研究者データベース

山口 聡一郎(ヤマグチ ソウイチロウ)
獣医学研究院 獣医学部門 基礎獣医科学分野
准教授

基本情報

所属

  • 獣医学研究院 獣医学部門 基礎獣医科学分野

職名

  • 准教授

学位

  • 博士(獣医学)(2009年03月 北海道大学)

論文上での記載著者名

  • Soichiro Yamaguchi
  • Souichirou Yamaguchi

科研費研究者番号

  • 50596864

J-Global ID

研究キーワード

  • トランスポーター   イオンチャネル   

研究分野

  • ライフサイエンス / 獣医学
  • ライフサイエンス / 獣医学
  • ライフサイエンス / 獣医学

職歴

  • 2018年08月 - 現在 北海道大学 大学院獣医学研究院 基礎獣医科学部門 生理学教室 准教授
  • 2013年11月 - 2018年08月 北海道大学大学院獣医学研究科 比較形態機能学講座薬理学教室 助教
  • 2012年10月 - 2013年10月 北海道大学大学院獣医学研究科比較形態機能学講座薬理学教室 特任助教
  • 2010年09月 - 2012年10月 新潟大学医歯学総合研究科生体機能調節医学専攻機能再建医学 助教
  • 2009年04月 - 2010年08月 テキサス大学サウスウェスタンメディカルセンター (米国) 博士研究員
  • 2010年 - Assistant Professor,Regenerative and Transplant Medicine,Biological Functions and Medical Control,Graduate School of Medical and Dental Sciences,Niigata University

学歴

  • 2005年04月 - 2009年03月   北海道大学   大学院獣医学研究科
  • 1999年04月 - 2005年03月   北海道大学   獣医学部

所属学協会

  • 日本薬理学会   日本生理学会   日本獣医学会   

研究活動情報

論文

  • Soichiro Yamaguchi, Maho Hamamura, Ken-Ichi Otsuguro
    International journal of molecular sciences 21 18 2020年09月04日 [査読有り][通常論文]
     
    Mechanical stimuli caused by sound waves are detected by hair cells in the cochlea through the opening of mechanoelectrical transduction (MET) channels. Transmembrane channel-like protein 1 (TMC1) has been revealed to be the pore-forming component of the MET channel. The two splice variants for mouse Tmc1 (mTmc1ex1 and mTmc1ex2) were reported to be expressed in the cochlea of infant mice, though only the sequence of mTmc1ex2 had been deposited in GenBank. However, due to the presence of an upstream open reading frame (uORF) and the absence of a typical Kozak sequence in mTmc1ex2, we questioned whether mTMC1 was translated from mTmc1ex2. Therefore, in this study, we evaluated which splice variant was protein-coding mRNA. Firstly, the results of RT-PCR and cDNA cloning of mTmc1 using mRNA isolated from the cochlea of five-week-old mice suggested that more Tmc1ex1 were expressed than mTmc1ex2. Secondly, mTMC1 was translated from mTmc1ex1 but not from mTmc1ex2 in a heterologous expression system. Finally, analyses using site-directed mutagenesis revealed that the uORF and the weak Kozak sequence in mTmc1ex2 prevented the translation of mTMC1 from mTmc1ex2. These results suggest that mTmc1ex1 plays a main role in the expression of mTMC1 in the mouse cochlea, and therefore, mTmc1ex1 should be the mRNA for mTMC1 hereafter.
  • Soichiro Yamaguchi, Akira Tanimoto, Shinsuke Iwasa, Ken-Ichi Otsuguro
    International journal of molecular sciences 20 8 2019年04月24日 [査読有り][通常論文]
     
    Transient receptor potential melastatin member 4 (TRPM4) and 5 (TRPM5) channels are Ca2+-activated nonselective cation channels. Intracellular Ca2+ is the most important regulator for them to open, though PI(4,5)P2, a membrane phosphoinositide, has been reported to regulate their Ca2+-sensitivities. We previously reported that negatively-charged amino acid residues near and in the TRP domain are necessary for the normal Ca2+ sensitivity of TRPM4. More recently, a cryo-electron microscopy structure of Ca2+-bound (but closed) TRPM4 was reported, proposing a Ca2+-binding site within an intracellular cavity formed by S2 and S3. Here, we examined the functional effects of mutations of the amino acid residues related to the proposed Ca2+-binding site on TRPM4 and also TRPM5 using mutagenesis and patch clamp techniques. The mutations of the amino acid residues of TRPM4 and TRPM5 reduced their Ca2+-sensitivities in a similar way. On the other hand, intracellular applications of PI(4,5)P2 recovered Ca2+-sensitivity of desensitized TRPM4, but its effect on TRPM5 was negligible. From these results, the Ca2+-binding sites of TRPM4 and TRPM5 were shown to be formed by the same amino acid residues by functional analyses, but the impact of PI(4,5)P2 on the regulation of TRPM5 seemed to be smaller than that on the regulation of TRPM4.
  • Kitano T, Kobayashi T, Yamaguchi S, Otsuguro KI
    Journal of veterinary pharmacology and therapeutics 42 2 243 - 247 2019年03月 [査読有り][通常論文]
  • Eguchi R, Yamaguchi S, Otsuguro KI
    Journal of pharmacological sciences 139 2 98 - 104 2019年02月 [査読有り][通常論文]
  • Ujike A, Kuraishi T, Yamaguchi S, Eguchi R, Kitano T, Kamise J, Ito S, Otsuguro KI
    European journal of pharmacology 821 88 - 96 2018年02月15日 [査読有り][通常論文]
     
    H2S has excitatory and inhibitory effects on Ca2+ signals via transient receptor potential ankyrin 1 (TRPA1) and ATP-sensitive K+ channels, respectively. H2S converts intracellularly to polysulfides, which are more potent agonists for TRPA1 than H2S. Under inflammatory conditions, changes in the expression and activity of these H2S target channels and/or the conversion of H2S to polysulfides may modulate H2S effects. Effects of proinflammatory cytokines on H2S-induced Ca2+ signals and polysulfide production in RIN14B cells were examined using fluorescence imaging with fura-2 and SSP4, respectively. Na2S, a H2S donor, induced 1) the inhibition of spontaneous Ca2+ signals, 2) inhibition followed by [Ca2+]i increase, and 3) rapid [Ca2+]i increase without inhibition in 50% (23/46), 22% (10/46), and 17% (8/46) of cells tested, respectively. IL-1β augmented H2S-induced [Ca2+]i increases, which were inhibited by TRPA1 and voltage-dependent L-type Ca2+ channel blockers. However, IL-1β treatment did not affect [Ca2+]i increases evoked by a TRPA1 agonist or high concentration of KCl. Na2S increased intracellular polysulfide levels, which were enhanced by IL-1β treatment. A NOS inhibitor suppressed the increased polysulfide production and [Ca2+]i increase in IL-1β-treated cells. These results suggest that IL-1β augments H2S-induced [Ca2+]i increases via the conversion of H2S to polysulfides through NO synthesis, but not via changes in the activity and expression of target channels. Polysulfides may play an important role in the effects of H2S during inflammation.
  • Soichiro Yamaguchi, Ken-ichi Otsuguro
    NEUROSCIENCE LETTERS 653 139 - 145 2017年07月 [査読有り][通常論文]
     
    Mas-related G-protein coupled receptor B4 (MrgprB4) has been reported to be expressed in the dorsal root ganglion (DRG) neurons which detect stroking of hairy skin of mice. However, the mechanisms by which the MrgprB4 positive (+) neurons respond to adequate stimulus remain unsolved as it was also reported that electrophysiological analysis of cultured MrgprB4+ neurons did not reveal responses to mechanical stimuli. Contrary to the observation, however, in this study we show that the MrgprB4+ neurons functionally express a mechanically activated channel using DRG neurons dissociated from genetically-modified mice whose MrgprB4+ neurons express a red fluorescent protein. Hypotonicity-induced cell swelling increased intracellular Ca2+ concentrations ([Ca2+](i)) of MrgprB4+ neurons. The [Ca2+](i) increases were prevented by extracellular Ca2+ removal and by applications of nonselective Piezo channel blockers. Patch clamp analysis revealed that the MrgprB4+ neurons exhibited rapidly-adapting mechanically-activated currents. The MrgprB4+ neurons were stained with anti-Piezo2 antibody. These results raise the possibility that the MrgprB4+ neurons directly detect the stroking-like stimuli of hairy skin. (C) 2017 Elsevier B.V. All rights reserved.
  • Dugar Delgermurun, Soichiro Yamaguchi, Osamu Ichii, Yasuhiro Kon, Shigeo Ito, Ken-ichi Otsuguro
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY C-TOXICOLOGY & PHARMACOLOGY 187 43 - 49 2016年09月 [査読有り][通常論文]
     
    Epithelioid cells in the chicken thoracic aorta are chemoreceptor cells that release 5-HT in response to hypoxia. It is likely that these cells play a role in chemoreception similar to that of glomus cells in the carotid bodies of mammals. Recently, H2S was reported to be a key mediator of carotid glomus cell responses to hypoxia. The aim of the present study was to reveal the mechanism of action of H2S on 5-HT outflow from chemoreceptor cells in the chicken thoracic aorta. The 5-HT outflow induced by NaHS, an H2S donor, and Na2S3, a polysulfide, was measured by using a HPLC equipped with an electrochemical detector. NaHS (0.3-3 mM) caused a concentration-dependent increase in 5-HT outflow, which was significantly inhibited by the removal of extracellular Ca2+. outflow induced by NaHS (0.3 mM) was also significantly inhibited by voltage-dependent L- and N-type Ca2+ channel blockers and a selective TRPA1 channel blocker. Cinnamaldehyde, a TRPA1 agonist, mimicked the secretory response to H2S. 5-HT outflow induced by Na2S3 (10 M) was also inhibited by the TRPA1 channel blocker. Furthermore, the expression of TRPA1 was localized to 5-HT-containing chemoreceptor cells in the aortic wall. These findings suggest that the activation of TRPA1 and voltage-dependent Ca2+ channels is involved in H2S-evoked 5-HT release from chemoreceptor cells in the chicken aorta. (C) 2016 Elsevier Inc. All rights reserved.
  • Dugar Delgermurun, Shigeo Ito, Toshio Ohta, Soichiro Yamaguchi, Ken-ichi Otsuguro
    JOURNAL OF VETERINARY MEDICAL SCIENCE 78 1 71 - 76 2016年01月 [査読有り][通常論文]
     
    Chemoreceptor cells aggregating in clusters in the chicken thoracic aorta contain 5-hydroxytryptamine (5-HT) and have voltage dependent ion channels and nicotinic acetylcholine receptors, which are characteristics typically associated with neurons. The aim of the present study was to investigate the effects of 5-HT uptake inhibitors, fluvoxamine, fluoxetine and clomipramine (CLM), and amphetamine derivatives, p-chloroamphetamine (PCA) and methamphetamine (MET), on endogenous 5-HT outflow from the isolated chick thoracic aorta in vitro. 5-HT was measured by using a HPLC system with electrochemical detection. The amphetamine derivatives and 5-HT uptake inhibitors caused concentration-dependent increases in endogenous 5-HT outflow. PCA was about ten times more effective in eliciting 5-HT outflow than MET. The 5-HT uptake inhibitors examined had similar potency for 5-HT outflow. PCA and CLM increased 5-HT outflow in a temperature-dependent manner. The outflow of 5-HT induced by PCA or 5-HT uptake inhibitors was independent of extracellular Ca2+ concentration. The 5-HT outflow induced by CLM, but not that by PCA, was dependent on the extracellular NaCl concentration. These results suggest that the 5-HT uptake system of 5-HT-containing chemoreceptor cells in the chicken thoracic aorta has characteristics similar to those of 5-HT-containing neurons in the mammalian central nervous system (CNS).
  • Ken-ichi Otsuguro, Yuki Tomonari, Saori Otsuka, Soichiro Yamaguchi, Yasuhiro Kon, Shigeo Ito
    NEUROPHARMACOLOGY 97 160 - 170 2015年10月 [査読有り][通常論文]
     
    Adenosine kinase (AK) inhibitor is a potential candidate for controlling pain, but some AK inhibitors have problems of adverse effects such as motor impairment. ABT-702, a non-nucleoside AK inhibitor, shows analgesic effect in animal models of pain. Here, we investigated the effects of ABT-702 on synaptic transmission via nociceptive and motor reflex pathways in the isolated spinal cord of neonatal rats. The release of adenosine from the spinal cord was measured by HPLC. ABT-702 inhibited slow ventral root potentials (sVRPs) in the nociceptive pathway more potently than monosynaptic reflex potentials (MSRs) in the motor reflex pathway. The inhibitory effects of ABT-702 were mimicked by exogenously applied adenosine, blocked by 8CPT (8-cyclopentyl-1,3-dipropylxanthine), an adenosine A(1) receptor antagonist, and augmented by EHNA (erythro-9-(2-hydroxy-3-nonyl) adenine), an adenosine deaminase (ADA) inhibitor. Equilibrative nucleoside transporter (ENT) inhibitors reversed the effects of ABT-702, but not those of adenosine. ABT-702 released adenosine from the spinal cord, an effect that was also reversed by ENT inhibitors. The ABT-702-facilitated release of adenosine by way of ENTs inhibits nociceptive pathways more potently than motor reflex pathways in the spinal cord via activation of A1 receptors. This feature is expected to lead to good analgesic effects, but, caution may be required for the use of AK inhibitors in the case of ADA dysfunction or a combination with ENT inhibitors. (C) 2015 Elsevier Ltd. All rights reserved.
  • Ayako Ujike, Ken-ichi Otsuguro, Ryo Miyamoto, Soichiro Yamaguchi, Shigeo Ito
    EUROPEAN JOURNAL OF PHARMACOLOGY 764 463 - 470 2015年10月 [査読有り][通常論文]
     
    Hydrogen sulfide (H2S) reportedly acts as a gasotransmitter because it mediates various cellular responses through several ion channels including ATP-sensitive K+ (K-ATP) channels and transient receptor potential (TRP) A1 channels. H2S can activate both K-ATP, and TRPA1 channels at a similar concentration range. In a single cell expressing both channels, however, it remains unknown what happens when both channels are simultaneously activated by H2S. In this study, we examined the effects of H2S on RIN14B cells that express both K-ATP and TRPA1 channels. RIN14B cells showed several intracellular Ca2+ concentration ([Ca2+](i)) responses to NaHS (300 mu M), an H2S donor, i.e., inhibition of spontaneous Ca2+ oscillations (37%), inhibition followed by [Ca2+](i) increase (24%), and a rapid increase in [Ca2+](i) (25%). K-ATP channel blockers, glibenclamide or tolbutamide, abolished any inhibitory effects of NaHS and enhanced NaHS-mediated [Ca2+](i) increases, which were inhibited by extracellular Ca2+ removal, HC030031 (a TRPA1 antagonist), and disulfide bond-reducing agents. NaHS induced 5-hydroxytryptamine (5-HT) release from RIN14B cells, which was also inhibited by TRPA1 antagonists. These results indicate that H2S has both inhibitory and excitatory effects by opening K-ATP and TRPA1 channels, respectively, in RIN14B cells, suggesting potential bidirectional modulation of secretory functions. (C) 2015 Elsevier B.V. All rights reserved.
  • Kobayashi T, Otsuguro K, Yamaguchi S, Ito S
    European journal of pharmacology 761 321 - 329 2015年08月 [査読有り][通常論文]
     
    Alpha-2A adrenergic receptor (AR) subtype plays an important role in the analgesic effect of alpha(2)-AR agonists. Here, we examined the effects of alpha(2)-AR agonists, dexmedetomidine and xylazine, on spinal synaptic transmission in newborn C57BL/6J and alpha(2A)-AR mutant mice. Spinal reflex potentials, the monosynaptic reflex potential (MSR) and the slow ventral root potential (sVRP), were measured in isolated spinal cords. The compound action potential was measured in isolated lumbar nerve. Dexmedetomidine and xylazine suppressed both the MSR and sVRP in a concentration-dependent manner. In alpha(2A)-AR mutant mice, sVRP suppression by dexmedetomidine was greatly weakened, while that by xylazine (30-100 mu M) showed only slight attenuation. A high concentration (300 mu M) of xylazine completely suppressed the sVRP, even in alpha(2A)-AR mutant mice spinal cords, and also suppressed the compound action potential. MSR suppression by these alpha(2)-AR agonists had no difference between wild-type and alpha(2A)-AR mutant mice. These results suggest that sVRP suppression by dexmerletomidine and xylazine is mainly mediated by alpha(2A)-AR. In addition, a high concentration of xylazine inhibits conduction of the action potential, which is not mediated by alpha(2A)-AR. alpha(2)-AR is not responsible for the dexmedetomidineand xylazine-mediated inhibition of the MSR. (C) 2015 Elsevier B.V. All rights reserved.
  • Miyamoto R, Otsuguro KI, Yamaguchi S, Ito S
    Neuroscience research 97 52 - 59 2015年08月 [査読有り][通常論文]
     
    Cystathionine beta-synthase (CBS), expressed in astrocytes, generates a gaseous neuromodulator, hydrogen sulfide (H2S) in the central nervous system (CNS). However, little is known about the regulatory mechanisms of astrocytic CBS expression and activity. This study evaluated the influence of neurons on astrocytic CBS expression by employing multiple culture systems. Substantial CBS expression was observed in the intact neonatal rat spinal cord, while CBS content was markedly reduced in an astrocyte-enriched culture prepared from the neonatal spinal cord. Immunofluorescence analysis confirmed the localization of spinal cord CBS in astrocytes, but not in neurons. Although CBS expression was weak in the embryonic rat spinal cord, enzyme levels were time-dependently increased in a neuron/astrocyte mixed culture originating from embryonic spinal cord. The reduced CBS expression in isolated neonatal astrocytes was restored by co-culture with embryonic neurons. Together with the observed CBS expression levels, H2S production was relatively low in astrocytes cultured alone, but was considerably higher in astrocytes cultured with neurons. These results indicate that neurons are essential for maintaining the expression and H2S-producing activity of astrocytic CBS in the rat spinal cord. (C) 2015 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
  • Ryota Eguchi, Sanae Akao, Ken-ichi Otsuguro, Soichiro Yamaguchi, Shigeo Ito
    JOURNAL OF PHARMACOLOGICAL SCIENCES 128 1 47 - 53 2015年05月 [査読有り][通常論文]
     
    Extracellular adenosine is a neuromodulator in the central nervous system. Astrocytes mainly participate in adenosine production, and extracellular adenosine accumulates under physiological and pathophysiological conditions. Inhibition of intracellular adenosine metabolism and reduction of the external Ca2+ concentration ([Ca2+](e)) participate in adenosine accumulation, but the precise mechanisms remain unclear. This study investigated the mechanisms underlying extracellular adenosine accumulation in cultured rat spinal astrocytes. The combination of adenosine kinase and deaminase (ADK/ADA) inhibition and a reduced [Ca2+](e) increased the extracellular adenosine level. ADK/ADA inhibitors increased the level of extracellular adenosine but not of adenine nucleotides, which was suppressed by inhibition of equilibrative nucleoside transporter (ENT) 2. Unlike ADK/ADA inhibition, a reduced [Ca2+](e) increased the extracellular level not only of adenosine but also of ATP. This adenosine increase was enhanced by ENT2 inhibition, and suppressed by sodium polyoxotungstate (ecto-nucleoside triphosphate diphosphohydrolase inhibitor). Gap junction inhibitors suppressed the increases in adenosine and adenine nucleotide levels by reduction of [Ca2+](e). These results indicate that extracellular adenosine accumulation by ADK/ADA inhibition is due to the adenosine release via ENT2, while that by reduction of [Ca2+] e is due to breakdown of ATP released via gap junction hemichannels, after which ENT2 incorporates adenosine into the cells. (C) 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society.
  • Soichiro Yamaguchi, Akira Tanimoto, Ken-ichi Otsuguro, Hiroshi Hibino, Shigeo Ito
    JOURNAL OF BIOLOGICAL CHEMISTRY 289 51 35265 - 35282 2014年12月 [査読有り][通常論文]
     
    Transient receptor potential (TRP) channel melastatin subfamily member 4 (TRPM4) is a broadly expressed nonselective monovalent cation channel. TRPM4 is activated by membrane depolarization and intracellular Ca2+, which is essential for the activation. The Ca2+ sensitivity is known to be regulated by calmodulin and membrane phosphoinositides, such as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P-2). Although these regulators must play important roles in controlling TRPM4 activity, mutation analyses of the calmodulin-binding sites have suggested that Ca2+ binds to TRPM4 directly. However, the intrinsic binding sites in TRPM4 remain to be elucidated. Here, by using patch clamp and molecular biological techniques, we show that there are at least two functionally different divalent cation-binding sites, and the negatively charged amino acids near and in the TRP domain in the C-terminal tail of TRPM4 (Asp-1049 and Glu-1062 of rat TRPM4) are required for maintaining the normal Ca2+ sensitivity of one of the binding sites. Applications of Co2+, Mn2+, or Ni2+ to the cytosolic side potentiated TRPM4 currents, increased the Ca2+ sensitivity, but were unable to evoke TRPM4 currents without Ca2+. Mutations of the acidic amino acids near and in the TRP domain, which are conserved in TRPM2, TRPM5, and TRPM8, deteriorated the Ca2+ sensitivity in the presence of Co2+ or PI(4,5)P-2 but hardly affected the sensitivity to Co2+ and PI(4,5)P-2. These results suggest a novel role of the TRP domain in TRPM4 as a site responsible for maintaining the normal Ca2+ sensitivity. These findings provide more insights into the molecular mechanisms of the regulation of TRPM4 by Ca2+.
  • Ryo Miyamoto, Ken-ichi Otsuguro, Soichiro Yamaguchi, Shigeo Ito
    JOURNAL OF NEUROCHEMISTRY 130 1 29 - 40 2014年07月 [査読有り][通常論文]
     
    Hydrogen sulfide (H2S) is a gaseous neuromodulator produced from L-cysteine. H2S is generated by three distinct enzymatic pathways mediated by cystathionine -lyase (CSE), cystathionine -synthase (CBS), and mercaptopyruvate sulfurtransferase (MPST) coupled with cysteine aminotransferase (CAT). This study investigated the relative contributions of these three pathways to H2S production in PC12 cells (rat pheochromocytoma-derived cells) and the rat dorsal root ganglion. CBS, CAT, and MPST, but not CSE, were expressed in the cells and tissues, and appreciable amounts of H2S were produced from L-cysteine in the presence of -ketoglutarate, together with dithiothreitol. The production of H2S was inhibited by a CAT inhibitor (aminooxyacetic acid), competitive CAT substrates (L-aspartate and oxaloacetate), and RNA interference (RNAi) against MPST. Immunocytochemistry revealed a mitochondrial localization of MPST in PC12 cells and dorsal root ganglion neurons, and the amount of H2S produced by CAT/MPST at pH 8.0, a physiological mitochondrial matrix pH, was comparable to that produced by CSE and CBS in the liver and the brain, respectively. Furthermore, H2S production was markedly increased by alkalization. These results indicate that CAT and MPST are primarily responsible for H2S production in peripheral neurons, and that the regulation of mitochondrial metabolism may influence neuronal H2S generation. In the peripheral nervous system, hydrogen sulfide (H2S) has been implicated in neurogenic pain or hyperalgesia. This study provides evidence that H2S is synthesized in peripheral neurons through two mitochondrial enzymes, cysteine aminotransferase (CAT) and mercaptopyruvate sulfurtransferase (MPST). We propose that mitochondrial metabolism plays key roles in the physiology and pathophysiology of the peripheral nervous system via regulation of neuronal H2S production.
  • Soichiro Yamaguchi, Toru Ishikawa
    FEBS LETTERS 588 5 672 - 677 2014年03月 [査読有り][通常論文]
     
    Although AHCYL2 (long-IRBIT) is highly homologous to IRBIT, which regulates ion-transporting proteins including the electrogenic Na+-HCO(3)(-)cotransporter NBCe1-B, its functions are poorly understood. Here, we found that AHCYL2 interacts with NBCe1-B in bovine parotid acinar cells using yeast two-hybrid, immunofluorescence confocal microscopy and co-immunoprecipitation analyses. Whole-cell patch-clamp experiments revealed that co-expression of AHCYL2 reduces the apparent affinity for intracellular Mg2+ in inhibition of NBCe1-B currents specifically in a HCO3- -deficient cellular condition. Our data unveil AHCYL2 as a potential regulator of NBCe1-B in mammalian cells. We propose that cytosolic ionic condition appropriate for AHCYL2 to function might be different from IRBIT. (C) 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • Naoko Adachi, Takamasa Yoshida, Fumiaki Nin, Genki Ogata, Soichiro Yamaguchi, Toshihiro Suzuki, Sizuo Komune, Yasuo Hisa, Hiroshi Hibino, Yoshihisa Kurachi
    JOURNAL OF PHYSIOLOGY-LONDON 591 18 4459 - 4472 2013年09月 [査読有り][通常論文]
     
    The endocochlear potential (EP) of +80 mV in the scala media, which is indispensable for audition, is controlled by K+ transport across the lateral cochlear wall. This wall includes two epithelial barriers, the syncytium and the marginal cells. The former contains multiple cell types, such as fibrocytes, which are exposed to perilymph on their basolateral surfaces. The apical surfaces of the marginal cells face endolymph. Between the two barriers lies the intrastrial space (IS), an extracellular space with a low K+ concentration ([K+]) and a potential similar to the EP. This intrastrial potential (ISP) dominates the EP and represents the sum of the diffusion potential elicited by a large K+ gradient across the apical surface of the syncytium and the syncytium's potential, which is slightly positive relative to perilymph. Although a K+ transport system in fibrocytes seems to contribute to the EP, the mechanism remains uncertain. We examined the electrochemical properties of the lateral wall of guinea pigs with electrodes sensitive to potential and K+ while perfusing into the perilymph of the scala tympani blockers of Na+,K+-ATPase, the K+ pump thought to be essential to the system. Inhibiting Na+,K+-ATPase barely affected [K+] in the IS but greatly decreased [K+] within the syncytium, reducing the K+ gradient across its apical surface. The treatment hyperpolarized the syncytium only moderately. Consequently, both the ISP and the EP declined. Fibrocytes evidently use the Na+,K+-ATPase to achieve local K+ transport, maintaining the syncytium's high [K+] that is crucial for the K+ diffusion underlying the positive ISP.
  • Yamaguchi S, Ishikawa T
    Biochemical and biophysical research communications 424 3 433 - 438 3 2012年08月 [査読有り][通常論文]
     
    The electrogenic Na+-HCO3- cotransporter NBCe1-B can be regulated by intracellular Mg2+ (Mg-i(2+)). We previously reported that under whole-cell voltage-clamp conditions, bovine NBCe1-B (bNBCe1-B) currents heterologously expressed in mammalian cells are strongly inhibited by Mg-i(2+), and the inhibition is likely mediated by electrostatic interaction and relieved by truncation of the cytosolic NBCe1-B specific N-terminal region. Intriguingly, NBCe1-B-like currents natively expressed in bovine parotid acinar (BPA) cells are much less sensitive to Mg-i(2+) inhibition than bNBCe1-B currents. Here, we hypothesized that this apparent discrepancy may involve IRBIT, a previously identified NBCe1-B-interacting protein. RT-PCR, Western blot and immunofluorescence confocal microscopy revealed that IRBIT was not only expressed in the cytosol, but also colocalized with NBCe1-B in the region of plasma membranes of BPA cells. IRBIT was coimmunoprecipitated with NBCe1-B by an anti-NBCe1 antibody in bovine parotid cell lysate. Whole-cell patch-clamp experiments showed that coexpression of IRBIT lowered the Mg-i(2+) sensitivity of bNBCe1-B currents stably expressed in HEK293 cells. Collectively, these results suggest that IRBIT may reduce the apparent affinity for Mg-i(2+) in inhibition of NBCe1-B activity in mammalian cells. (C) 2012 Elsevier Inc. All rights reserved.
  • Soichiro Yamaguchi, Archana Jha, Qin Li, Abigail A. Soyombo, George D. Dickinson, Dev Churamani, Eugen Brailoiu, Sandip Patel, Shmuel Muallem
    JOURNAL OF BIOLOGICAL CHEMISTRY 286 26 22934 - 22942 2011年07月 [査読有り][通常論文]
     
    NAADP is a potent second messenger that mobilizes Ca(2+) from acidic organelles such as endosomes and lysosomes. The molecular basis for Ca(2+) release by NAADP, however, is uncertain. TRP mucolipins (TRPMLs) and two-pore channels (TPCs) are Ca(2+)-permeable ion channels present within the endolysosomal system. Both have been proposed as targets for NAADP. In the present study, we probed possible physical and functional association of these ion channels. Exogenously expressed TRPML1 showed near complete colocalization with TPC2 and partial colocalization with TPC1. TRPML3 overlap with TPC2 was more modest. TRPML1 and to some extent TRPML3 co-immuno-precipitated with TPC2 but less so with TPC1. Current recording, however, showed that TPC1 and TPC2 did not affect the activity of wild-type TRPML1 or constitutively active TRPML1(V432P). N-terminally truncated TPC2 (TPC2delN), which is targeted to the plasma membrane, also failed to affect TRPML1 and TRPML1(V432P) channel function or TRPML1(V432P)-mediated Ca(2+) influx. Whereas overexpression of TPCs enhanced NAADP-mediated Ca(2+) signals, overexpression of TRPML1 did not, and the dominant negative TRPML1(D471K) was without affect on endogenous NAADP-mediated Ca(2+) signals. Furthermore, the single channel properties of NAADP-activated TPC2delN were not affected by TRPML1. Finally, NAADP-evoked Ca(2+) oscillations in pancreatic acinar cells were identical in wild-type and TRPML1(-/-) cells. We conclude that although TRPML1 and TPCs are present in the same complex, they function as two independent organellar ion channels and that TPCs, not TRPMLs, are the targets for NAADP.
  • Hyun Jin Kim, Soichiro Yamaguchi, Qin Li, Insuk So, Shmuel Muallem
    JOURNAL OF BIOLOGICAL CHEMISTRY 285 22 16513 - 16520 2010年05月 [査読有り][通常論文]
     
    TRPML3 is a H(+)-regulated Ca(2+) channel that shuttles between intracellular compartments and the plasma membrane. The A419P mutation causes the varitint-waddler phenotype as a result of gain-of-function (GOF). The mechanism by which A419P leads to GOF is not known. Here, we show that the TRPML3 pore is dynamic when conducting Ca(2+) to change its conductance and permeability, which appears to be mediated by trapping Ca(2+) within the pore. The pore properties can be restored by strong depolarization or by conducting Na(+) through the pore. The A419P mutation results in expanded channel pore with altered permeability that limits modulation of the pore by Ca(2+). This effect is specific for the A419P mutation and is not reproduced by other GOF mutations, including A419G, H283A, and proline mutations in the fifth transmembrane domain. These findings describe a novel mode of a transient receptor potential channel behavior and suggest that pore expansion by the A419P mutation may contribute to the varitint-waddler phenotype.
  • Akihiro Inagaki, Soichiro Yamaguchi, Hiromi Takahashi-Iwanaga, Toshihiko Iwanaga, Toru Ishikawa
    JOURNAL OF MEMBRANE BIOLOGY 235 1 27 - 41 2010年05月 [査読有り][通常論文]
     
    ClC-2, a member of the voltage-gated Cl- channel family, is expressed in the distal colonic surface epithelial cells of various species, but its functional significance remains unclear. Here, by means of electrophysiological and molecular biological techniques, we have identified and characterized a ClC-2-like conductance naturally expressed by surface epithelial cells acutely dissociated from rectal colon of rats fed a standard diet. Whole-cell patch-clamp experiments showed that the surface cells, whether an amiloride-sensitive Na+ conductance was present or not, displayed a strong hyperpolarization-activated, inwardly rectifying Cl- current. Analysis both by in situ hybridization and immunohistochemistry confirmed the expression of ClC-2 in the rectal surface epithelium. The native Cl- current shared common electrophysiological properties including voltage-dependent activation, anion selectivity sequence, and Zn2+ sensitivity with that recorded from HEK293 cells transfected with ClC-2 cloned from rat rectal colon (rClC-2). Cell-attached patch recordings on the surface cells revealed that native ClC-2-like currents activated only at potentials at least 40 mV more negative than resting membrane potentials. In Ussing chamber experiments with rat rectal mucosa, either basolateral or apical application of Zn2+ (0.1 mM), which inhibited both native ClC-2-like currents and recombinant rClC-2 currents, had little, if any, effects on basal amiloride-sensitive short-circuit current. Collectively, these results not only demonstrate that a functional ClC-2-type Cl- channel is expressed in rat rectal surface epithelium, but also suggest that the channel activity may be negligible and thus nonessential for controlling electrogenic Na+ transport in this surface epithelium under basal physiological conditions.
  • Soichiro Yamaguchi, Shmuel Muallem
    CHEMISTRY & BIOLOGY 17 3 209 - 210 2010年03月 [査読有り][通常論文]
     
    The intracellular TRPML channels have multiple biological roles, but the physiological stimuli that open them remained unknown. In a previous issue of Chemistry & Biology, Grimm et al. report a high-throughput chemical screen that identified a plethora of selective activators of TRPML3 that should open the way to fully characterize these channels and their physiological roles.
  • Kirill Kiselyov, Soichiro Yamaguchi, Christopher W. Lyons, Shmuel Muallem
    CELL CALCIUM 47 2 103 - 111 2010年02月 [査読有り][通常論文]
     
    Lysosomal storage diseases (LSDs) are caused by inability of cells to process the material captured during endocytosis. While they are essentially diseases of cellular "indigestion", LSDs affect large number of cellular activities and, as such, they teach us about the integrative function of the cell, as well as about the gaps ill our knowledge of the endocytic pathway and membrane transport. The present review summarizes recent findings on Ca2+ handling in LSDs and attempts to identify the key questions on alterations in Ca2+ signaling and membrane transport in this group of diseases, answers to which may lie in delineating the cellular pathogeneses of LSDs. (C) 2009 Elsevier Ltd. All rights reserved.
  • Soichiro Yamaguchi, Toru Ishikawa
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 376 1 100 - 104 2008年11月 [査読有り][通常論文]
     
    NBCe1-B, a major splice variant of the electrogenic Na+-HCO3- cotransporter (NBCe1) fulfills basic cellular functions including regulation of intracellular pH and epithelial HCO3- secretion. However, its cellular regulatory mechanism still remains elusive. Here, we provide evidence for the first time that NBCe1-B activity can be controlled by intracellular Mg2+ (Mg-i(2+)), the physiologically most abundant intracellular divalent cation. Using the whole-cell patch-clamp technique, we found that recombinant NBCe1-B currents expressed in HEK293 and NIH3T3 cells were inhibited voltage-independently by Mg-i(2+) in a concentration-dependent manner (K-i approximate to 0.01 mM). The Mg-i(2+) inhibition was partially relieved by truncation of the NBCe1-B specific N-terminal region (K-i approximate to 0.3 mM), and was also observed for native electrogenic Na+-HCO3- cotransporter current in bovine parotid acinar cells that endogenously express NBCe1-B (K-i approximate to 1 mM). These results suggest that Mg2+ may be a cytosolic factor that limits intrinsic cotransport activity of NBCe1-B in mammalian cells. (C) 2008 Elsevier Inc. All Fights reserved.
  • S Yamaguchi, T Ishikawa
    JOURNAL OF PHYSIOLOGY-LONDON 568 1 181 - 197 2005年10月 [査読有り][通常論文]
     
    Using patch-clamp and molecular biological techniques, we identified and characterized membrane currents most likely generated by an electrogenic Na+-HCO3- cotransporter (NBCe) in acutely dissociated bovine parotid acinar (BPA) cells. When BPA cells were dialysed with a N-methyl-D-glucamine (NMDG)-glutamate-rich pipette solution, switching a Na-glutamate-rich, nominally HCO3- -free bath solution to the one containing 25 mM HCO3-, but not Cl-, elicited a whole-cell current with a linear current-voltage relation. The HCO3- evoked current was abolished by total replacement of extracellular Na+ (Na-0(+)) with NMDG or by 0.5 mM 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS), and was only partially supported by Li-0(+), but not by K-0(+), Cs-0(+), and cholinle(0). The reversal potential shift of DIDS (0.5 mm)-sensitive current induced by a change of [Na+](0) corresponded to an apparent coupling ratio of HCO3- to Na+ of 2. RT-PCR analysis showed the presence of transcripts of NBCel-B, but not NBCel-A in BPA cells. Electrophysiological and pharmacological properties of whole-cell currents recorded from HEK293 cells transfected with the NBCel-B, which was cloned from BPA cells resembled those of the native currents. Non-invasive measurements of membrane potential changes in the cell-attached patch configuration indicated that an NBCe activity is present in intact unstimulated BPA cells bathed in a 25 mM HCO3--containing solution. Collectively, these results not only suggest that an NBCe is present, functional and may be mediated, at least in part, by NBCel-B in BPA cells, but also provide the first electrophysiological characterization of transport properties of NBCe expressed in native exocrine glands.
  • A Inagaki, S Yamaguchi, T Ishikawa
    AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY 286 2 C380 - C390 2004年02月 [査読有り][通常論文]
     
    Surface cells of the mammalian distal colon are shown to molecularly express the amiloride-sensitive epithelial Na+ channel composed of three homologous subunits (alpha-, beta-, and gamma-ENaC). However, because basic electrophysiological properties of amiloride-sensitive Na+ channels expressed in these cells are largely unknown at the cellular level, functional evidence for the involvement of the subunits in the native channels is incomplete. Using electrophysiological techniques, we have now characterized functional properties of native ENaC in surface cells of rectal colon ( RC) of rats fed a normal Na+ diet. Ussing chamber experiments showed that apical amiloride inhibited a basal short-circuit current in mucosal preparation of RC with an apparent half-inhibition constant (K-i) value of 0.20 muM. RT-PCR analysis confirmed the presence of transcripts of alpha-, beta-, and gamma-rENaC in rectal mucosa. Whole cell patch-clamp experiments in surface cells of intact crypts acutely isolated from rectal mucosa identified an inward cationic current, which was inhibited by amiloride with a K-i value of 0.12 muM at a membrane potential of -64 mV, the inhibition being weakly voltage dependent. Conductance ratios of the currents were Li+ (1.8)>Na+ (1)much greater thanK(+) (approximate to0), respectively. Amiloride-sensitive current amplitude was almost the same at 15 or 150 mM extracellular Na+, suggesting a high Na+ affinity for current activation. These results are consistent with the hypothesis that a heterooligomer composed of alpha-, beta-, and gamma-ENaC may be the molecular basis of the native channels, which are responsible for amiloride-sensitive electrogenic Na+ absorption in rat rectal colon.

その他活動・業績

受賞

  • 2011年09月 日本獣医学会 生理学・生化学分科会 生理学・生化学分科会奨励賞
     
    受賞者: 山口 聡一郎

共同研究・競争的資金等の研究課題

  • 膜タンパク質による新しい転写調節機構の実証
    日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2016年04月 -2020年03月 
    代表者 : 山口 聡一郎
     
    本研究はTMC1の細胞内N端領域の機能解明を目的とする。本年度は、western blot法による、切断部位についての解析が進んだ。昨年度までにN端側から二か所の切断部位を示唆する断片が二つ確認できていた。ここで、N端側から順に断片1、2、あるいは切断部位1、2とする。切断部位1の領域のアミノ酸を欠損させたTMC1では、新たに、さらにC端側の切断部位3と切断部位4の存在を示唆する断片が増加し、よりはっきりと検出された。切断部位4の場所を、推定されるアミノ酸を含む断片を作製し、電気泳動上の位置を比較する、あるいは周囲のアミノ酸を欠損させた変異体により解析したところ、第一膜貫通領域内で切断されている可能性が浮上した。これは、膜関連タンパク分解という機構が働き、第一膜貫通領域が切断されることによって、遊離した細胞内断片が、さらに切断されてN端の断片が作成されている可能性を示唆する。次年度は、膜関連タンパク分解の機構の関与も調べていく。 また、転写調節への影響を調べるため、断片1あるいは断片2を安定的に発現するHEK293細胞の作成を試みたところ、断片2を少ないながら発現する細胞クローンを作成することができた。この細胞からmRNAを抽出し、次世代シーケンサーにかけるためのcDNAライブラリーの作成も行った。シーケンスおよび解析を次年度行う。 一方で、TMC1を発現する蝸牛や精巣の抽出物からTMC1タンパク質のwestern blotによる解析を試みたが、切断されていない全長のタンパク質も検出できなかった。これは発現する有毛細胞が蝸牛内では数が少ないことと、精巣では発現量が全体として低いことに起因すると思われる。内因性細胞での切断現象の有無を検討するため、マウス有毛細胞を分離して、免疫染色によりTMC1を検出することを考慮している。
  • 文部科学省:科学研究費補助金(若手研究(B))
    研究期間 : 2014年 -2015年 
    代表者 : 山口 聡一郎
  • 文部科学省:科学研究費補助金(若手研究(B))
    研究期間 : 2011年 -2011年 
    代表者 : 山口 聡一郎
  • ウシ耳下腺でのナトリウム重炭酸イオン共輸送体の分子基盤の解明:複合体形成の可能性
    日本学術振興会:科学研究費助成事業 特別研究員奨励費
    研究期間 : 2006年 -2008年 
    代表者 : 山口 聡一郎
     
    Yeast two-hybrid法を用いたウシ耳下腺cDNAライブラリーの探索によりNBCe1-BのN端領域との結合タンパク質候補として浮上した2種類のタンパク質(以下、タンパク質Aとタンパク質B)について、以下の実験を行った。共焦点顕微鏡を用いたimmunocytochemistryの実験により、分離したウシ耳下腺腺房細胞において、NBCe1-B、タンパク質A、およびタンパク質Bが発現し、形質膜上にそれらが集積し共局在している傾向が見られた。導管細胞と思われる細胞からはいずれのシグナルも検出されなかった。また、タンパク質AあるいはBをNBCe1-Bと共発現させたHEK293細胞を用いた実験でも、タンパク質AあるいはBとNBCe1-Bの共局在を示唆する結果が得られた。さらに、関連する実験テーマとしてNBCe1-Bの輸送活性が細胞内Mg^<2+>によって抑制され、その抑制作用にNBCe1-Bの特異的N端領域が関与することを明らかにしたことから、N端領域に結合するタンパク質AあるいはBの共発現によるNBCe1-Bに対する細胞内Mg^<2+>の抑制作用への影響を調べた。HEK293細胞にタンパク質AあるいはBとNBCe1-Bを共発現させ、パッチクランプ法によりNBCe1-B電流を測定したところ、条件によっては共発現により細胞内Mg^<2+>による抑制作用が減弱することが明らかとなった。これはタンパク質AあるいはBの結合によるNBCe1-Bの活性化のメカニズムに細胞内Mg^<2+>による抑制作用が関与する可能性を示唆する結果である。以上、本研究によりNBCe1-Bに対する新たな相互作用タンパク質が明らかとなり、それらによる機能的修飾のメカニズムの一つの可能性を提唱することができた。現在、これらの成果を論文としてまとめているところである。

教育活動情報

主要な担当授業

  • ケミカルハザード対策専門特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 薬理学実習
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 獣医学部
    キーワード : 体内動態、標的分子、用量反応関係、作動薬、拮抗薬、オータコイド、ホルモン、神経伝達物質、抗炎症薬、利尿薬,麻酔薬、鎮痛薬、自律神経作動薬、筋弛緩薬
  • 先端獣医科学特論B 生命科学特論Ⅲ:細胞膜の興奮性
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 生理学実習
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 獣医学部
    キーワード : 血液、循環系、筋収縮、消化と吸収、膵機能、恒常性維持機構
  • 獣医科学基礎科目 生命科学特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • 先端獣医科学科目 先端生命科学特論Ⅳ:薬理学
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • ケミカルハザード対策専門特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • アドバンスト演習
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 獣医学部


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