelator, EIPA, an inhibitor of Na⁺/H⁺ exchanger, or H-89, an inhibitor of protein kinase A. Finally, COX-2 gene expression was significantly increased in the presence of forskolin (normal solution), whereas it was reversed by H-89. In conclusion, low [Cl⁻]-induced COX-2 gene expression may be regulated by intracellular cAMP, pH, Ca²⁺, and [NaCl]. [J Physiol Sci. 2007;57 Suppl:S218]

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Our previous studies revealed that orexin-A and neuropeptide Y, which are potent orexigenic peptides, induced relaxation of the proximal stomach. This relaxation probably facilitates reservoir function of the stomach. Ghrelin is a recently identified endogenous legand for the grows hormone secretagogue receptor (GHS-R) and has orexigenic effect. Up to the present, the effect of ghrelin on the proximal stomach motility is not known. In the present study, the effect of ghrelin on motility of the proximal stomach was examined in anaesthetized rats. Intragastric pressure (IGP) was measured using a balloon situated in the proximal part of the stomach. The administration of ghrelin into the fourth ventricle induced relaxation of the proximal stomach in a dose-dependent manner. Simultaneous administration of ghrelin and GHS-R antagonist ([D-Lys³] GHRP-6) did not induce significant change in IGP. Microinjections of ghrelin into the dorsal vagal complex (DVC) also induced relaxation of the proximal stomach. Immunohistochemical staining for GHS-R showed labeled cells in the DVC, where most of cells were appeared in the nucleus of the solitary tract and the dorsal motor nucleus of the vagus. The bilateral sectioning of the vagi at the cervical level abolished the relaxation induced by the administration of ghrelin into the fourth ventricle. These results indicate that ghrelin induces the relaxation of the proximal stomach via the GHS-R situated in the DVC. [J Physiol Sci. 2007;57 Suppl:S218]

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Hydra, a member of phylum Cnidaria, undergoes three types of digestive movements (Shimizu et al., 1984), esophagal reflex-like, peristaltic reflex-like and defecation reflex-like movements. Since Hydra belongs to Hydrozoa, the most latent class of Cnidaria, we extended our analysis to a more ancestral class of the phylum, the class Anthozoa that includes popular organisms e.g. corals and sea anemone. Nematostella vectensis is a well studied Anthozoa. We found no movements related to digestion. Instead, we find from archival sources that peristalsis unrelated to digestion occurs in this organism. Unlike peristalsis in Hydra, peristalsis in Nematostella was characterized by (1) very narrow bands of constriction of the tissue, (2) very drastic constriction of the tissue which can never be seen in intestinal tissue. Also, we find from recent development in genome project in Hydra and Nematostella that Hydra has gap junction communication and related genes whrereas Nematostella has no gap junction communication or related genes. To examine the effect of gap junction communication in Hydra, we tried to block the communication by treatment with heptanol. We found that the digestive movements are all blocked by the treatment. From this and other observations, we propose a possibility that the gain of gap junction communication was an evolutionary cue for the appearance of digestion related peristaltic movements. [J Physiol Sci. 2007;57 Suppl:S218]

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This report deals with effects of gastro-intestinal infusion of amino acids on the activity of the vagal afferents in anesthetized rats. Intragastric infusion of isotonicMSG solution facilitated afferent activity of vagal gastric nerve, however, gastric infusion of other essential and non-essential amino acid showed no effect. On the other hand, intraduodenal infusion of isotonic essential amino acid solutions such as valine, leucine, isoleucine, lysine, tryptophane and phenylalanine and most of non-essential amino acid including MSG resulted in activation of vagal celiac afferents. On the contrary, infusion of two esential amino acids, methionine and threonine,and non-essential amino acids, glycine and histidine, suppressed afferent activity of vagal celiac nerve. These observations indicate that amino acid sensors in the gastric wall send MSG information in the gastric canal to the cenral nervous system through vagal gastric afferents, and that amino acid sensors in the duodeno-intestinal wall send information on each essential and non-essential amino acid tothe central nervous system through vagal celiac afferents. These observations suggest that amino acid sensors in the intestinal wall may play an important role in regulation of metabolic functions including protein metabolism through afferent system in vagal celiac nerve. [J Physiol Sci. 2007;57 Suppl:S219]

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A Cl⁻/HCO₃⁻ exchanger slc26a3 is expressed in the large intestine. To determine whether slc26a3 is involved in HCO₃⁻ secretion in the large intestine, we examined Cl⁻/HCO₃⁻ exchange activity of HEK293 cells transfected with mouse slc26a3 by microfluorometry using a pH-sensitive dye BCECF. The result showed that slc26a3 was largely inhibited by 0.1 mM glibenclamide while it was hardly inhibited by 30 &muM NPPB. In contrast, the Cl⁻-dependent bicarbonate secretion in the mouse large intestine (cecum) as determined by using pH-stat method was not inhibited by 0.1 mM glibenclamide while it was largely inhibited by 30 &muM NPPB. Therefore, slc26a3 may not play a major role in the Cl⁻-dependent bicarbonate secretion in the cecum. [J Physiol Sci. 2007;57 Suppl:S219]

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[Background] Recently, embryonic stem (ES) cells were shown to spontaneously (without LIF) give rise to a functional organ-like unit, the "ES gut " which undergoes rhythmic contractions and is comprised of enteric derivatives of all three embryonic germ layers: epithelial cells (endoderm), smooth muscle cells and interstitial cells of Cajal (ICCs) (mesoderm), and a small number of enteric neurons (ectoderm), but no ganglia. The ICC network within the GI tract is responsible for the generation of electrical pacemaker activity. [Results] After 6-7 day hanging drop culture of embryoid body without LIF, the ES gut began to exhibit phasic contractions on about day-21 of outgrowth culture. The ES gut with ICC network generated pacemaker potentials and exhibited Ca²⁺ oscillation, thereby generated phasic contractions. On the other hand, when imanitib 10⁻⁵ M, a tyrosine kinase receptor c-KIT inhibitor, was added to hanging drop culture medium, it inhibited differentiation of ICCs and thus depressed spontaneous motility, although it appeared that imatinib 10⁻⁶ -10⁻⁷ M rather enhanced the differentiation of ICCs. [Conclusion]The present results revealed the role of a tyrosine kinase receptor c-KIT in differentiating ICCs and the role of ICCs in generating spontaneous motility in ES gut. This work was supported by Grants-in-aid for Scientific Research (14657311,16650090,17300130 and 18630007 for MT and 15300134 for SN) from the Ministry of Education, Science, Sports and Culture of Japan. [J Physiol Sci. 2007;57 Suppl:S219]

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Growing evidence has been accumulated that myofibroblasts may play a pivotal role in inflammatory and neoplastic processes in the gastrointestinal tract, especially via release of an important inflammatory mediator, prostaglandin E2. Part of the release has been shown to be dependent on Ca²⁺, but what source contributes thereto remains entirely unclear. In this study, we explored the potential role of transient receptor potential (TRP) proteins for this process by evaluating their expression profile and function in the human colonic myofibroblast cell line CCD-18Co. RT-PCR amplification with specific primers detected the expression of seven TRP isoforms in CCD-18Co cells, including TRPC1, TRPC5, TRPC6, TRPV2, TRPV4, TRPM3 and TRPM7. Activators so far known for these TRP isoforms such as OAG, hypoosmolarity, 2-APB and sphinganine evoked elevation of the intracellular Ca²⁺ concentration ([Ca²⁺]i). Whole-cell current recording confirmed that all these activators could activate non-selective cationic currents, although their magnitudes and pattern of activation were highly variable. The observed I-V relationship and pharmacological sensitivity suggest that at least five out of the seven TRP isoforms, i.e. TRPC6, TRPV2, TRPV4, TRPM3 and TRPM7, are functional. In addition, one of well-established inflammatory cytokines in the gut, TNFα, was also able to evoke [Ca²⁺]i rise and a concomitant nonselective cationic current with properties reminiscent of TRPC or TRPV subfamily members. [J Physiol Sci. 2007;57 Suppl:S219]

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In hypoxia, pulmonary arteries undergo vasocontriction (HPV). As a plausible mediator of the HPV, we focused on ATP which is released from RBC during hypoxia. As for pulmonary artery, an application of intravascular ATP caused constriction both in endothelium-denuded and ?intact ones. In contrast, intravascular ATP dilated coronary artery both in endothelium-denuded and -intact ones. The ATP-induced dilation of coronary artery was mimicked by ATPγS and 2-meS-ATP but not by UTP or UDP. Next, ATP-induced membrane current and intracellular Ca²⁺ signals ([Ca²⁺]_i) were compared in pulmonary (PASMCs) and coronary (CASMCs) myocytes. In PASMCs, extracellular ATP activated P2X currents while not in CASMCs. Extracellular ATP to PASMCs induced a sharp increase in [Ca²⁺]_i which was composed of both Ca²⁺ influx and store release. Both the P2X and P2Y responses were greater in PASMCs from the small (200-500 μm o.d.) arteries than the large (0.8–1.0 mm o.d.) ones. In CASMCs, however, extracellular ATP activated sparse P2X currents and intracellular Ca²⁺ signals. To explore the physiological relevance of these differences, we examined the effect of ATP on HPV in isolated rat lung model. In isolated lung model, application of ATP caused dose-dependent constriction and suramin, P2 antagonist, inhibited the HPV. Taken together, these results suggest that pulmonary and coronary arteries respond differently to extracellular ATP via purinergic receptors. This difference may explain the mechanism of opposite behavior of these arteries in hypoxia. [J Physiol Sci. 2007;57 Suppl:S220]

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Under chronic hypoxia, tumour cells undergo adaptive changes involving hypoxia-inducible factors (HIFs). Here we show that ion currents mediated by Ca²⁺-activated K⁺ (K_Ca) channels in human melanoma IGR1 cells are increased by chronic hypoxia (3% O₂), as well as by hypoxia mimetics. This increase involves the HIF system as confirmed by overexpression of HIF-1α. Furthermore, overexpression of the von Hippel-Lindau tumour suppressor gene (VHL) leads to decrease in K_Ca currents. Under normoxic conditions the K_Ca channels in IGR1 cells showed pharmacological characteristics of intermediate conductance K_Ca subtype IK channels, whereas the subtype SK2 channels were up-regulated under hypoxia, shown with pharmacological tools and with mRNA analysis. Hypoxia increased cell proliferation, but the K_Ca channel blockers apamin and charybdotoxin slowed down cell growth, particularly under hypoxic conditions. Similar results were obtained for the human melanoma cell line IGR39 and for acutely isolated cells from a biopsy of a melanoma metastasis. Thus, up-regulation of K_Ca channels may be a novel mechanism by which HIFs can contribute to the malignant phenotype of human tumour cells. [J Physiol Sci. 2007;57 Suppl:S220]

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In the ciliary muscle, the tonic component of the contraction produced by muscarinic stimulation is highly dependent on Ca²⁺ provided by influx through receptor-operated cation channels (ROCs). Here we have examined effects of YM-254890, a G_q/11-specific inhibitor, on contraction, ROC currents and [Ca²⁺]_i elevation induced by carbachol (CCh). Methods Bovine eyes were obtained from a local slaughterhouse. Isometric tension was recorded from excised ciliary muscle bundles using a strain-gauge transducer. For the other experiments, ciliary muscle cells were dispersed with collagenase and cultured for 1-5 days. Whole-cell currents were recorded by voltage clamp. The intracellular free Ca²⁺ [Ca²⁺]_i concentration was recorded using Fluo-4 (Invitrogen) as the indicator. Results In the muscle bundles, superfusion of 2 μM-CCh evoked a sustained contraction. Both phasic and tonic components of this contraction were inhibited by YM-254890 (3-10 μM) in a dose-dependent manner. In the cultured cells, CCh (0.05-10 μM) evoked a ROC current as well as an elevation of the [Ca²⁺]_i. Both initial and sustained phases of these CCh-evoked responses were abrogated by YM-254890 (3-10 μM). Immunostaining revealed a dense distribution of G_q/11 in the vicinity of the cytoplasmic side of the plasma membrane of the cultured cells. Conclusion G_q/11 appears to be critically involved in Ca²⁺ mobilization in the tonic as well as phasic component of the contraction in the bovine ciliary muscle. [J Physiol Sci. 2007;57 Suppl:S220]

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In bovine ciliary muscle (BCM) cells, stimulation of M₃-muscarinic receptor (MR₃) opens two types of receptor-operated non-selective cation channel (ROC) which serve as major pathways for Ca²⁺ entry during the tonic phase of contraction. It has been shown by RT-PCR that BCM contains mRNA for four TRP channel homologues (TRPC1, 3, 4, 6) commonly regarded as molecular candidates for ROCs. Here we tried to visually identify these TRPCs in the plasma membrane of BCM by immunofluorescence microscopy. After 3-5 days culture of BCM cells on fibronectin-coated glass in a serum-free media, the body of the cells was removed by gentle sonication under a hypotonic condition. The plasma membrane remaining attached on the glass was treated with primary antibodies against putative cytoplasmic segments of the four TRPCs and visualized with secondary antibodies labeled with a fluorescent dye. The membrane preparations were also similarly immunostained with antibodies against MR₃, α-actin and G_αq/11. All the anti-TRPC antibodies used gave many microscopically visible spots of immunofluorescence (roughly 1 spots/μm²) in the cell-free membrane preparation which was also positively stained with antibodies against MR₃ and α-actin. Anti-G_αq/11 antibodies used showed the dense distribution G_αq/11 proteins in or in the vicinity of the plasma membrane. The present results clearly demonstrate the existence of the four TRPC homologues, MR₃, and G_αq/11 in the plasma membrane of the BCM. [J Physiol Sci. 2007;57 Suppl:S221]

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A voltage-regulated phosphatase idenfieid from Ciona intestinalis (Ci-VSP) has ion channel-like voltage sensor with its downstream domain of the cytoplasmic phosphatase (Murata et al, Nature, 2005). A Ci-VSP orthologue from zebrafish (Z-VSP) shows similar properties: phosphoinositide phosphatase activities are voltage-dependent (this meeting, last year),. Interactions among modules are often bidirectional. To address whether the phosphatase domain (PD) modifies properties of voltage sensor domain, we compared gating currents between enzyme-active condition and enzyme-defective condition. When the critical amino acid residue, cysteine, in the active center of PD of Z-VSP, corresponding to the same residue conserved among all protein tyrosine phosphatases (PTP) and PTENs, was mutated to serine, its phosphatase activity was abolished. In this mutant, gating current measured in heterologous expression in HEK tsA201 cells showed unchanged charge versus current (Q-V) curve whereas kinetics was remarkably faster than the wild type protein. In addition, pervanadate, an inhibitor of phosphatase activity of PTP or PTEN, that binds to the phosphatase active site, mimicked the phenotypes of the C-S mutant both of Z-VSP and Ci-VSP. These findings implicate that interactions between VSD and effector domain are bidirectional. [J Physiol Sci. 2007;57 Suppl:S221]

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It is reported that thermosensitive-TRP channels (thermo-TRPs) are sensitive to a wide range of temperatures and involved in nociception in terminals of dorsal root ganglia (DRG) neurons. Especially, TRPV1, TRPV2, TRPA1 and TRPM8 are expressed in adult mouse DRG neurons to exert nociception and/or thermosensation. In general, it is well known that ion channels have distinct roles between embryonic and adult stages. For example, voltage-gated potassium channels regulate cell proliferations, cell migrations or axon extensions during neural development. Analogous with these observations, the thermo-TRPs also might have unexpected specific roles in embryonic neurons compared with those in adult stage. Interestingly, no investigation has been performed to identify when the thermo-TRPs were expressed in developing DRG by focusing on their specific roles in the embryonic stages. Identifications of expression timing of the channels are necessary to clarify the specific roles of the thermo-TRPs in embryo. Therefore, we examined the expression profiles of TRPV1, TRPV2, TRPA1 and TRPM8 in developing mouse DRG and spinal cord by RT-PCR, in situ hybridization, Western blotting, immunohistochemistry and calcium imaging. Surprisingly, the expressions of those thermo-TRPs in DRG were already observed in early embryonic stages, and some of them were observed in spinal cord as well, suggesting that those channels, might have specific roles in the embryonic stages. [J Physiol Sci. 2007;57 Suppl:S221]

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Objective: Although Ca homeostasis is essential to the maintenance of lens transparency and the control of lens fiber cell differentiation, the molecular mechanism of Ca regulation remains unclear. Na/Ca exchanger (NCX) catalyzed reversible exchange of Na for Ca across the plasma membrane. To reveal the role of NCX in the control of lens Ca homeostasis, we characterized NCX isoforms expressed in developing rat lens. Methods: The lens from neonatal (post natal day 5, 7 and 9) and adult Wistar rats were homogenized in the lysis buffer containing 20mM HEPES (pH 7.4), 150mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 2μg/ml leupeptin, 1μg/ml aprotinin, 200μM phenylmethylsulfonyl fluoride, and 200mM benzamidine hydrochroride. The lysates were centrifuged and resultant supernatants were subjected to SDS-PAGE on 8.5% gel and then to immunoblotting with anti-NCX1, 2, and 3 antibody. Results and Discussion: Anti-NCX1 antibody recognized the molecule of 120KDa, which is mature NCX1, in both neonatal and adult rat lens, while Anti-NCX2 and 3 antibodies did not detect any band. In rat lens, including developmental period, NCX1 acts a role as a Ca extrusion system to modulate the intracellular Ca concentration. To establish the causal relationship between NCX activity and lens transparency, we are trying a characterization and biochemical analysis of NCX1 using the lens of experimental rat cataract models. [J Physiol Sci. 2007;57 Suppl:S221]

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The aim of this study is to elucidate the functional role of large conductance, stretch-activated, Ca²⁺-activated K⁺ (SAKca) channel originally cloned from cultured chick ventricular myocytes (CCVM). We previously demonstrated that SAKca channel can be activated by membrane stretch as well as membrane depolarization and intracellular calcium. However, the physiological importance of SAKca channel in cardiovascular system remains unknown. To pursue the functional role of cardiac SAKca channel, conventional whole-cell patch-clamp techniques were applied to human embryonic kidney (HEK293) cell lines transfecting cloned SAKca genes and CCVM (isolated from chick embryos 10 day old). These recordings show that the voltage dependent outward currents in CCVM as well as the HEK 293 cell lines were inhibited by BK channel specific blocker charybdotoxin (ChTX, 10 nM). Thus, we hypothesized that ChTX could prolong the repolarization phase of action potentials (APs) of CCVM. To clarify this hypothesis, we recorded evoked APs of CCVM in the whole-cell current clamp technique, and examined the effects of ChTX on the APs. The results show that repolarization phases of evoked APs were not affected by ChTX (30 nM). The series of results implied that SAKca channels in CCVM could not at least activated in physiological conditions but possibly in pathophysiological conditions such as overloading of mechanical stimuli and/or hypoxia, and might play some defensive roles against prolongation of repolarization phase on APs in these conditions. [J Physiol Sci. 2007;57 Suppl:S222]

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The Role of TRP Channels in Mechanotransduction of HUVECYuki Katanosaka, Tomohiko Suemori, Keiji Naruse. Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University.Human umbilical vein endothelial cells (HUVEC) at the interface between the bloodstream and the vessel wall are continuously subjected to mechanical stimulations in vivo, such as shear stress, hydrostatic pressure and stretch, and it is widely recognized that they are playing an important physiological role in cardiovascular system. In response to stretch, we reported that intracellular Ca²⁺ increased transiently through the activation of mechanosensitive (MS) cation channel in HUVEC. However, the molecular entities that form the MS cation channel in the cell remain unknown. Recent papers report some members of the transient receptor potential (TRP) channels have been suggested as one of components of MS channels. To identify the molecules forming MS in HUVEC, we investigated the role of TRP channels in this cell. We observed the expression of several isoforms of TRP channels in HUVEC using immunoblotting method. Among these channels, hTRPV2 was localized very close to the mesh-work structure of the cortical-actin in semi-intact plane membrane of HUVEC. It was noteworthy that stretch-induced change in [Ca²⁺]_i measured by fura2 was significantly higher in hTRPV2 transfected HUVEC and that it was significantly inhibited by the treatment of morphorino oligo against hTRPV2. From these results, we discuss molecular complex that forms MS channel, above all the possible involvement of TRPV2 in HUVEC. [J Physiol Sci. 2007;57 Suppl:S222]

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The bacterial mechanosensitive channel MscS (mechanosensitive channel of small conductance) is known to function as a defensive device against cell rupture by hypotonic downshock. However, structure basis of its mechano-gating is little known. MscS is composed of a transmembrane (TM) part and a large cytoplasmic vestibule (CV). In this study, we investigated the role of interaction between these tow parts in MscS gating by focusing on the electrostatic interaction between charged residues in the respective parts. D62 is a negatively charged residue located in the loop connecting the TM1 and the TM2 helices and has been proposed to interact with R128 that is located at the upper surface of the CV. We examined whether D62 interacts with the CV, and if so, how the interaction contributes to MscS gating. Neutralization (D62C) or substitution (D62R) with a positively charged residue of D62 increased dramatically the threshold level for activation of MscS. In contrast, substitution with negatively charged residue (D62E) did not change the threshold. Interestingly the negative effect of D62R on mechano-activation was largely complemented by reversing the charge sign of the residue at 131 (R131D) but not with the residue at 128 (R128D), both of the residues are located in the CV being close to D62. Above findings suggest that the CV affects crucially the mechano-gating of MscS through the electrostatic interaction between D62 and R131. [J Physiol Sci. 2007;57 Suppl:S222]

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Removal of external K⁺ ions abolishes not only inward currents but also outward currents through inwardly rectifying K⁺ channels which determine the resting potential. Negatively charged amino acid residues located near the external entrance are expected to attract and stabilize K⁺ ions to modulate channel gating or block. To understand the significance of negative charges (D112, D114, E125, D152 and E153) in the extracellular loops of the inwardly rectifying K⁺ (Kir2.1) channel, single or double point mutants were constructed and transfected into COS-1 and HEK293 cells. Each single point mutant expressed functional channels. Cells transfected with the D152N/E153Q construct did not show any inwardly rectifying K⁺ currents, although fluorescence images confirmed that the channel proteins produced by the D152N/E153Q construct were transported to the cell surface. While a tandem tetramer with one D152N subunit and three double mutant subunits, D152N-(D152N/E153Q)3, did not express functional channels, a tandem tetramer with one E153Q subunit and three double mutant subunits, E153Q-(D152N/E153Q)3, and that with two D152N subunits and two double mutant subunits, (D152N)2-(D152N/E153Q)2 expressed Kir2.1 channels. These results suggest that one negative charge of D152 or two negative charges of E153 are required for Kir2.1 channels to function. It is suggested that K⁺ ions may be stabilized at D152 and/or E153 site and K⁺ stabilization at this site sets the channel to function. [J Physiol Sci. 2007;57 Suppl:S222]

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Rat bone marrow stromal cells express G protein-coupled purinergic receptor (P2Y₂ receptor). We investigated intracellular Ca²⁺ signals in these cells using Fura 2-AM and found that UTP, an agonist of P2Y₂ receptor, induced Ca²⁺ rise (Ichikawa, J. and Gemba, H. (2005) Jpn.J.Physiol. 55(Suppl.): S124, Ichikawa, J. and Gemba, H. (2006) J.Physiol.Sci. 56(Suppl.): S155). In several types of cells, it was reported that the activation of purinergic receptors induced cell proliferation (J.Neurochem. (2006) 99(3): 913-923, Br.J.Pharmacol. (2003) 139(6): 1119-1126). Here, we investigated the effect of the activation of P2Y₂ receptor on cell proliferation of rat bone marrow stromal cells. We added the agonist of P2Y₂ receptor (UTP or ATP) to culture media, and assayed cell number by measuring Calcein-AM fluorencence intensity. In the presence of 10% FCS, the addition of UTP (or ATP, 1-100μM) enhanced cell proliferation. At higher concentration (1000μM), UTP (or ATP) induced cell death. In serum-free condition, UTP (or ATP, 1-1000μM) did not induce the remarkable cell proliferation although they expressed functional P2Y₂ receptor in these cultures. It is suggested that UTP (or ATP) itself does not induce cell proliferation but assists it in serum-existing condition. [J Physiol Sci. 2007;57 Suppl:S223]

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It has been thought that the etiology of bladder cooling reflex is related to unmyelinated C-afferent fibers in pelvic nerve that is not involved in the normal micturition reflex. Some investigators reported that the bladder mucosa have the thermoreceptor and can detect cold sensation that generally occurred below 30°C.) Recently, TRPM8 has been identified in urogenital organ, but it is unknown the function of this receptor in these area. We investigated the relationship between TRPM8 and voiding behavior. A catheter was inserted into wistar female rat bladder dome, and the cystometry was performed postoperative 72 hours later. After saline (37°C) was infused into the bladder for two hours, menthol (Sigma-0.06 mM, 0.6 mM, 6 mM) was infused applied for 15 min. Menthol increased a voiding pressure in a dose-dependent manner but no change in menthol decreased a voiding interval. The immunohistochemistry was done to define the distribution of TRPM8 in lower urinary tract. Fluorogold was injected into bladder wall, after 5 days, the bladder and L6-S1 DRG was extirpated and enbeded in OCT and sectioned on a cryostat. In DRG, TRPM8 positive neurons contained fluorogold, while in bladder, urotherial layer was positive to TRPM8, and nerve fibers were merged with NF200. [J Physiol Sci. 2007;57 Suppl:S223]

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Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels contribute to rhythmic activity, membrane excitability, and synaptic transmission. We previously isolated cDNA for HCN4. HCN1 showed the fastest activation kinetics while HCN4 showed the slowest. We previously reported that the different kinetics among subtypes are generated mainly by S1 and the S1-S2 loop in HCN channels. Here, we investigated the structural basis of the ability of S1 to affect the voltage-dependency by introducing mutations in S1. We found that one Tryptophan mutation introduced in the middle of S1 (L139W) induced a positive voltage-shift of activation by 87 mV with an incomplete closure even at an extremely depolarizing potential. Further mutations at position 139 also disrupted channel closure and shifted the voltage-dependency. These results suggest that position 139 in S1 likely faces a mobile part of the S4 voltage sensor and may interact with it. [J Physiol Sci. 2007;57 Suppl:S223]

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The dependence of mitochondrial Na⁺/Ca²⁺ exchanger (mNCX) on mitochondrial membrane potential (MMP) has been controversial. We investigated the MMP dependence of mNCX by measuring MMP and mitochondrial Ca²⁺ (Ca²⁺_m) using TMRE and Rhod-2, respectively, in saponin-permeabilized rat ventricular myocytes. The mNCX activity was measured as a decline of Ca²⁺_m upon decreasing cytoplasmic Ca²⁺ (Ca²⁺ _c) from 300 to 0 nM in the presence of Na⁺. The half maximum concentration for Na+ was ∼1 mM. The activation of Ca²⁺ efflux via mNCX by removing Ca²⁺ _c did not significantly affect the MMP. Eliminating mitochondrial substrates (ADP, K-pyruvate, K₂HPO₄, succinic acid, malic acid, and K-glutamic acid) depolarized MMP but did not significantly affect the rate of Ca²⁺ efflux via mNCX. Mitochondrial uncoupler, FCCP (1μM), also depolarized MMP but only slightly slowed the Ca²⁺ efflux rate. These data suggested that mNCX has only a weak dependence on MMP. [J Physiol Sci. 2007;57 Suppl:S223]

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Intracellular Ca²⁺ concentration elevation is essential for activation of Ca²⁺-dependent NO synthases (NOSs). However, molecular components eliciting this mechanism remain unclear. Here, we report a novel activation mechanism mediated by cysteine S-nitrosylation in transient receptor potential (TRP) channels. Recombinant TRPC5 and TRPV1, which are commonly classified as receptor-activated channel and thermosensor channel respectively, induce entry of Ca²⁺ into cells in response to nitric oxide (NO). Labeling and functional assays using cysteine mutants, together with membrane sidedness in activating reactive disulfides, show that cytoplasmically accessible Cys553 and nearby Cys558 are nitrosylation sites mediating NO sensitivity in TRPC5. The responsive TRP proteins have conserved cysteines on the same N-terminal side of the pore region. Notably, nitrosylation of native TRPC5 upon G protein-coupled ATP receptor stimulation elicits entry of Ca²⁺ into endothelial cells. These findings suggest that native TRPC5 is essential for Ca²⁺ influx activated by NO via eNOS upon receptor stimulation in endothelial cells. Forthermore, the group of native S-nitrosylation-sensitive TRP channels mediates a ubiquitous mechanism that is critical for feedback regulation of Ca²⁺ signals by NO. [J Physiol Sci. 2007;57 Suppl:S224]

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Active zones (AZs) are highly specialized sites for release of neurotransmitter in presynaptic nerve terminals. The spacing between voltage-dependent calcium channels (VDCCs) and synaptic vesicles at AZs is thought to influence the dynamic properties of synaptic transmission. However, molecular determinants that maintain or regulate appropriate distance between vesicles and VDCCs have been elusive. Recently we have demonstrated a novel molecular interaction between an AZ scaffolding protein and VDCCs. To elucidate the functional significance of this direct coupling, we characterized whole-cell Ba²⁺ currents through recombinant P/Q-type VDCC expressed as α_1Aα₂/δβ_1a complex in baby hamster kidney cells. The AZ protein induced a pronounced deceleration of inactivation rate and a depolarizing shift of the inactivation curve. During 2-s voltage-displacement to -30 mV, which is the threshold of the P/Q-type VDCC activation, almost all of the channels were inactivated in the absence of the AZ protein (closed-state inactivation), but less than 20% of the channels were inactivated in the presence of the AZ protein. Currents evoked by trains of action potential waveforms, a more physiological voltage-clamp protocol used particularly to reveal closed-state inactivation, further support profound suppression of voltage-dependent inactivation by the AZ protein. Thus, the AZ protein coordinates calcium signaling and spatial organization of molecular constituents in presynaptic AZ. [J Physiol Sci. 2007;57 Suppl:S224]

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The spacing between voltage-dependent calcium channels (VDCCs) and vesicles at active zones is thought to influence the dynamic properties of synaptic transmission. However, molecular determinants that maintain or regulate synaptic vesicles and VDCCs within a physiologically appropriate distance have been elusive. Here, we demonstrate a novel molecular interaction between an active zone protein and VDCCs, that controls neurotransmitter release. GST-pull down and immunoprecipitation assay showed that the active zone protein associated with VDCC β-subunits via its C-terminus. In neuroendocrine PC12 cells, acetylcholine release was significantly potentiated by the full-length, N-terminus, and C-terminus constructs, but the proportion of membrane-docked vesicles was enhanced only by the full-length. Thus, the active zone protein associated with VDCC β-subunits near the active zone supports neurotransmitter release via anchoring of neurotransmitter-containing vesicles in the vicinity of VDCCs. [J Physiol Sci. 2007;57 Suppl:S224]

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The tight junction known as a barrier between apical and basolateral sides has the paracellular conductance (Gp) selective for ions, water and macromolecules. However, the regulatory mechanism of Gp is still unknown. To clarify the mechanism, we studied the effect of water fluxes on the paracellular ion conductance in renal A6 cells, measuring the transepithelial potential and the Gp under the condition that the transcellular ion transport was abolished with applying channel blockers. We estimated the Na⁺-selective Gp (G_Na) and the Cl⁻-selective Gp (G_Cl) based on the equilibrium potentials for Na⁺ and Cl⁻ between apical and basolateral solutions. The basolateral hypotonicity or the apical hypertonicity significantly increased G_Na, indicating that the secretion-directed water flux generated by the osmotic gradient between apical and basolateral sides increases G_Na. In contrast, the apical hypotonicity increased Gp with no discrimination between G_Na and G_Cl, indicating the absorption-directed water flux increases Gp with no ion selectivity. The secretion-directed water flux without NaCl gradients itself had little effects on Gp, but produced precondition generating Gp elevation observed with NaCl gradients. These results indicate that an inherited system in paracellular pathway regulates the Gp with ion selectivity by sensing the water flux including the direction. Supported by JSPS 18890157, 17590191, 17390057, and 18659056. [J Physiol Sci. 2007;57 Suppl:S224]

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The amygdala and dopaminergic innervation thereonto are considered to cooperatively regulate emotional states and behaviors. In the present experiments, we investigated the effects of dopamine on lateral amygdala (LA) neurons by whole cell recordings in rat brain slices. Bath-application of dopamine induced slow afterdepolarization (sADP) in LA neurons. This sADP lasted for more than 5 seconds and was induced by dopamine in a concentration-dependent manner. D1 receptor agonists induced the same sADP. Previous reports have repeatedly suggested that sADP could be triggered by calcium influx. Consistently, calcium channel blockers or chelating intracellular calcium inhibited the present dopamine-induced sADP. A membrane conductance decreased at the peak of sADP current (I_sADP). Also, the present I_sADP was suppressed by including cesium in the pipette solution. The I-V relationship of net I_sADP induced by dopamine was shifted as the external concentration of potassium was raised. The reversal potential of the net I_sADP was identical to that of potassium. These results suggest that the present dopamine-induced modulation of LA neuron excitability may depend on decrease in potassium current. [J Physiol Sci. 2007;57 Suppl:S225]

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The aim of our present study was to clarify the presence and the functional importance of ATP-sensitive K⁺ channel (K_ATP channel) in pig detrusor since the poor studies of K_ATP channel in pig detrusor smooth muscle cells.The patch clamp technique were used to investigate the properties of K_ATP channel. In the whole cell configuration, outward currents were induced by levcromakalim ( greater than 1 μM). The outward currents evoked by levcromakalim were abolished by glibenclamide, however CGRP, VIP, adenosine and somatostatin failed to induce simultaneous outward currents. Levcromaklim activated a channel with an unitary conductance of 12 pS in the cell-attached patch configuration but not in the cell-free patch configuration. An application of 1 mM guanosine 5'-diphosphate (GDP) re-activated the 12 pS channel in the cell-free patch configuration. These openings of 12 pS channel were inhibited by ATP ( 0.1 and 1 mM) and 1 μM glibenclamide. Our molecular study showed that K_ATP channel in pig detrusor is composed of Kir6.1 coupled with SUR 2A. It is concluded that the 12 pS K⁺ channel in pig urinary bladder smooth muscle cells is K_ATP channel composed of Kir 6.1 and SUR 2A. [J Physiol Sci. 2007;57 Suppl:S225]

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Molecular and behavioral studies have identified heterodimers of the T1R family as receptors for detecting the tastes of sweet (T1R2/T1R3) and umami (T1R1/T1R3). Previous studies demonstrated that T1R3-KO mice showed greatly reduced but not abolished behavioral preferences for sweet compounds. It is possible that there may be T1R3-independent sweet-responsive receptor components in mice. In the present study, to further examine behavioral taste responses of T1R3-KO mice, we used a short-term (10s) lick test after conditioning to avoid to each of sucrose, glucose, glycine and D-phenylalanine in T1R3-KO mice. We found that T1R3-KO mice were conditioned to learn to avoid 1.0M sucrose. The aversion generalized to 0.3M sucrose but not to the other various sweet compounds. An aversion conditioned to 0.5M glucose generalized to 1.0M sucrose, 0.5M maltose, 1.0M sorbitol, 0.1M NaCl and 0.1M MSG, whereas an aversion to 0.3M glycine did not generalize the other sweet compounds. Conditioned aversion to 0.1M D-phenylalanine was generalized to 0.5M fructose, 1.0M sorbitol, 0.3M glycine and 30mM D-tryptophan. Our results demonstrate that T1R3-KO mice still possess the ability to learn the conditioned avoidance to various sweet compounds with different generalization patterns. This suggests that there may exist multiple T1R3-independent sweet-responsive components in mice. [J Physiol Sci. 2007;57 Suppl:S225]

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Signaling through receptor tyrosine kinases (RTKs) is implicated in the control of many cellular functions. Immediately following activation of RTKs, these receptors are rapidly translocated from cell surface into the endosomal compartment. Then, these are sorted into lysosomes for degradation. Recently, evidence is accumulating that numerous adaptor proteins are involved in RTKs downregulation by internalization and endocytosis. CIN85 (Cbl-interacting protein of 85 kDa) is a multiadaptor protein containing three Src homology 3 (SH3) domains, a proline-rich region and a coiled-coil domain. Here we show that CIN85 is involved in regulation of ligand-induced endocytosis of epidermal growth factor (EGF) receptors. CIN85 is monoubiquitinated by Cbl/Cbl-b after EGF stimulation. Monoubiquitination is thought to regulate receptor internalization and endosomal sorting. Monoubiquitination of CIN85 required direct interaction between CIN85 and Cbl, the intact RING finger domain of Cbl and a ubiquitin acceptor site present in the carboxyl terminus of CIN85. CIN85 was also found in the complex with Cbl and polyubiquitinated EGF receptors, leading to their common degradation in the lysosome. Taken together, our results indicate that Cbl can mediate monoubiquitination of CIN85 to control endosomal sorting and degradation of RTKs. To analyze the biological function of CIN85 in vivo, we generated mutant mice deficient in the expression of CIN85. The phenotype of CIN85 knock-out mice is under investigation. [J Physiol Sci. 2007;57 Suppl:S225]

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In the early developing stage of the central nervous system, proliferation of newly produced cells from the neuroepithelial cells were maintained with the survival signals from tropic factors such as fibroblast growth factors (FGFs) and endothelial growth factors (EGFs) in addition with other signals, e.g. TGFβ signal, Wnt-singal, Delta-Notch signal, LIF signal, etc. In the later stage, action potential- dependent survival signals take part, known as "firing rule". Assuming that the action potential- dependent survival signals are participated, at least in a part, by the activation of glutamate receptors, we have examined the function of glutamate receptors in proliferative cells in the central nervous system. In the present study, we performed genome-wide gene expression analysis using a human glioblastoma cell line, CGNH, of which proliferation and migration are glutamate dependent. Total RNA was prepared and biotinylated antisense RNA was synthesized according to Affymetrix's one-cycle target labeling protocol. Using a GeneChip Human Genome U133 plus 2.0 oligonucleotide array, which contains 54,655 probes, expression of mRNA in CGNH cells was examined. Expression profiles of glutamate receptor subunits, GluR1-4, NMDAR1 show good agreements with quantitative RT-PCR. Whole expression profiles were compared with other glioblastoma cell lines in MIAME data base (http://www.ncbi.nlm.nih.gov/geo). The result shows that glutamate survival signals and tropic factors signals might be in trade-off relationships. [J Physiol Sci. 2007;57 Suppl:S226]

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The methyl group present in tramadol but not mono-O-demethyl-tramadol (M1), both of which exhibit a high affinity for μ-opioid receptors, is suggested to play an important role in producing nerve conduction block. To address further this issue, we examined the effects of opioids (morphine, codeine and ethylmorphine) on compound action potentials (CAPs) by applying the air-gap method to frog sciatic nerves. All of the drugs at concentrations less than 10 mM depressed the peak amplitude of CAP in a reversible manner. When compared in efficacy among the opioids, codeine more effectively reduced CAP peak amplitudes than morphine (30 and 15%, respectively, at 5 mM) did, where their chemical structures are distinct in only a substituent in the benzene ring in such that codeine and morphine have -OCH₃ and -OH, respectively, in the ring. This structure-activity relationship was the same as that for tramadol and M1. Ethylmorphine having -O-CH₂-CH₃ in the benzene ring was more effective in reducing CAP amplitudes (61% at 5 mM; IC₅₀ = 3.7 mM) than morphine and codeine. All of the opioid actions were resistant to an opioid-receptor antagonist naloxone (10 μM). It is concluded that opioids reduce CAP peak amplitudes in a manner which is independent of opioid receptor activation. It is suggested that the substituted groups of -OH bound to the benzene ring of codeine, morphine and ethylmorphine may play an important role in producing nerve conduction block. [J Physiol Sci. 2007;57 Suppl:S226]

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The outward current of the strong inward rectifier K⁺ currents (I_KIR) plays an important role in hyperpolarizing the membrane potential in many types of excitable and non-excitable cells. The detailed mechanism of outward I_KIR has not been well understood, because the time-dependent gating mechanism reflecting the block and unblock of the channels by intracellular spermine maximally "closes" the channels at voltages where outward I_KIR flows. In our previous study on the Kir2.1 channel, which is considered to be the predominant channel contributing to I_KIR, we showed that high-affinity block by spermine causes the time-dependent gating, but that the channels mediating ∼10% of the maximum conductance show lower sensitivity to the spermine block and the outward currents flow through these channels. In this study, we analyzed the Kir2.1 channels harboring mutations of the negatively charged residues on the wall of the pore, which are reportedly involved in the block by spermine. Neutralization of Asp174 in the transmembrane pore region made the channel susceptible only to the low-affinity spermine block, while that of Glu224 in the cytoplasmic pore region greatly reduced the sensitivity of the channel to the low-affinity spermine block without markedly altering the fractional conductance attributed to the low-affinity channels. These findings support our view that there are two populations of Kir2.1 channels susceptible to different modes of spermine block. [J Physiol Sci. 2007;57 Suppl:S226]

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Although tramadol is known to exhibit a local anesthetic effect following intradermal injection, this action has not been examined yet thoroughly. The present study examined the effects of tramadol and its metabolite mono-O-demethyl-tramadol (M1) on compound action potentials (CAPs) by applying the air-gap method to frog sciatic nerves; these results were compared with those of well-known local anesthetics, lidocaine and ropivacaine. Tramadol dose-dependently reduced the peak amplitude of the CAP (IC₅₀ = 2.3 mM). On the other hand, M1, which exhibits a higher affinity for μ-opioid receptors than tramadol does, at concentrations less than 2 mM did not affect CAPs, albeit the peak amplitude was reduced at 5 mM by 9%. Such an effect of tramadol was resistant to a nonspecific opioid-receptor antagonist naloxone (10 μM); a μ-opioid receptor agonist DAMGO (1 μM) did not affect CAPs. The CAP peak amplitude reduction produced by tramadol was less in extent than those of lidocaine and ropivacaine (IC₅₀: 0.74 mM and 0.34 mM, respectively), and the former action was slower in recovery than the latter two ones. These results indicate that tramadol reduces CAP peak amplitudes in peripheral nerve fibers in a manner independent of μ-opioid receptors with a potency which is less than those of lidocaine and ropivacaine, and with a slower time course compared to them, whereas M1 has much less effect on CAPs. The methyl group present in tramadol but not M1 is suggested to play an important role in producing nerve conduction block. [J Physiol Sci. 2007;57 Suppl:S226]

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The action of phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 on human Kv1.5 (hKv1.5) channels expressed in Chinese hamster ovary (CHO) cells was investigated using the whole cell patch-clamp technique. LY294002 rapidly and reversibly inhibited hKv1.5 current in a concentration-dependent manner with an IC₅₀ of 7.3 μM. However, another PI3K inhibitor wortmannin had no inhibitory effect on hKv1.5 current. The LY294002 block of hKv1.5 progressed with time during depolarizing voltage with a more rapid block at higher concentrations. The LY294002 action was voltage-dependent with a steep increase over the voltage range of channel opening however, its inhibition changed little when the channels were fully activated. LY294002 markedly shifted the voltage dependence of channel opening to more hyperpolarizing potentials and significantly accelerated the deactivation time course. Inhibition of hKv1.5 by LY294002 was use-dependent. In addition, a mutation of arginine 487 to valine in the outer pore region of Kv1.5 (R487V) significantly reduced the degree of LY294002 block (with an IC₅₀ of 16.9 μM), whereas other outer pore mutations (T462C and H463C) had no effect on LY294002 action. The present results suggest that (i) LY294002 acts directly on Kv1.5 currents as an open channel blocker and independently of the effects of LY294002 on PI3K activity; and (ii) the inhibitory target of LY294002 is present in the channel outer pore region, probably related to arginine 487. [J Physiol Sci. 2007;57 Suppl:S227]

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I_Ks current modulation through stretch or swelling of single cells was reported, although exact mechanism of this regulation is still not clarified. It was demonstrated that AT₁ receptor could be activated by stretch in the absence of angiotensin II. Therefore, we attempted to elucidate the involvement of AT₁ receptor in swelling-activated I_Ks increase in guinea pig atrial myocytes by the whole-cell patch-clamp method. Hypotonic solution (HS) with osmolality 70% enhanced guinea pig atrial I_Ks tail current by 84% (n = 12). Pretreatment with specific AT₁ receptor antagonist candesartan (1 or 5 μM) attenuated I_Ks increase to 48% (n = 10) and 43% (n = 9), respectively. I_Ks enhancement coincided with increase of the cells width by 19% (n = 12), which was completely reversible during readministration of isotonic solution and candesartan has no influence on the changes of cell dimensions. PLC inhibitor U73122 (3 μM) in the pipette solution or superfusion with PKC inhibitor BIS I (200 nM) did not affect the extent of I_Ks increase by HS. On the contrary, tyrosine kinase (TK) inhibitors A23 (20 μM) and A25 (20 μM) reduced I_Ks enhancement to 42 and 52%, respectively. Tyrosine phosphatase inactivation by orthovanadate (500 μM) loaded into the cells through the pipette solution stimulated I_Ks magnification to 124%. The above results suggest involvement of AT₁ receptor and TKs in the swelling-activated I_Ks enhancement in guinea pig atrium. [J Physiol Sci. 2007;57 Suppl:S227]

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Hyperpolarization activated, cyclic nucleotide sensitive cation channels (Ih) of cardiac myocytes are composed of HCN1, HCN2 and HCN4. These molecules are highly expressed in the pacemaker cells in the heart. Low level of HCN2 are expressed in ventricular myocytes, and is upregulated in cardiac hypertrophy. In the present study, we aimed to clarify the transcriptional mechanism of HCN2 gene. Fisrt, We carried out in-silico search for enhancer elements in mouse hcn2 gene. By comparing rat, mouse, chicken, dog and human genome sequences using VISTA browser, we identified only one conserved region (–200 bps) in 5'region between neighboring gene, and designated it CNS#1. Proximal promoter region of hcn2 was also conserved, although its homology was lower than that of CNS#1. Proximal promoter lacked consensus TATA motif, but contained multiple CpG sites. We next carried out luciferase reporter assay using proximal promoter and CNS#1 of mouse hcn2 gene. Genomic fragments were subcloned into pGL4.10 vector, and transfected into primary cultured, neonatal cardiac myocytes. Deletion study of demonstrated that CNS#1 contained both positive- and negative regulatory function on the activity of proximal promoter. The genomic region other than CNS#1 and proximal promoter did not possess significant regulatory role. Thus, in silico approach appeared very useful to determine cis-element of ion channel genes. [J Physiol Sci. 2007;57 Suppl:S227]

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Acid sensation is a critical cue to avoid spoiled foods or harmful solutions in animals. It has been reported that several ion channels activated by proton such as ASIC and HCN channels are involved in sour taste sensation. However, molecular mechanisms underlying sour taste sensation remain largely unclear. Here, we report a novel class of acid-activated ion channel complex PKD1L3/PKD2L1, as a candidate of sour taste receptor molecules. PKD1L3 and PKD2L1 are similar to members of polycistin family polycystin-1 and -2, whose defects are thought to cause autosomal dominant polycystic kidney diseases. TRP2L1 also belongs to transient receptor potential (TRP) channel family. In mouse tongue, PKD1L3 and PKD2L1 are co-expressed in a subset of taste receptor cells, which are distinct from taste cells having receptors for bitter, sweet, or umami tastants. PKD1L3 and PKD2L1 proteins can interact with each other, and co-expression of the PKD1L3 and PKD2L1 is necessary for their functional cell surface expression. Large currents are evoked by both weak and strong acids (citric acid and HCl, respectively) in the PKD1L3/PKD2L1 expressed HEK293 cells. EC₅₀ is pH 2.9 for citric acid and pH 2.7 for HCl, respectively. These results suggest that PKD1L3/PKD2L1 may function as sour taste receptors. [J Physiol Sci. 2007;57 Suppl:S227]

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We previously reported an ascidian protein Ci-VSP which has a transmembrane voltage sensor motif and a phosphatase domain. We showed that the voltage sensor domain functionally couples with the phosphatase domain and the phosphatase activity arises by depolarization. We also showed that the linker region between the voltage sensor domain and phosphatase domain plays an important role in this coupling. However, these residues found in Ci-VSP are not completely conserved in zebrafish VSP (z-VSP) that also shows activities of voltage-regulated phosphatase. We studied structure-function relationship of the coupling.First, to test if the coupling mechanism is common to other species, residues in the linker of Ci-VSP that are distinct from those of z-VSP were changed into z-VSP-based ones. Next, we tested whether the length of the linker affects coupling by introducing additional residues or deleting some residues around the linker. If the length of the linker is important, these mutants may not express the voltage-dependent change. Moreover, we tested whether there is interaction between the linker and the enzymatic domain by swapping the linker and the proximal enzymatic domain between Ciona and zebrafish VSPs. Wild-type and mutant VSPs were coexpressed with phosphoinositide-sensitive GIRK2 channels into Xenopus oocytes, and changes of the phosphatase activity with membrane potentials were examined by measuring GIRK2 channel activity using the two-electrode voltage clamp recording. [J Physiol Sci. 2007;57 Suppl:S228]

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Voltage-gated ion channels are composed of six transmembrane segments (S1 to S6). S5 and S6 form the hydrophilic pore, while S1 to S4 constitute the voltage-sensor domain (VSD). We have recently identified a novel protein that consists only of the four-transmembrane segments, corresponding to VSD, and found that it is long-sought molecular correlate for voltage-gated proton channel (VSOP) (Sasaki et al, Science 2006). Voltage-gated potassium channels are known to assemble as tetramers. However, it remains unknown whether VSOP functions as multimers or monomers. In this study, we have investigated the protein stoichiometry of VSOP labeled with green fluorescent protein (GFP-VSOP) by total internal reflection fluorescence microscopy. GFP-VSOP was overexpressed in Xenopus oocyte and fluorescence intensity and whole-cell current were simultaneously measured under the two-electrode voltage clamp. This protein showed depolarization-induced outward currents and voltage-dependent fluorescence change probably due to local change of pH. In this chimeric protein, activation kinetics was faster and deactivation was slower than in the wild-type protein. This suggests that fusion of GFP to VSOP slightly modifies properties of channel activities. We are currently using this molecule to identify the stoichiometry of GFP-VSOP by the photobleaching of single GFP molecules. [J Physiol Sci. 2007;57 Suppl:S228]

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L-type Ca²⁺ channel activity is diminished in inside-out patches (run-down). We have previously reported that calmodulin (CaM) and calpastatin (CS, an endogenous inhibitor of Ca²⁺ activated protease, calpain) recover channel activity. CaM can produce channel activity of more than 100% of control, whereas CS domain L (CSL), the effective region of CS, produces channel activity of ∼20% of control. However, interactions between CaM and CSL have not yet been investigated. In this study, we recorded the channel activity in the presence of both CaM and CSL using inside-out patch-clamp technique in guinea-pig cardiac myocytes. Unexpectedly, the channel activity recovered by 1 μM CaM was suppressed by 10 μM CSL (inhibition 78.3%). This inhibition was consistent with a model, in which CSL acted as a partial agonist for the CaM binding site. We also examined the interaction between CaM and CSL in binding to the channel by pull-down assay using GST fusion peptides derived from the intracellular regions of the channel. CaM and CSL competed for the same binding site located in the C-terminal proximal region. These results suggested that CSL and CaM competitively interact at a site in the channel and thereby modulate the channel activity. [J Physiol Sci. 2007;57 Suppl:S228]

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Calpastatin (CS), an endogenous inhibitor of the calcium-activated protease calpain, is composed of N-terminal domain L (CS_L) and four repetitive calpain inhibitory domains 1-4, and expressed in virtually all cell types. We have previously found that CS modulates the cardiac CaV1.2 Ca²⁺ channels in inside-out patch mode of patch-clamp technique in guinea-pig cardiomyocytes and that the Ca²⁺ channel regulatory function is located in CS_L. However, how does CS_L modulate the channels activity is not known. In this study, we have determined the interaction site of CS_Lin the channel by the pull-down binding assay using GST-fused fragment peptides of the α₁ subunit. CS_L interacted directly with IQ domain of the proximal C-terminal region of α₁ subunit (a. a. number of guinea-pig α₁: 1648-1672), which is also one of the calmodulin (CaM) binding sites. CS_L bound to IQ domain with a higher affinity in the presence of Ca²⁺ than its absence. CS_L binding to IQ also showed a competitive manner with CaM. These results suggest that CS_L directly interacts with IQ domain of α₁ subunit of the voltage dependant Ca²⁺ channel and inhibits the CaM binding to this domain. A role of CS_L in the regulation of the Ca²⁺ channels will be discussed. [J Physiol Sci. 2007;57 Suppl:S228]

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Heating of cells produce various immediate cellular events including activation of ion channels. However, the effects of heat on ion channels in cardiac myocytes remain unclear. In this study, we examined the effect of heat on L-type Ca²⁺ channels in guinea-pig ventricular myocytes, using the patch clamp method. Heating of the myocytes (45°C, 30 min) increased the L-type Ca²⁺ current about 4-fold. After the heat treatment, forskolin failed to further increase the L-type Ca²⁺ current. The increase of L-type Ca²⁺ current induced by heating was greatly attenuated (to 1.5-2 fold) by pre-treatment of the myocytes with an inhibitor of protein kinase A (PKA), H-89 (5 μM) or a non-specific protein kinase inhibitor, K-252a (0.5 μM). Furthermore, the heat treatment shifted the I-V curve of the L-type Ca²⁺ current to the hyperpolarizing direction by about 8 mV. This shift of the I-V curve was prevented by the pre-treatment with H-89 or K-252a. These results suggested that heating of the myocytes may promote phosphorylation of the L-type Ca²⁺ channels through activation of PKA and/or inactivation of counteracting protein phosphatases. [J Physiol Sci. 2007;57 Suppl:S229]

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Hydrogen peroxide (H₂O₂) is produced during post ischemic reperfusion. It is reported that H₂O₂ increases L-type Ca²⁺ current (I_Ca,L), while catalase, a scavenger of H₂O₂, can alter response to β-stimulation of I_Ca,L. L-type Ca²⁺ channels (LCC) are mainly modified via cAMP/PKA pathway, however, it is not yet clear about the mechanisms of the effects of H₂O₂ on LCC. The purpose of the present study is to examine the mechanism that involved in H₂O₂-induced modification of LCC. We applied H₂O₂ to isolated rabbit ventricular myocytes and measured I_Ca,L by whole-cell patch clamp technique. We examined to H₂O₂-induced increase of I_Ca,L in the presence of a PKA activator, a PKA inhibitor and a non-selective PKC inhibitor (BIS). We also observed the effect of PKC inhibitor peptide specific to each subtypes through the pipette solution. In control group (CT), H₂O₂ increased I_Ca,L to 1.20±0.04 folds. Neither activation nor inhibition of PKA affected the H₂O₂-induced increase in I_Ca,L. In contract, BIS inhibited the H₂O₂-induced increase in I_Ca,L (0.80±0.07 folds). When non-selective PKC inhibitor peptide dialyzed intracellularly, H₂O₂-induced increase in I_Ca,L was suppressed (1.00±0.05 folds). PKCε inhibitor peptide, but not PKCδ inhibitor peptide dialyzed to pipette solution similarly inhibited the H₂O₂-induced increase in I_Ca,L(1.04±0.05 folds). These results suggest that modification of LCC by H₂O₂ is not through the PKA pathway, but through PKC, especially through PKCε pathway. [J Physiol Sci. 2007;57 Suppl:S229]

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Volatile anesthetics suppress cardiac contractility. On the other hand, volatile anesthetics, isoflurane, sevoflurane and halothane, were reported to enhance the positive inotropic action of β-adrenoceptor stimulation. In this study, these modulations of volatile anesthetics to β-adrenoceptor stimulation were examined by electrophysiological method. L-type Ca²⁺ channel current (I_Ca,L) of isolated rabbit ventricular myocytes were measured with the whole-cell configuration of patch clamp technique. The β-adrenoceptor stimulation was induced by 30 nM isoproterenol (ISO). Isoflurane (1.15%), sevoflurane (1.7%) or halothane (0.74%) were bubbled in experimental solution and air was bubbled in control group. Isoflurane, sevoflurane and halothane decreased I _Ca,L by 21.3±1.3%, 19.8±2.4%, and 31.7±3.1%, respectively. In control group, ISO increased I _Ca,L to 153.0±7.0%. While ISO increased I _Ca,L to 241.8±14.9%, 237.2±25.4%, and 247.6±16.3% in the presence of isoflurane, sevoflurane, and halothane respectively. These results show that isoflurane, sevoflurane and halothane enhance the effect of β-adrenoceptor stimulation on I _Ca,L. This study suggests that positive inotropic effect of β-adrenoceptor stimulation is enhanced in the presence of volatile anesthetics through increase in I_Ca,L. [J Physiol Sci. 2007;57 Suppl:S229]

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Activation and inactivation properties of the large conductance Ca²⁺-and voltage-activated K⁺ channel (BK_i channel) in rat chromaffin cells were modeled based on the experimental data provided by M. Prakriya et al., J. Neurosci. 16: 4344, 1996) and J. P. Ding et al., Biophys. J. 82: 2448, 2002). The BK_i channel model was tested in simulation by incorporating it into a previously developed chromaffin cell model which comprises mechanisms of Ca²⁺ influx through voltage-activated Ca²⁺ channels, Ca²⁺ diffusion and extrusion. The BK_i channel model is designed to sense the submembrane [Ca²⁺]_i increase with two overlapped domains; one is derived from a rapidly formed Ca²⁺ microdomain around a pore of open Ca²⁺ channel positioned at 50-60 nm distance from individual BK_i channel, and the other develops slowly in a cell-averaged fashion as the result of buffered intracellular Ca²⁺ diffusion. Calculations with the model appropriately reproduced characteristic details shown in experimental records, which are as follows: 1) BK_i current profiles induced by imposing a two-step voltage clamp, first pulses to -9 mV for varying durations to load the cell with various amounts of Ca²⁺ followed by a test pulse to +81 mV; 2) Distinct influences of EDTA and BAPTA on the BK_i current profiles during the two-step voltage pulses due to different Ca²⁺-binding rates of the buffers; 3) A change in the profile of action potential. with inhibition of BK_i channel; 4) Role of BK_i channels on the generation of repetitive firing. Thus, the proposed BK_i channel model may be useful as a mechanism implemented in chromaffin cell model. [J Physiol Sci. 2007;57 Suppl:S229]

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Previously we found that the activation of P2Y receptors elicits a long-term enhancement of GABAergic transmission between cerebellar interneurons and Purkinje cells (PCs). We here employed peak-scaled non-stationary analysis (PS-NSFA) to further examine the mechanisms underlying the P2Y receptor-mediated enhancement of GABAergic synapses. Stimulation-evoked IPSCs (eIPSCs) were recorded from PCs in neonatal rat cerebellar slices. ATP (100μM) applied by perfusion increased the amplitude of eIPSCs to 132±4.4% of the control. Using PS-NSFA, the number and single channel conductance of GABA_A receptors responsible for eIPSCs in PCs were determined. The single channel conductance increased significantly (159±10%, n=6) during the ATP-induced enhancement of eIPSCs, whereas there was no discernible change in the number of the receptors (93±6.3%). Furthermore, pretreatment with the protein kinase A inhibitor H-89, but not the calmodulin kinase II inhibitor KN-62, abolished the ATP-induced enhancement of eIPSCs. These results suggest that P2Y receptor activation enhances GABAergic transmission to cerebellar Purkinje cells through a long-term increase in the postsynaptic GABA_A receptor conductance via cyclic AMP-protein kinase A-coupled signaling cascade. This is in accordance with the previous finding that a P2Y11 receptor subtype that stimulates the adenylyl cyclase-cyclic AMP-dependent pathway occurs in the mammalian cerebellum. [J Physiol Sci. 2007;57 Suppl:S230]

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