研究者データベース

乙黒 兼一(オツグロ ケンイチ)
獣医学研究院 獣医学部門 基礎獣医科学分野
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

  • 教授

学位

  • 博士(獣医学)(北海道大学)

ホームページURL

科研費研究者番号

  • 40344494

J-Global ID

研究キーワード

  • プリン受容体   アドレナリン受容体   TRPA1   硫化水素   アデノシン   アストロサイト   低酸素   イノシン   薬理学   Cell biology   Pharmacology   

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04月 - 現在 北海道大学 大学院獣医学研究院 教授
  • 2009年06月 - 2018年03月 北海道大学 大学院獣医学研究科 准教授
  • 2007年04月 - 2009年05月 北海道大学 大学院獣医学研究科 助教
  • 2006年06月 - 2007年03月 Queen's University of Belfast (UK) 客員研究員
  • 2002年06月 - 2007年03月 北海道大学 大学院獣医学研究科 助手
  • 1999年04月 - 2002年05月 三共㈱ 研究員
  • 1998年04月 - 1999年03月 北海道大学 日本学術振興会特別研究員(PD)

学歴

  • 2001年05月 -   浜松医科大学   医学部 研究生
  • 1998年03月 -   北海道大学   大学院獣医学研究科   博士課程修了
  • 1994年03月 -   北海道大学   獣医学部   獣医学科 卒業

所属学協会

  • 日本神経化学会   日本獣医学会   日本薬理学会   

研究活動情報

論文

  • Nii T, Eguchi R, Yamaguchi S, Otsuguro K
    European Journal of Pharmacology 2020年10月29日 [査読有り][通常論文]
  • Eguchi R, Kitano T, Otsuguro K
    Purinergic Signalling 2020年10月06日 [査読有り][通常論文]
  • Yamaguchi S, Hamamura M, Otsuguro K
    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.
  • Morimoto K, Kitano T, Eguchi R, Otsuguro K
    Biochemical and Biophysical Research Communications 528 78 - 84 2020年05月22日 [査読有り][通常論文]
     
    Noradrenaline (NA) suppresses TNF-α production via β-adrenoceptors (ARs) in brain astrocytes. However, the downstream pathways from β-ARs, and the involvement of α-ARs, remains unknown. In this study, we investigated the AR-mediated regulation of TNF-α mRNA levels in cultured astrocytes from rat spinal cord. NA, the α1-agonist phenylephrine, and the β-agonist isoproterenol decreased the TNF-α mRNA level, while the α2-agonist dexmedetomidine increased it. The isoproterenol-induced TNF-α mRNA decrease was accompanied by a decrease in ERK phosphorylation. An adenylyl cyclase activator and an ERK inhibitor mimicked these effects. These results indicate that the transcriptional regulation of TNF-α by β-ARs is mediated via cAMP pathways followed by the ERK pathway inhibition. The dexmedetomidine-induced TNF-α mRNA increase was accompanied by phosphorylation of JNK and ERK, which was blocked by a JNK inhibitor. Furthermore, the LPS-induced increase in the TNF-α mRNA level was accompanied by NF-κB nuclear translocation, and both these effects were blocked by phenylephrine. An NF-κB inhibitor suppressed the LPS-induced increase in the TNF-α mRNA level. These results suggest that α1-ARs suppress the LPS-induced increase in the TNF-α mRNA level via inhibition of NF-κB nuclear translocation. Taken together, our study reveals that both α- and β-ARs are involved in the transcriptional regulation of TNF-α in astrocytes.
  • Yamaguchi S, Tanimoto A, Iwasa S, Otsuguro K
    Int J Mol Sci 20 8 E2012  2019年04月 [査読有り][通常論文]
  • Kitano T, Kobayashi T, Yamaguchi S, Otsuguro K
    J Vet Pharmacol Ther 42 2 243 - 247 2019年03月 [査読有り][通常論文]
  • Ujike A, Kuraishi T, Yamaguchi S, Eguchi R, Kitano T, Kamise J, Ito S, Otsuguro K
    Eur J Pharmacol 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.
  • Yamasaki M, Watanabe N, Idaka N, Yamamori T, Otsuguro K, Uchida N, Iguchi A, Ohta H, Takiguchi M
    Exp Parasitol 183 92 - 98 2017年12月 [査読有り][通常論文]
     
    The mechanism of the development of diminazene aceturate (DA) resistance in Babesia gibsoni is still unknown even though DA-resistant B. gibsoni isolate was previously developed in vitro. To clarify the mechanisms of DA-resistance in B. gibsoni, we initially examined the intracellular DA content in the DA resistant isolate using high-performance liquid chromatography, and compared it with that in the wild type. As a result, the intracellular DA content in the DA-resistant isolate was significantly lower than that in the wild-type, suggesting that the decreased DA content may contribute to DA-resistance. Additionally, the glucose consumption of the DA-resistant isolate was significantly higher than that of the wild-type, indicating that a large amount of glucose is utilized to maintain DA-resistance. It is possible that a large amount of energy is utilized to maintain the mechanisms of DA-resistance. It was reported that as the structure of DA is similar with that of adenosine, DA may be taken up by the P2 transporter, which contributes to the uptake of adenosine, in Trypanosoma brucei brucei, and that the uptake of adenosine is decreased in DA-resistant T brucei brucei. In the present study, the adenosine incorporation in the DA-resistant B. gibsoni isolate was higher than in the wild-type. Moreover, the adenosine incorporation in the wild-type was not inhibited by the presence of DA. These results suggest that adenosine transport in B. gibsoni is not affected by DA and may not mediate DA-resistance. To clarify the mechanism of the development of DA resistance in B. gibsoni, we should investigate the cause of the decreased DA content in the DA-resistant isolate in the future. (C) 2017 Elsevier Inc. All rights reserved.
  • 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
    Eur J Pharmacol 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 K, 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.
  • Takeyuki Iwasaki, Ken-ichi Otsuguro, Takeshi Kobayashi, Toshio Ohta, Shigeo Ito
    EUROPEAN JOURNAL OF PHARMACOLOGY 702 1-3 149 - 157 2013年02月 [査読有り][通常論文]
     
    Serotonin (5-HT) released from descending fibers plays important roles in spinal functions such as locomotion and nociception. 5-HT2A and 5-HT3 receptors are suggested to contribute to spinal antinociception, although their activation also contributes to neuronal excitation. In the neonatal spinal cord, DL-p-chloroamphetamine (pCA), a 5-HT releaser, inhibited both A fiber-evoked monosynaptic reflex potential (MSR) and C fiber-evoked slow ventral root potential (sVRP). The pCA-mediated inhibition was reversed by ketanserin (a 5-HT2A receptor antagonist) and tropisetron (a 5-HT3 receptor antagonist). Bath-applied 5-HT also inhibited MSR and sVRP; in this case, the actions of 5-HT were antagonized by ketanserin, but not by tropisetron. The pCA-evoked inhibition of sVRP was reduced by bicuculline (a GABA(A) receptor antagonist) and strychnine (a glycine receptor antagonist). Furthermore, ketanserin inhibited the pCA-evoked release of gamma-aminobutyric acid (GABA) and glycine, while tropisetron inhibited the pCA-evoked release of 5-HT. These results suggest that 5-HT released by pCA activates 5-HT2A receptors, which in turn stimulates the release of GABA/glycine and thereby blocks the spinal nociceptive pathway. 5-HT3 receptors may be involved in the facilitation of 5-HT release via a positive feedback process. (C) 2013 Elsevier B.V. All rights reserved.
  • K. Kawamoto, K. Otsuguro, M. Ishizuka, S. Ito
    BRITISH JOURNAL OF PHARMACOLOGY 166 2 788 - 800 2012年05月 [査読有り][通常論文]
     
    BACKGROUND AND PURPOSE Dopamine released from the endings of descending dopaminergic nerve fibres in the spinal cord may be involved in modulating functions such as locomotion and nociception. Here, we examined the effects of dopamine on spinal synaptic transmissions in rats. EXPERIMENTAL APPROACH Spinal reflex potentials, monosynaptic reflex potential (MSR) and slow ventral root potential (sVRP), were measured in the isolated spinal cord of the neonatal rat. Dopamine release was measured by HPLC. KEY RESULTS Dopamine at lower concentrations (< 1 mu M) depressed sVRP, which is a C fibre-evoked polysynaptic response and believed to reflect nociceptive transmission. At higher concentrations (> 1 mu M), in addition to a potent sVRP depression, dopamine depolarized baseline potential and slightly depressed MSR. Depression of sVRP by dopamine was partially reversed by dopamine D-1-like but not by D-2-like receptor antagonists. SKF83959 and SKF81297, D-1-like receptor agonists, and methamphetamine, an endogenous dopamine releaser, also caused the inhibition of sVRP. Methamphetamine also depressed MSR, which was inhibited by ketanserin, a 5-HT2A/2C receptor antagonist. Methamphetamine induced the release of dopamine and 5-HT from spinal cords, indicating that the release of endogenous dopamine and 5-HT depresses sVRP and MSR respectively. CONCLUSION AND IMPLICATIONS These results suggested that dopamine at lower concentrations preferentially inhibited sVRP, which is mediated via dopamine D-1-like and other unidentified receptors. The dopamine-evoked depression is involved in modulating the spinal functions by the descending dopaminergic pathways.
  • K. Otsuguro, M. Wada, S. Ito
    BRITISH JOURNAL OF PHARMACOLOGY 164 1 132 - 144 2011年09月 [査読有り][通常論文]
     
    BACKGROUND AND PURPOSE Hypoxic effects on neuronal functions vary significantly with experimental conditions, but the mechanism for this is unclear. Adenosine has been reported to play a key role in depression of neuronal activities in the CNS during acute hypoxia. Hence, we examined the effect of acute hypoxia on different spinal reflex potentials and the contribution of adenosine to them. EXPERIMENTAL APPROACH Spinal reflex potentials, monosynaptic reflex potential (MSR), slow ventral root potential (sVRP) and dorsal root potential (DRP), were measured in the isolated spinal cord of the neonatal rat. Adenosine release was measured by using enzymatic biosensors. KEY RESULTS In the spinal cord preparation isolated from postnatal day 5-8 rats at 27 degrees C, acute hypoxia induced adenosine release and depressed three reflex potentials. However, in postnatal day 0-3 rats at 27 degrees C, the hypoxic-induced adenosine release and depression of MSR were negligible, while the depression of sVRP and DRP were perceptible responses. In postnatal day 0-3 rats at 33 degrees C, hypoxia evoked adenosine release and depression of MSR. An adenosine A(1) receptor selective antagonist and a high [Ca2+](o), which suppressed adenosine release, abolished the hypoxic-induced depression of MSR but not those of sVRP and DRP. CONCLUSIONS AND IMPLICATIONS Hypoxic-induced depression of MSR depends on adenosine release, which is highly susceptible to age, temperature and [Ca2+](o). However, a large part of the depressions of DRP and sVRP are mediated via adenosine-independent mechanisms. This differential contribution of adenosine to depression is suggested to be an important factor for the variable effects of hypoxia on neuronal functions.
  • Ryo Miyamoto, Ken-ichi Otsuguro, Shigeo Ito
    NEUROSCIENCE LETTERS 499 2 137 - 142 2011年07月 [査読有り][通常論文]
     
    Hydrogen sulfide (H(2)S) is considered as a gasotransmitter. Although several reports have shown that H(2)S stimulates sensory neurons, the primary targets of H(2)S remain controversial. We investigated the effects of H(2)S on cultured sensory neurons isolated from rat dorsal root ganglion (DRG) using Ca(2+) imaging and whole-cell voltage-clamp techniques. Brief (2 min) application of NaHS (1 mM), a donor of H(2)S, evoked marked increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) in a subset of DRG neurons. These neurons also responded to both capsaicin and mustard oil (MO), transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1) agonists, respectively. The NaHS-evoked [Ca(2+)](i) increases were inhibited by a removal of external Ca(2+) and antagonists for TRPA1, but not for TRPV1 or voltage-dependent Ca(2+) channels. At -80 mV, NaHS evoked inward currents in MO-sensitive neurons, which were also inhibited by a TRPA1 antagonist. Even at lower concentration (<= 1 mu M), the 10-min application of NaHS increased [Ca(2+)](i) in a time- and concentration-dependent manner. These results suggest that H(2)S stimulates sensory neurons via activation of TRPA1. Endogenous H(2)S may be involved in physiological processes through TRPA1. (C) 2011 Elsevier Ireland Ltd. All rights reserved.
  • T. Takahashi, K. Otsuguro, T. Ohta, S. Ito
    BRITISH JOURNAL OF PHARMACOLOGY 161 8 1806 - 1816 2010年12月 [査読有り][通常論文]
     
    BACKGROUND AND PURPOSE Adenosine and inosine accumulate extracellularly during hypoxia/ischaemia in the brain and may act as neuroprotectants. In spinal cord, there is pharmacological evidence for increases in extracellular adenosine during hypoxia, but no direct measurements of purine release. Furthermore, the efflux pathways and origin of extracellular purines are not defined. To characterize hypoxia-evoked purine accumulation, we examined the effect of acute hypoxia on the extracellular levels of adenosine and inosine in isolated spinal cords from rats. EXPERIMENTAL APPROACH Extracellular adenosine and inosine concentrations were assayed in an in vitro preparation of the isolated spinal cord of the neonatal rat by HPLC. KEY RESULTS The extracellular level of inosine was about 10-fold higher than that of adenosine. Acute hypoxia (10 min) caused a temperature-dependent increase in these two purines, which were inhibited by an increase in external Ca2+, but not by several inhibitors of efflux pathways or metabolic enzymes of adenine nucleotides. Inhibitors of adenosine deaminase or the equilibrative nucleoside transporter (ENT) abolished the hypoxia-evoked increase in inosine but not adenosine. The inhibition of glial metabolism abolished the increase of both purines evoked by hypoxia but not by oxygen-glucose deprivation, hypercapnia or an adenosine kinase inhibitor. CONCLUSIONS AND IMPLICATIONS Our data suggest that hypoxia releases adenosine itself from intracellular sources. Inosine formed intracellularly may be released through ENTs. During hypoxia, astrocytes appear to play a key role in purine release from neonatal rat spinal cord.
  • Y. Kajihara, M. Murakami, T. Imagawa, K. Otsuguro, S. Ito, T. Ohta
    NEUROSCIENCE 166 1 292 - 304 2010年03月 [査読有り][通常論文]
     
    In inflamed tissues, extracellular pH decreases and acidosis is an important source of pain. Histamine is released from mast cells under inflammatory conditions and evokes the pain sensation in vivo, but the cellular mechanism of histamine-induced pain has not been well understood. In the present study, we examined the effects of histamine on [Ca(2+)](i) and membrane potential responses to acid in isolated mouse dorsal root ganglion (DRG) neurons. In capsaicin-sensitive DRG neurons from wild-type mice, acid (>pH 5.0) evoked [Ca(2+)](i) increases, but not in DRG neurons from transient receptor potential V1 (TRPV1) (-/-) mice. Regardless of isolectin GS-IB4 (IB4)-staining, histamine potentiated [Ca(2+)](i) responses to acid (>= pH 6.0) that were mediated by TRPV1 activation. Histamine increased membrane depolarization induced by acid and evoked spike discharges. RT-PCR indicated the expression of all four histamine receptors (H1R, H2R, H3R, H4R) in mouse DRG. The potentiating effect of histamine was mimicked by an H1R agonist, but not H2R-H4R agonists and was inhibited only by an H1R antagonist. Histamine failed to potentiate the [Ca(2+)](i) response to acid in the presence of inhibitors for phospholipase C (PLC) and protein kinase C (PKC). A lipoxygenase inhibitor and protein kinase A inhibitor did not affect the potentiating effects of histamine. Carrageenan and complete Freund's adjuvant produced inflammatory hyperalgesia, but these inflammatory conditions did not change the potentiating effects of histamine in DRG neurons. The present results suggest that histamine sensitizes acid-induced responses through TRPV1 activation via H1R coupled with PLC/PKC pathways, the action of which may be involved in the generation of inflammatory pain. (C) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.
  • Taketoshi Ogawa, Masami Hashimoto, Yoichi Niitsu, Joseph A. Jakubowski, Yoshiro Tani, Ken-ichi Otsuguro, Fumitoshi Asai, Atsuhiro Sugidachi
    EUROPEAN JOURNAL OF PHARMACOLOGY 612 1-3 29 - 34 2009年06月 [査読有り][通常論文]
     
    Prasugrel is an orally available thienopyridyl prodrug with more potent in vivo antiplatelet effects compared to clopidogrel. In the present study, we examined the effects of prasugrel in rat models of cerebral and peripheral arterial occlusive diseases. Cerebral arterial thrombosis was induced by photochemical irradiation of the middle cerebral artery. Prasugrel (3 and 10 mg/kg) dose-relatedly and significantly reduced thrombus-mediated cerebral infarction 24 h after the irradiation. The effect of prasugrel was further examined in ail embolic infarction model. Four h after an oral administration of prasugrel, non-occlusive thrombus formation in the right common carotid artery was initiated. In this model, prasugrel (0.3-3 mg/kg) reduced incidence, total area, and total number of cerebral infarcts in a dose-related manner 24 h after the vascular injury. Clopidogrel (10 or 30 mg/kg) was less potent than prasugrel at the doses tested oil these thrombotic and embolic infarctions. Finally, the effect of prasugrel on lauric acid-induced peripheral arterial occlusive diseases was evaluated. After injection of lauric acid into the femoral artery, the lesions were scored for the following 10 days as they gradually progressed from the toe throughout the leg. Prasugrel (0.03-3 mg/kg/day) administered from the day before the lauric acid injection for 11 successive days inhibited the progression of the disease in a close-related manner. Clopidogrel (3-30 mg/kg/day) showed similar effect but its effect was less potent than prasugrel. These results suggest that prasugrel could be a useful drug for preventing thromboembolic diseases including cerebral infarction and peripheral arterial occlusive diseases. (C) 2009 Elsevier B.V. All rights reserved.
  • K. Otsuguro, M. Ban, T. Ohta, S. Ito
    BRITISH JOURNAL OF PHARMACOLOGY 156 7 1167 - 1177 2009年04月 [査読有り][通常論文]
     
    The purine compounds, adenosine 5'-triphosphate (ATP) and adenosine, are known to accumulate in the extracellular space and to elicit various cellular responses during hypoxia/ischemia, whereas the roles of purines during hypercapnia are poorly understood. In this study, we examined the effects of various drugs affecting purine turnover on the responses to hypercapnia in the spinal cord. Electrically evoked reflex potentials were measured in an in vitro preparation of the isolated spinal cord of the neonatal rat by extracellular recording. Extracellular adenosine concentrations were assayed by high performance liquid chromatography (HPLC) methods. Hypercapnia (20% CO(2)) depressed the reflex potentials, which were partially reversed by an adenosine A(1) receptor antagonist, 8-cyclopentyl theophylline, but not by a P2 receptor antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid. Exogenous adenosine and ATP also depressed the reflex potentials via adenosine A(1) receptors. The hypercapnia-evoked depression was not reversed by inhibitors of gap junction hemichannels, anion channels, P2X(7) receptors or equilibrative nucleoside transporters, all of which might be involved in purine efflux pathways. The adenosine accumulation evoked by hypercapnia was not inhibited by tetrodotoxin, ethylene glycol-bis(beta-amino ethyl ether) tetraacetic acid (EGTA) or an ecto-ATPase inhibitor, ARL 67156. Homocysteine thiolactone, used to trap intracellular adenosine, significantly reduced extracellular adenosine accumulation during hypercapnia. These results suggest that hypercapnia released adenosine itself from intracellular sources, using pathways different from the conventional exocytotic mechanism, and that this adenosine depressed spinal synaptic transmission via adenosine A(1) receptors.
  • Tetsuya Ishida, Ryo Takei, Shree Hari Gautam, Ken-ichi Otsuguro, Toshio Ohta, Shigeo Ito, Yoshiaki Habara, Toshiyuki Saito
    NEUROSCIENCE LETTERS 441 3 277 - 281 2008年08月 [査読有り][通常論文]
     
    Bipolar vomeronasal sensory neurons (VSNs) in the vomeronasal organ (VNO) are believed to detect pheromones in most mammals. The vomeronasal sensory epithelium (VSE) is composed of VSNs and supporting cells. There are morphological differences in VNOs between species. Many electrophysiological experiments have been performed on rodent VSEs but few on other mammals. We therefore investigated voltage-gated channel properties of cells in the porcine VSE using slice whole-cell voltage-clamp techniques. In immunohistochemical study of the porcine VSE, most PGP9.5-immunoreactive cells were found between the middle and basal region, and negative cells were distributed in the apical to middle region. Depolarizing pulses to epithelial cells from -90 mV produced transient inward Na+ channel currents and sustained outward K+ channel currents with various amplitudes. The distribution of cells having high and low Na+ current densities was mostly consistent with the histological distribution of VSNs and supporting cells, respectively. The half-inactivation voltage of voltage-gated Na+ channels in supporting cells was 26 mV more negative than that in VSNs. Voltage-gated K+ channel currents in both cell types were suppressed by tetraethylammonium to the same extent. VSNs possessed TTX-sensitive voltage-gated Na+ channels and Ni2+-sensitive T-type Ca2+ channels. These results suggest that the histological distribution of porcine vomeronasal epithelial cells is more similar to the dog and goat than to rodents, and that the electrophysiological characteristics of porcine vomeronasal epithelial cells are similar to those of rodents. it is also suggested that porcine VSNs detecting pheromones generate action potentials through these channels. (c) 2008 Elsevier Ireland Ltd. All rights reserved.
  • Ken-ichi Otsuguro, Jisen Tang, Yufang Tang, Rui Xiao, Marc Freichel, Volodymyr Tsvilovskyy, Shigeo Ito, Veit Flockerzi, Michael X. Zhu, Alexander V. Zholos
    JOURNAL OF BIOLOGICAL CHEMISTRY 283 15 10026 - 10036 2008年04月 [査読有り][通常論文]
     
    Full-length transient receptor potential (TRP) cation channel TRPC4 alpha and shorter TRPC4 beta lacking 84 amino acids in the cytosolic C terminus are expressed in smooth muscle and endothelial cells where they regulate membrane potential and Ca(2+) influx. In common with other "classical" TRPCs, TRPC4 is activated by G(q)/phospholipase C-coupled receptors, but the underlying mechanism remains elusive. Little is also known about any isoform-specific channel regulation. Here we show that TRPC4 alpha but not TRPC4 beta was strongly inhibited by intracellularly applied phosphatidylinositol 4,5-bisphosphate (PIP(2)). In contrast, several other phosphoinositides (PI), including PI(3,4)P(2), PI(3,5)P(2), and PI(3,4,5)P(3), had no effect or even potentiated TRPC4 alpha indicating that PIP(2) inhibits TRPC4 alpha in a highly selective manner. We show that PIP2 binds to the C terminus of TRPC4 & but not that of TRPC4 beta in vitro. Its inhibitory action was dependent on the association of TRPC4 alpha with actin cytoskeleton as it was prevented by cytochalasin D treatment or by the deletion of the C-terminal PDZ-binding motif (Thr-Thr-Arg-Leu) that links TRPC4 to F-actin through the sodium-hydrogen exchanger regulatory factor and ezrin. PIP(2) breakdown appears to be a required step in TRPC4 alpha channel activation as PIP(2) depletion alone was insufficient for channel opening, which additionally required Ca(2+) and pertussis toxin-sensitive G(i/o) proteins. Thus, TRPC4 channels integrate a variety of G-protein-dependent stimuli, including a PIP(2)/cytoskeleton dependence reminiscent of the TRPC4-like muscarinic agonist activated cation channels in ileal myocytes.
  • Yoichi Niitsu, Atsuhiro Sugidachi, Taketoshi Ogawa, Joseph A. Jakubowski, Masami Hashimoto, Takashi Isobe, Ken-ichi Otsuguro, Fumitoshi Asai
    EUROPEAN JOURNAL OF PHARMACOLOGY 579 1-3 276 - 282 2008年01月 [査読有り][通常論文]
     
    (A)ntiplatelet and antithrombotic activity of multiple oral dosing of prasugrel were evaluated in several animal species. Prasugrel's active metabolite concentration-relatedly inhibited in vitro ADP-induced aggregation of rat, rabbit, dog, monkey and human platelets. Oral administration of prasugrel to dogs (0.03-0.3 mg/kg/day) and monkeys (0.1 and 0.3 mg/kg/day) once a day for 14 days resulted in potent, dose-related and cumulative inhibition of ADP-induced platelet aggregation. The inhibitory effects reached a plateau on days 3 to :5 and thereafter were maintained during dosing. Inhibition decreased gradually after cessation of dosing with near full recovery by 7 days after last dose. Antiplatelet and antithrombotic activity of prasugrel and clopidogrel were further examined in rats. Multiple oral dosing of prasugrel (0.3-3 mg/kg/day) to rats resulted in more potent inhibition of platelet aggregation compared to clopidogrel (3-30 mg/kg/day) and ticlopidine (30-300 mg/kg/day). Separate experiments confirmed that platelet inhibition was associated with inhibition of [H-3]-2-methylthio-ADP binding to rat platelets. In a rat model of electrically-induced arterial thrombosis, prasugrel (0.1-1 mg/kg/day, p.o.) significantly prolonged the time to arterial occlusion and increased the duration of arterial patency. The inhibition of platelet aggregation of prasugrel was about 10 and 300 times more potent than clopidogrel and ticlopidine, respectively. Overall these results show that in several species multiple oral administration of prasugrel results in more potent inhibition of platelet aggregation and thrombus formation than clopidogrel and ticlopidine, and that these effects are mediated by inhibition of platelet ADP receptors. (c) 2007 Elsevier B.V. All rights reserved.
  • M. Murakami, T. Ohta, K. -I. Otsuguro, S. Ito
    NEUROSCIENCE 145 2 642 - 653 2007年03月 [査読有り][通常論文]
     
    We characterized bradykinin (BK)-induced changes in the intracellular Ca2+ concentration ([Ca2+](i)) and membrane potential in cultured rat myehteric neurons using ratiometric Ca2+ imaging with fura-2 and the whole-cell patchclamp technique, respectively. BK evoked a dose-dependent increase of [Ca2+](i) that was abolished by HOE 140, a 82 receptor antagonist but not by [Lys-des-Arg(9)]-BK, a 81 receptor antagonist. [Lys-des-Arg(9)]-HOE140, a B1 receptor agonist, failed to cause a [Ca2+](i) response. Double staining with antibodies against the 82 receptor together with PGP9.5 or S100 indicated that 82 receptors were expressed in neurons and glial cells. The BK-evoked [Ca2+](i) increase was suppressed by indomethacin, a non-selective cyclooxygenase (COX) inhibitor, and potentiated by prostaglandin E-2 (PGE(2)). The release of PGE2 from cultured myenteric plexus cells was increased by BK. BK induced a large increase in [Ca2+](i) in neurons when myenteric plexus cells were cultured at the high density but not at the low density, and caused a small increase in [Ca2+](i) in neurons when proliferation of enteric glial cells was suppressed. BK evoked a slow and sustained depolarization in myenteric neurons, which was sensitive to indomethacin.These results indicated that BK caused a [Ca2+](i) increase and depolarization in rat myenteric neurons through the activation of 132 receptors, which was partly associated with PGE(2) released from glial cells in response to BK. It is suggested that a neuron-glial interaction plays an important role in the effect of BK in the rat myenteric plexus. (c) 2007 IBRO. Published by Elsevier Ltd. All rights reserved.
  • S. H. Gautam, K. -I. Otsuguro, S. Ito, T. Saito, Y. Habara
    NEUROSCIENCE 144 2 702 - 713 2007年01月 [査読有り][通常論文]
     
    Propagation of odor-induced Ca2+ transients from the cilia/knob to the soma in mammalian olfactory receptor neurons (ORNs) is thought to be mediated exclusively by high-voltage-activated Ca2+ channels. However, using confocal Ca2+ imaging and immunocytochemistry we identified functional T-type Ca2+ channels in rat ORNs. Here we show that T-type Ca2+ channels in ORNs also mediate propagation of odor-induced Ca2+ transients from the knob to the soma. In the presence of the selective inhibitor of T-type Ca2+ channels mibefradil (10-15 mu M) or Ni2+ (100 mu M), odor- and forskolin/3-isobutyl-1-methyl-xanthine (IBMX)-induced Ca2+ transients in the soma and dendrite were either strongly inhibited or abolished. The percentage of inhibition of the Ca2+ transients in the knob, however, was 40-50% less than that in the soma. Ca2+ transients induced by 30 mM K+ were partially inhibited by mibefradil, but without a significant difference in the extent of inhibition between the knob and soma. Furthermore, an increase of as little as 2.5 mM in the extracellular K+ concentration (7.5 mM K+) was found to induce Ca2+ transients in ORNs, and such responses were completely inhibited by mibefradil or Ni2+. Total replacement of extracellular Na+ with N-methyl-D-glutamate inhibited none of the odor-, forskolin/IBMX- or 7.5 mM K+-induced Ca2+ transients. Positive immunoreactivity to the Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 subunits of the T-type Ca2+ channel was observed throughout the soma, dendrite and knob. These data suggest that involvement of T-type Ca2+ channels in the propagation of odor-induced Ca2+ transients in ORNs may contribute to signal transduction and odor sensitivity. (c) 2006 IBRO. Published by Elsevier Ltd. All rights reserved.
  • Shree Hari Gautam, Ken-ichi Otsuguro, Shigeo Ito, Toshiyuki Saito, Yoshiaki Habara
    NEUROSCIENCE RESEARCH 57 1 129 - 139 2007年01月 [査読有り][通常論文]
     
    T-type Ca2+ channels are low-voltage-activated Ca2+ channels that control Ca2+ entry in excitable cells during small depolarization above resting potentials. Using Ca2+ imaging with a laser scanning confocal microscope we investigated the involvement of T-type Ca2+ channels in IBMX/forskolin- and sparingly elevated extracellular K+-induced Ca2+ transients in freshly isolated porcine olfactory receptor neurons (ORNs). In the presence of mibefradil (10 mu M) or Ni2+ (100 mu M), the selective T-type Ca2+ channel inhibitors, IBMX/forskolin-induced Ca2+ transients in the soma were either strongly (> 60%) inhibited or abolished completely. However, the Ca2+ transients in the knob were only partially (< 60%) inhibited. Ca2+ transients induced by 30 mM K+ were also partially (similar to 60%) inhibited at both the knob and soma. Furthermore, ORNs responded to as little as a 2.5 mM increase in the extracellular K+ concentration (7.5 mM K+), and such responses were completely inhibited by mibefradil or Ni2+. These results reveal functional expression of T-type Ca2+ channels in porcine ORNs, and suggest a role for these channels in the spread Ca2+ transients from the knob to the soma during activation of the cAMP cascade following odorant binding to G-protein-coupled receptors on the cilia/knob of ORNs. (c) 2006 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
  • Toshio Ohta, Yuki Ikemi, Matsuka Murakami, Toshiaki Imagawa, Ken-ichi Otsuguro, Shigeo Ito
    JOURNAL OF PHYSIOLOGY-LONDON 576 3 809 - 822 2006年11月 [査読有り][通常論文]
     
    5-Hydroxytryptamine (5-HT) is one of the major chemical mediators released in injured and inflamed tissue and is capable of inducing hyperalgesia in vivo. However, the cellular mechanisms of 5-HT-induced hyperalgesia remain unclear. Transient receptor potential V1 (TRPV1) plays a pivotal role in nociceptive receptors. In the present study, we determined whether 5-HT changes TRPV1 functions in cultured dorsal root ganglion (DRG) neurons isolated from neonatal rats, using Ca2+ imaging and whole-cell patch-clamp techniques. In more than 70% of DRG neurons, 5-HT potentiated the increases of [Ca2+](i) induced by capsaicin, protons and noxious heat. Capsaicin-induced current and depolarizing responses, and proton-induced currents were also augmented by 5-HT. RT-PCR analysis revealed the expression of 5-HT2A and 5-HT7 receptors in rat DRG neurons. Agonists for 5-HT2A and 5-HT7 receptors mimicked the potentiating effect of 5-HT, and their antagonists decreased it. In DRG ipsilateral to the complete Freund's adjuvant-injected inflammation side, expression levels of 5-HT2A and 5-HT7 mRNAs increased, and the potentiating effect of 5-HT was more prominent than in the contralateral control side. These results suggest that the PKC- and PKA-mediated signalling pathways are involved in the potentiating effect of 5-HT on TRPV1 functions through the activation of 5-HT2A and 5-HT7 receptors, respectively. Under inflammatory conditions, the increases of the biosynthesis of these 5-HT receptors may lead to further potentiation of TRPV1 functions, resulting in the generation of inflammatory hyperalgesia in vivo.
  • Ken-ichi Otsuguro, Yoshihiko Yamaji, Masaaki Ban, Toshio Ohta, Shigeo Ito
    JOURNAL OF PHYSIOLOGY-LONDON 574 3 835 - 847 2006年08月 [査読有り][通常論文]
     
    Adenosine is one of the most important neuromodulators in the CNS, both under physiological and pathological conditions. In the isolated spinal cord of the neonatal rat in vitro, acute hypercapnic acidosis (20% CO2, pH 6.7) reversibly depressed electrically evoked spinal reflex potentials. This depression was partially reversed by 8-cyclopentlyl-1,3-dimethylxanthine (CPT), a selective A(1) adenosine receptor antagonist. Isohydric hypercapnia (20% CO2, pH 7.3), but not isocapnic acidosis (5% CO2, pH 6.7), depressed the reflex potentials, which were also reversed by CPT. An ecto-5'-nucleotidase inhibitor did not affect the hypercapnic acidosis-evoked depression. An inhibitor of adenosine kinase, but not deaminase, mimicked the inhibitory effect of hypercapnic acidosis on the spinal reflex potentials. Accumulation of extracellular adenosine and inhibition of adenosine kinase activity were caused by hypercapnic acidosis and isohydric hypercapnia, but not isohydric acidosis. These results indicate that the activation of adenosine A(1) receptors is involved in the hypercapnia-evoked depression of reflex potentials in the isolated spinal cord of the neonatal rat. The inhibition of adenosine kinase activity is suggested to cause the accumulation of extracellular adenosine during hypercapnia.
  • Shree Hari Gautam, Ken-ichi Otsuguro, Shigeo Ito, Toshiyuki Saito, Yoshiaki Habara
    NEUROSCIENCE RESEARCH 55 4 410 - 420 2006年08月 [査読有り][通常論文]
     
    We investigated the relation between the intensity of odorant stimulation and the mode of spatiotemporal Ca2+ dynamics in Fluo-4-loaded rat olfactory receptor neurons (ORNs) using a confocal laser scanning microscope. We found that relatively smaller Ca2+ transients remained confined to the knob while larger ones spread to the soma with latency. Prolonged odor exposure ensured the spread of Ca2+ transients from the knob to the soma. Upon exposing ORNs to progressively increasing concentrations of odor, the Ca2+ transients that were confined to the knob at lower concentrations extended to the soma at higher concentrations. Stimulation with progressively increasing concentrations of forskolin plus IBMX yielded identical results. Partial inhibition of adenylyl cyclase by MDL12330A changed the odor response extending to the soma to a response confined to the knob. Blocking of L-type Ca2+ channels by nifedipine reduced the magnitude of the response extending to the soma but had no effect on the response confined to the knob. It is thus suggested that Ca2+ transients confined to the knob represent weak stimulation, and, speculatively, such responses either constitute inhibitory responses or indicate weak excitatory responses that fail to outstand the spontaneous electrical noise of ORNs. (c) 2006 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
  • K Otsuguro, T Ohta, S Ito
    NEUROSCIENCE 138 1 281 - 291 2006年 [査読有り][通常論文]
     
    Zinc ions (Zn2+) are known to modulate the functions of a variety of channels, receptors and transporters. We examined the effects of Zn2+ on the reflex potentials evoked by electrical stimulation and responses to depolarizing agents in the isolated spinal cord of the neonatal rat in vitro. Zn2+ at low concentrations (0.5-2 mu M) inhibited, but at high concentrations (5 and 10 mu M) augmented, a slow depolarizing component (slow ventral root potential). Zn2+ had no effect on fast components (monosynaptic reflex potential; fast polysynaptic reflex potential). Unlike Zn2+, strychnine (5 mu M), a glycine receptor antagonist, and (S),9(R)-(-)-bicuculline methobromide (10 mu M), a GABA, receptor antagonist, potentiated both fast polysynaptic reflex potential and slow ventral root potential. Zn2+ (5 mu M) did not affect depolarizing responses to glutamate and N-methyl-D-aspartate. Zn2+ enhanced the substance P-evoked depolarization in the absence of tetrodotoxin (0.3 mu M) but not in its presence. The dorsal root potential was inhibited by (S),9(R)-(-)-bicuculline methobromide (10 mu M) but not by Zn2+ (5 gNl). The Zn2+- potentiated slow ventral root potential was inhibited by the N-methyl-D-aspartate receptor antagonists, ketamine (10 mu M) and DL-2-amino-5-phosphaonovaleric acid (50 mu M) but not by P2X receptor antagonists, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (30 mu M) and 2',3'-O-(2,4,6-trinitrophenyl)ATP (10 mu M). Ketamine (10 mu M) and DL-2-amino-5-phosphaonovaleric acid (50 mu M) almost abolished spontaneous activities increased by Zn2+. It is concluded that Zn2+ potentiated slow ventral root potential induced by primary afferent stimulation, which was mediated by the activation of N-methyl-D-aspartate receptors but not by activation of P2X receptors or blockade of glycinergic and GABAergic inhibition. Zn2+ does not seem to directly affect N-methyl-D-aspartate receptors. The release of glutamate from interneurons may play an important role in Zn2+-induced potentiation of slow ventral root potential in the spinal cord of the neonatal rat. (c) 2005 Published by Elsevier Ltd on behalf of IBRO.
  • T Ohta, R Komatsu, T Imagawa, K Otsuguro, S Ito
    BIOCHEMICAL PHARMACOLOGY 71 1-2 173 - 187 2005年12月 [査読有り][通常論文]
     
    In the present study, we cloned a porcine orthologue of transient receptor potential V1 (pTRPV1) and heterologously expressed it in human embryonic kidney (HEK) 293 cells to characterize its pharmacological properties. At the amino acid level, pTRPV1 was highly homologous (83-90%) to other orthologues of TRPV1. The expression of receptors was examined with current and [Ca2+] responses to capsaicin using whole-cell patch-clamp and fura-2 ratio imaging techniques, respectively, and by immunostaining with an anti-TRPV1 antibody. The receptors were characterized by changes in [Ca2+], in response to various vanilloid agonists, low pH and heat and by the effects of TRPV1 antagonists on them. The various TRPV1 agonists activated pTRPV1 in a dose-dependent manner in the order of potency of resiniferatoxin (RTX) > olvanil > capsaicin > phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV), phorbol 12,13-dinonanoate 20-homovanillate (PDNHV). Isovelleral and scutigeral had no effect. Endogenous vanilloids (anandamide > 15 (s)-HPETE >> NADA), low pH and noxious heat (> 42 degrees C) activated pTRPV1. Comparison of amino acid sequences with various mammalian TRPV1 homologues suggested some novel putative vanilloid recognition sites. TRPV1 antagonists, iodoRTX, ruthenium red and capsazepine suppressed capsaicin-induced responses. Similar to human TRPV1, but not rodent TRPV1, capsazepine was effective in blocking pH- and heat-induced responses. Similar pharmacological profiles were observed in cultured porcine dorsal root ganglion neurons. We discuss putative amino acid residues related to pharmacological differences among mammalian TRPV1 homologues. (c) 2005 Elsevier Inc. All rights reserved.
  • T Ohta, A Kubota, M Murakami, K Otsuguro, S Ito
    AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY 289 5 G935 - G948 2005年11月 [査読有り][通常論文]
     
    We characterized ATP-induced changes in intracellular Ca2+ concentration ([Ca2+](i)) and membrane current in cultured rat myenteric neurons using ratiometric Ca2+ imaging with fura-2 and the whole cell patch-clamp technique, respectively. Neuronal cells were functionally identified by [Ca2+](i) responses to high K+ and nicotine, which occurred only in cells positive for neuron-specific protein gene product 9.5 immunoreactivity. ATP evoked a dose-dependent increase of [Ca2+](i) that was greatly decreased by the removal of extracellular Ca2+ concentration ([ Ca2+](o)). The amplitude of the [Ca2+](i) response to ATP was reduced by half in the presence of voltage-dependent Ca2+ channel blockers. In [Ca2+](o)-free solution, ATP produced a small transient rise in [Ca2+](i) similar to that induced by P2Y agonists. At -60 mV, ATP evoked a slowly inactivating inward current that was suppressed by the removal of extracellular Na+ concentration. The current-voltage relation for ATP showed an inward rectification with the reversal potential of about 0 mV. The apparent rank order of potency for the purinoceptor agonist-induced increases of [Ca2+](i) was ATP >= adenosine 5'-O-3-triphosphate >= CTP >= 2-methylthio-ATP > benzoylbenzoyl-ATP. A similar potency order was obtained with current responses to these agonists. P2 antagonists inhibited inward currents induced by ATP. Ca2+ and Mg2+ suppressed the ATP-induced current, and Zn2+, Cu2+, and protons potentiated it. RT-PCR and immunocytochemical studies showed the expression of P2X(2) receptors in cultured rat myenteric neurons. These results suggest that ATP mainly activates ionotropic P2X(2) receptors, resulting in a [Ca2+](i) increase dependent on [Ca2+](o) in rat myenteric neurons. A small part of the ATP-induced [Ca2+](i) increase may be also mediated via a P2Y receptor-related mechanism.
  • K Otsuguro, S Yasutake, T Ohta, S Ito
    DEVELOPMENTAL BRAIN RESEARCH 158 1-2 50 - 58 2005年08月 [査読有り][通常論文]
     
    The inhibitory effects of morphine and alpha(2)-adrenoceptor agonists on slow ventral root potentials (slow VRP) following ipsilateral dorsal root stimulation in neonatal rat spinal cord were compared with the analgesic effects of these drugs on formalin and capsaicin tests in neonatal rats. Morphine, (D-Phe(2), D-Pen(5))-enkephalin (DPDPE), dexmedetomidine, clonidine and xylazine showed concentration-related inhibition of slow VRP. The order of potency was dexmedetomidine > morphine = DPDPE > clorridine > xylazine. The inhibitory effects of opioid agonists and alpha(2)-adrenoceptor agonists were abolished by naloxone, an opioid antagonist, and atipamezole, an alpha(2)-adrenoceptor antagonist, respectively. There was no cross antagonism. Morphine, dexmedetomidine and xylazine dose-dependently inhibited body movement induced by formalin or capsaicin. The order of potency was dexmedetomidine > morphine > xylazine. Although morphine and dexmedetomidine inhibited formalin- and capsaicin-induced body movement in the same dose range, xylazine inhibited formalin-induced body movement at lower concentrations than capsaicin-induced one. The inhibitory potency for slow VRP by these drugs seems to be correlated with that for capsaicin-induced body movement but not that for formalin-induced one. Dexmcdetomidine and morphine in combination inhibited slow VRP and body movement induced by capsaicin in an additive manner. It is suggested that the antinociceptive effects of dexmedetomidine and morphine but not xylazine on the capsaicin test are mainly due to spinal effects and that there is no synergistic interaction between dexmedetomidine and morphine in the neonatal rat. (c) 2005 Elsevier B.V. All rights reserved.
  • K Otsuguro, SH Gautam, S Ito, Y Habara, T Saito
    JOURNAL OF PHARMACOLOGICAL SCIENCES 97 4 510 - 518 2005年04月 [査読有り][通常論文]
     
    Forskolin-induced Ca2+ signals were examined in isolated rat olfactory receptor neurons (ORNs) using a Ca2+ indicator, fura-2. In the soma of the ORNs, forskolin caused an increase in the intracellular Ca(2+)concentration ([Ca2+](i)) that was enhanced by a phosphodiesterase(PDE)1 inhibitor, 8-methoxymethyl-3-isobutyl-1-methyl-xanthine, but not a PDE4 inhibitor, rolipram. Forskolin-induced Ca2+ signals were abolished with the removal of extracellular Ca2+ and un-affected by treatment with thapsigargin or caffeine plus ryanodine. Niflumic acid, a Ca2+-activated Cl- channel inhibitor, or nifedipine, an L-type Ca2+ channel inhibitor, slowed the initial rate of the increase in [Ca2+]i in response to forskolin. Nifedipine did not affect the increase in [Ca2+](i) that was slowed by niflumic acid. In Ca2+ measurements with a confocal microscope and a calcium indicator, Fluo-4, the onset of the response to forskolin in the knob region occurred simultaneously or earlier, but not later, than that in the soma. It is suggested that the forskolin-induced Ca2+ signals are due to Ca2+ influx, but not the release of Ca2+ from Ca2+ stores, and that the initial rapid increase in [Ca2+](i) is associated with the activation of the voltage-dependent Ca2+ channels in rat ORNs.
  • Y Suzuki, K Kondo, Y Matsumoto, BQ Zhao, K Otsuguro, T Maeda, Y Tsukamoto, T Urano, K Umemura
    LIFE SCIENCES 73 10 1289 - 1298 2003年07月 [査読有り][通常論文]
     
    We have previously demonstrated that natto-extracts containing nattokinase (NK) inactivates plasminogen activator inhibitor type 1 and then potentiates fibrinolytic activity. In the present study, we investigated the effects of dietary supplementation with natto-extracts on neointima formation and on thrombolysis at the site of endothelial injury. Endothelial damage in the rat femoral artery was induced by intravenous injection of rose bengal followed by focal irradiation by transluminal green light. Dietary natto-extracts supplementation containing NK of 50 or 100 CU/body was started 3 weeks before endothelial injury and then continued for another 3 weeks. Intimal thickening in animals given supplementation was significantly (P < 0.01) suppressed compared with controls and the intima/media ratio in animals with 50 and 100 CU/body NK and control group was 0.09 +/- 0.03, 0.09 +/- 0.06 and 0.16 +/- 0.12, respectively. Although femoral arteries were reopened both in control animals and those treated with NK within 8 hours after endothelial injury, mural thrombi were histologically observed at the site of endothelial injury. In the control group, the center of vessel lumen was reopened and mural thrombi were attached on the surface of vessel walls. In contrast, in NK-treated groups, thrombi near the vessel wall showed lysis and most of them detached from the surface of vessel walls. In conclusion, dietary natto-extracts supplementation suppressed intimal thickening produced by endothelial injury in rat femoral artery. These effects may partially be attributable to NK, which showed enhanced thrombolysis near the vessel wall. (C) 2003 Elsevier Science Inc. All rights reserved.
  • T Ogawa, A Sugidachi, K Otsuguro, T Isobe, F Asai
    BIOCHEMICAL PHARMACOLOGY 63 10 1911 - 1918 2002年05月 [査読有り][通常論文]
     
    Platelet-agonist interaction results in aggregatory and secretory responses. While the activation of glycoprotein (GP) IIb/IIIa plays an essential role in platelet aggregation, its role in granule secretion is not clear. The present study was performed to examine the effect of 3-[[[[1-[4-(aminoiminomethyl) phenyl] -2-oxo-3S-pyrrolidinyl] amino] carbonyl] amino] -propanoate monohydrochloride salt (SC-57101A), a GPIIb/IIIa antagonist, on platelet alpha-granule secretion responses to collagen, ADP, and thrombin receptor activating peptide (TRAP). Both SC-57101A and prostaglandin E-1 (PGE(1)) inhibited collagen-, ADP-, and TRAP-induced platelet aggregation in a concentration-dependent manner. SC-57101A inhibited the collagen- and ADP-induced release of platelet-derived growth factor (PDGF) and beta-thromboglobulin (beta-TG) from platelets, but not TRAP-induced secretion of these granule contents. On the other hand, PGE(1) inhibited the release of PDGF and beta-TG from platelets activated with all the agonists used. ADP and TRAP elicited P-selectin expression in the absence of platelet aggregation, while collagen produced no such reaction. SC-57101A only moderately inhibited P-selectin expression induced by ADP and had no inhibitory effect on that induced by TRAP. The inhibition of ADP-induced secretion of alpha-granule contents by SC-57101A was abolished when platelets were pretreated with aspirin. These results suggest that GPIIb/IIIa activation plays a minor role, if any, in a granule secretion in human platelets. (C) 2002 Elsevier Science Inc. All rights reserved.
  • A Sugidachi, F Asai, K Yoneda, R Iwamura, T Ogawa, K Otsuguro, H Koike
    BRITISH JOURNAL OF PHARMACOLOGY 132 1 47 - 54 2001年01月 [査読有り][通常論文]
     
    1 CS-747 is a novel thienopyridine-type platelet ADP inhibitor which lacks in vitro activity. This study examined pharmacological profiles of R-99224, a hepatic metabolite of CS-747. 2 R-99224 produced a concentration-dependent inhibition of in vitro platelet aggregation in washed human platelets (0.03-1 mug ml(-1)), which was relatively specific to ADP compared to collagen and thrombin. 3 R-99224 (0.1-3 mug ml(-1)) also elicited a similar inhibition of ADP-induced aggregation in rat platelets. The inhibition by R-99224 (10 mug ml(-1)) persisted even after platelets were washed three times. Intravenous injection of R-99224 (0.1-3 mg kg(-1)) to rats resulted in a dose-dependent inhibition of ex vivo ADP-induced platelet aggregation. 4 R-99224 (0.1-100 muM) decreased binding of [H-3]-2-methylthio-ADP ([H-3]-2-MeS-ADP), a stable ligand for platelet ADP receptors, to washed human platelets. The inhibition by R-99224 reached a plateau at a concentration of 3 muM (1.4 mug ml(-1)), but complete inhibition was not achieved even at the highest concentration used (100 muM). 5 R-99224 (10 muM) in combination with ARL-66096 (0.3 muM), an ATP analogue-type Gi-linked P2T receptor antagonist, produced no additional inhibition of [H-3]-2-MeS-ADP binding. In contrast, [H-3]-2-MeS-ADP binding was completely abolished by R-99224 (10 muM) in combination with A3P5PS (300 muM), a selective P2Y(1) antagonist, suggesting that R-99224 selectively binds to the G(i)-linked P2T receptor. 6 R-99224 (0.01-3 mug ml(-1)) inhibited ADP-induced [I-125]-fibrinogen binding to human platelets in a concentration-dependent manner. R-99224 (0.1-1 mug ml(-1)) also inhibited the ADP-induced decrease in cyclic AMP levels in PGE(1)-stimulated platelets, whereas the agent did not affect ADP (10 muM)-induced Ca2+ mobilization. 7 These findings suggest that R-99224 is a selective and irreversible antagonist of Gi-linked P2T receptors and that R-99224 is a responsible molecule for in vivo actions of CS-747.
  • K Otsuguro, T Ohta, S Ito, Y Nakazato
    JAPANESE JOURNAL OF PHARMACOLOGY 78 2 209 - 215 1998年10月 [査読有り][通常論文]
     
    Effects of purinoceptor antagonists on the relaxant responses to adenine nucleotides were examined to characterize the subtypes of Pt-receptor in rat gastric circular muscle. In tissues contracted by acetylcholine, a Pt-receptor antagonist, suramin (100 mu M), inhibited the relaxant responses to ATP, adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) and alpha,beta-methylene ATP but not that to adenosine, while a Pi-receptor antagonist, 8-phenyltheophylline (3 mu M) did vice versa. The inhibitory effect of suramin was more potent for the relaxant responses to cr,il-methylene ATP than those to ATP or ADP beta S. Pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (3-30 mu M) and 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) (30 and 100 mu M) inhibited the relaxation caused by a,alpha,beta-methylene ATP but not by ATP, ADP beta S or adenosine. These results suggest that ATP and ADP beta S cause relaxation via the classical P-2Y receptors resistant to PPADS and DIDS. In addition, alpha,beta-methylene ATP causes relaxation via the distinct P-2 receptors sensitive to PPADS and DIDS in rat gastric circular muscle.
  • K Otsuguro, S Ito, T Ohta, Y Nakazato
    EUROPEAN JOURNAL OF PHARMACOLOGY 317 1 97 - 105 1996年12月 [査読有り][通常論文]
     
    The effects of UTP were examined to characterize the receptor subtypes for UTP in the circular smooth muscle of the rat proximal stomach. The rank order of potency for contraction was 2-methylthio ATP much greater than ATP greater than or equal to UDP = UTP greater than or equal to adenosine 5'-O-(3-thiotriphosphate)(ATP-gamma-S) much greater than UMP > CTP = alpha,beta-methylene ATP > adenosine = uridine. In tissues contracted by acetylcholine, ATP, 2-methylthio ATP, alpha,beta-methylene ATP and adenosine each caused relaxation. alpha,beta-Methylene ATP had the most potent effect and UTP caused only a small relaxation. Suramin inhibited ATP- and UTP-induced contractions. The contractile responses to ATP decreased in tissues desensitized with UTP, ATP-gamma-S and 2-methylthio ATP, but not with alpha,beta-methylene ATP. However, UTP-induced contraction was not inhibited by desensitization with ATP, alpha,beta-methylene ATP, ATP-gamma-S and 2-methylthio ATP. These results suggest that UTP causes contraction via receptors different from common P-2 purinoceptors. These receptors are blocked by suramin in the rat proximal stomach.
  • K Otsuguro, T Ohta, S Ito, Y Nakazato
    PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY 431 3 402 - 407 1996年01月 [査読有り][通常論文]
     
    The effects of extracellular adenosine 5'-triphosphate (ATP) on currents were examined using the: whole-cell voltage-clamp technique in guinea-pig isolated adrenal chromaffin cells. ATP (500 mu M) reversibly suppresssd Ca2+ currents ill the presence of 5 mM Ca2+ in the extracellular solution. The inhibitory effect of ATP on Ca2+ currents tended to increase with increases in the peak amplitude of ATP-evoked current when the intracellular solution contained 0.1 or 1 mM ethylenebis(oxonitrilo)tetraacetate(EGTA). Using the intracellular solution containing 10 mM EGTA, on the other hand, the inhibitory efftect did not change regardless of the amplitude of current responses to ATP. In the presence of 10 mM Ba2+, ATP (100 mu M) reduced Ba2+ currents in a mariner similar to Ca2+ currents. This reduction was decreased by dialysis of cells with the internal solution containing guanosine 5'-O-(2-thiodiphosphate) (GDP [beta-S]; 1 mM) or guanosine 5'-O-(3-thiotriphosphate) (GTP [gamma-S]. 100 mu M). A depolarizing prepulse to + 100 mV partly relieved ATP-induced reduction of Ba2+ currents. ADP, AMP and adenosine also reduced Ba2+ currents and the effect of adenosine was the most potent. Adenosine (0.5 and 1 mM) significantly inhibited adrenaline secretion induced by nicotine (50 mu M). Ca2+ entry through ATP-activated non-selective cation channels results in the inactivation of voltage-dependent Ca2+ channels. In addition, ATP seems to modulate Ca2+ channels via the pathway related to G-protein. Adenine nucleotides and adenosine may play a role in controlling secretory activity in guinea-pig adrenal chromaffin cells.
  • T Asano, T Ohta, K Otsuguro, S Ito, Y Nakazato
    JOURNAL OF VETERINARY MEDICAL SCIENCE 57 6 1067 - 1071 1995年12月 [査読有り][通常論文]
     
    Muscarinic receptor subtypes mediating catecholamine secretion and increase in the intracellular concentration of Ca2+ ([Ca2+](i)) were examined using muscarinic agonists and antagonists in dispersed adrenal chromaffin cells of the guinea pig. All muscarinic agonists (1-1,000 mu M) tested caused increases in adrenaline secretion in a dose-dependent manner. Muscarine and methacholine were more effective than bethanechol, oxotremorine and pilocarpine. Muscarine and oxotremorine caused a small increase in adrenaline secretion even in the absence of extracellular Ca2+. Both 4-DAMP (0.1 mu M) and pirenzepine (0.1 mu M), but not methoctramine (0.1 mu M), shifted the dose-response curve for muscarine-induced adrenaline secretion to the right. These muscarinic agonists also caused increase in [Ca2+](i) in the presence of extracellular Ca2+. Muscarine-induced [Ca2+](i) rises were reduced, but not abolished, by removal of extracellular Ca2+. These results suggest that catecholamine secretion induced by muscarinic agonists is mediated through M(1), or M(1) and M(3) muscarinic receptor subtypes in adrenal chromaffin cells of the guinea pig.
  • T Asano, K Otsuguro, T Ohta, T Sugawara, S Ito S, Y Nakazato
    Comp Biochem Physiol C 112 101 - 108 2 1995年10月 [査読有り][通常論文]
  • K OTSUGURO, T ASANO, T OHTA, S ITO, Y NAKAZATO
    NEUROSCIENCE LETTERS 187 3 145 - 148 1995年03月 [査読有り][通常論文]
     
    Adenosine 5'-triphosphate (ATP) evoked an inward current in whole-cell voltage-clamped adrenal chromaffin cells of the guinea pig. The reversal potential (E(rev)) of ATP-evoked current was about 0 mV in normal external solution and was shifted towards negative potentials by substituting Tris(+) or sucrose, but not Ca2+, for the extracellular Na+. This current was mediated by the activation of nonselective cation channels and had some different properties from nicotinic current. It is suggested that these channels may function as a part of the ATP-induced Ca2+ influx pathway in guinea pig chromaffin cells.

書籍

  • 獣医臨床薬理学
    日本比較薬理学, 毒性学会 (担当:共編者(共編著者))
    近代出版 2017年12月
  • 薬理学・毒性学実験 第3版
    日本比較薬理学, 毒性学会 (担当:分担執筆)
    文栄堂出版 2008年04月

講演・口頭発表等

  • Investigation of analgesic effect of carbon dioxide in isolated spinal cord preparation - Alternative to in vivo pain tests- Section "Alternatives to animal use"  [招待講演]
    Ken-ichi OTSUGURO
    The 2nd Joint Meeting of Veterinary Science in East Asia 2019年04月 シンポジウム・ワークショップパネル(指名)
  • 北海道大学の動物実験教育の取り組み  [招待講演]
    乙黒 兼一
    第161回日本獣医学会学術集会 第14回獣医学教育改革シンポジウム 2018年09月 シンポジウム・ワークショップパネル(指名)
  • Effects of adenosine on spinal neuronal activities: its contribution to hypercapnia-evoked depression  [招待講演]
    Ken-ichi OTSUGURO
    The 14th Seoul National University – Hokkaido University Joint Symposium 2011年11月 シンポジウム・ワークショップパネル(指名)
  • 脊髄反射電位に対するアデノシンの作用と低酸素惹起シナプス伝達抑制への関与 シンポジウム「虚血病態の病態と治療」  [招待講演]
    乙黒 兼一
    第38回 薬物活性シンポジウム 2010年11月 シンポジウム・ワークショップパネル(指名)
  • 二酸化炭素の薬理作用  [招待講演]
    乙黒 兼一
    第146回 日本獣医学会学術集会 日本実験動物医学会教育シンポジウム 2008年09月 シンポジウム・ワークショップパネル(指名)

その他活動・業績

共同研究・競争的資金等の研究課題

  • ベクター媒介性感染症におけるベクター唾液の役割の解明
    文部科学省:基盤研究B
    研究期間 : 2015年 -2018年 
    代表者 : 加藤 大智
  • 細胞外アデノシンデアミナーゼの分泌機構の解明
    内藤記念科学振興財団:奨励金・研究助成
    研究期間 : 2018年 
    代表者 : 乙黒 兼一
  • グリア伝達物質による脊髄アストロサイト機能の調節機構と病態下における役割の解明
    北海道大学:科研費種目ステップアップ支援事業
    研究期間 : 2017年 
    代表者 : 乙黒 兼一
  • シスタチオニンβ合成酵素の発現調節を介した新規中枢疾患治療ターゲットの探索
    秋山記念生命科学振興財団:研究奨励(一般)
    研究期間 : 2017年 
    代表者 : 乙黒 兼一
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 2014年 -2016年 
    代表者 : 乙黒 兼一
  • 中枢神経組織における硫化水素産生機構とアストロサイトの役割の解明
    寿原記念財団:研究助成金
    研究期間 : 2015年 
    代表者 : 乙黒 兼一
  • 文部科学省:科学研究費補助金(挑戦的萌芽研究)
    研究期間 : 2011年 -2012年 
    代表者 : 乙黒 兼一
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 2008年 -2010年 
    代表者 : 乙黒 兼一
     
    虚血病態下の低酸素やアシドーシスにより神経機能は大きく影響を受ける。本研究課題では、脊髄神経経路の電気的活動の記録とともに、細胞外プリン化合物濃度を測定することで、脊髄組織から低酸素やアシドーシスによってアデノシンと共にイノシンが放出されること、アデノシンはアデノシンA1受容体を介して神経活動を抑制することを示した。さらに、低酸素の神経活動への影響は、日齢や温度、神経経路によって大きく異なることが示され、この要因として抑制反応に対するアデノシンの関与の有無が重要であることが示唆された。
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2007年 -2009年 
    代表者 : 伊藤 茂男, 乙黒 兼一
     
    痛みの発生に重要なタンパク質であるバニロイド受容体チャネルは、カプサイシンに加えてマスタードオイルやメチルサリチル酸などにも反応すること、さらに外液Na^+イオンがチャネル開口を制御していることを明らかにした。また、炎症により放出されるヒスタミンや5-ヒドロキシトリプタミンは、それぞれの受容体に作用して痛みを増悪させること、高炭酸暴露による鎮痛作用にはアデノシンが関与していることを示した。
  • 文部科学省:科学研究費補助金(萌芽研究)
    研究期間 : 2007年 -2008年 
    代表者 : 伊藤 茂男, 梅村 孝司, 乙黒 兼一, 生駒 俊之
     
    1) 徐放担体の開発 : 以下の3点を調べた。(1) ヒドロキシアパタイト(HAP直径5μm)含有金属 : ポリ乳酸処理インスリン含有亜鉛(Zn)-HAP投与による血中インスリン濃度の増加は投与後2日で投与前のレベルに回復したが、投与したHAP中にインスリンは残留していた。鉄(Fe)-HAPとマグネシウム(Mg)-HAPを用いて、インスリンの徐放性を調べたが、Zn-HAPと同様、一過性の血糖低下作用と血中インスリン濃度増加作用しか示さなかった。(2)HAP粒子の大きさ : 直径2.5μmの脚と25μmのHAPをラットに皮下投与し、その消失速度を調べた。HAPの粒子径が小さくなると皮下投与部位からの消失速度が速まった(2.5μm ; 10日、5μm ; 14日、25μm ; 20日)。糖尿病ウサギにこれらインスリン含有HAPを投与して、血糖値と血中インスリン濃度変化を調べた結果、血糖低下反応とインスリン増加反応は投与後2日目で回復した。2.5μmの方が、インスリン増加は僅かに持続した。(3)薬物の併用 : インスリン-HAPをアルギン酸、カルボキシメチルセルロース(CMC)、デキサメサゾン(Dex)とともに投与したが、インスリン増加と血糖低下作用は一過性であった。2)製剤の毒性評価 : HAPのみ、CMC+HAP、アルギン酸+HAP、Dex+HAP、Fe-HAP、Mg-HAPを...
  • アシドーシスによる脊髄の機能変化
    上原記念生命科学財団:研究奨励金
    研究期間 : 2008年 
    代表者 : 乙黒 兼一
  • 病態動物モデルを用いた血管変性機序の解明
    文部科学省:大学教育の国際化推進プログラム(海外先進研究実践支援)
    研究期間 : 2006年04月 -2007年03月 
    代表者 : 乙黒 兼一
  • 文部科学省:科学研究費補助金(若手研究(B))
    研究期間 : 2005年 -2006年 
    代表者 : 乙黒 兼一
     
    本研究の目的である摘出脊髄標本を用い、亜鉛の脊髄神経に与える影響を明らかにするため、新生ラットから摘出した脊髄半裁標本を用いて脊髄反射電位に対する亜鉛の効果を検討し、以下の結果を得た。1.脊髄後根を電気刺激すると、対応する前根からMSR(monosynaptic reflex potential)、fPSR(fast polysynaptic reflex potential)及びsVRP(slow ventral root potential)が、また隣接する後根からDRP(dorsal root potential)記録された。2.亜鉛は、MSR、fPSRには影響を与えず、sVRPに対しては低濃度(0.5-2μM)では抑制、高濃度(5及び10μM)では増強作用を示した。また高濃度亜鉛によってベース前根電位のわずかな脱分極が観察され、激しい自発活性の増加が生じる標本もあった。3.グリシン受容体拮抗薬ストリキニーネとGABA_A受容体拮抗薬ビククリンは、亜鉛と異なりsVRPとともにfPSRも増強した。4.GABA_A受容体拮抗薬ビククリンは、DRPを抑制したが、亜鉛はDRPに影響を与えなかった。5.亜鉛による増強効果は、NMDA受容体拮抗薬ケタミン及びAP-5によって抑制されたが、ATP受容体拮抗薬PPADS及びTNP-ATPは亜鉛の効果に影響を与えなかった。以上の結果から、...
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2002年 -2004年 
    代表者 : 伊藤 茂男, 太田 利男, 乙黒 兼一
     
    (1)培養ラット後根神経節(DRG)細胞には5-HT_<2A>(5-hydroxytryptamine)と5-HT_7受容体が発現し、5-HTはカプサイシンによるCa^<2+>反応を増強した。この増強反応はそれぞれCキナーゼとAキナーゼ阻害薬により抑制された。(2)新生ラット摘出脊髄後根の電気刺激は10秒以上続く遅発性前根電位を引き起こした。この電位はアドレナリンα2受容体刺激薬、デクスメデトミジン(DEC)、キシラジン(XY)とクロニジン(CLO)もしくはオピオイド受容体刺激薬、モルヒネ(MOR)とDPDPEにより抑制された。抑制作用の力価はDEC>DPDPE=MOR>CLO>XYの順であった。(3)高炭酸(20%CO_2)を通気した栄養液で摘出脊髄を灌流すると、単シナプス反射電位と遅発性前根電位が抑制された。この抑制はアデノシンA1受容体拮抗薬で減弱した。アデノシンキナーゼ阻害薬は高炭酸の通気と類似した抑制作用を示した。(4)亜鉛は遅発性前根電位を選択的に増強した。亜鉛による増強反応はグルタミン酸NMDA受容体拮抗薬であるケタミンとAP-5により抑制された。(5)新生ラットにおいて、ホルマリン誘発体動はDEC、XYおよびMORにより抑制された。カプサイシン誘発体動はDECとMORで抑制されたが、XYでは抑制されなかった。(6)ラットバニロイド受容体の塩基配列に基づきブタD...
  • ラット嗅細胞の匂い応答におけるイオンチャネルと細胞内情報伝達系の関与に関する研究
    秋山記念生命科学振興財団:研究助成(奨励)
    研究期間 : 2003年 
    代表者 : 乙黒 兼一

教育活動情報

主要な担当授業

  • 獣医科学特別研究
    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
  • 基礎薬理学
    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 : 作用機序、作用点、細胞内情報伝達、構造活性相関、競合的拮抗、非競合的拮抗、シグモイド曲線、用量反応曲線、50%有効量、50%致死量、治療係数、PD2、内活性、第Ⅰ相反応、チトクロームP450、第2相反応、グルクロン酸抱合、硫酸抱合、半減期、分布容積、血中濃度―曲線下面積、コリン誘導体、アルカロイド、ムスカリン受容体、ニコチン受容体、脱分極性遮断、競合拮抗、α受容体、β受容体、筋弛緩薬、局所麻酔薬、オータコイド、アンギオテンシン変換酵素阻害薬、アラキドン酸代謝物、プロスタグランジン、エイコサノイド、ロイコトリエン、ステロイド性抗炎症薬、非ステロイド性抗炎症薬、H1受容体、H2受容体、プロトンポンプ阻害薬
  • 専門獣医科学特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 臨床薬理学
    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 : 剤形、薬物動態(ADME)、PK/PD解析、吸収、半減期、分布容積、クリアランス、血漿タンパク質結合率、生体内利用率(BA)、定常状態、治療的薬物モニタリング(TDM)、薬物相互作用、酵素誘導、休薬期間、トランスポーター
  • 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
  • 動物福祉学
    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 : 動物福祉、伴侶動物、産業動物、実験動物、野生動物、展示動物
  • 獣医科学特論演習
    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A 行動解析学特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 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
  • 獣医科学特別研究
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • 獣医科学特論演習
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • 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
  • 専門獣医科学特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • 動物愛護福祉論
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 獣医学部
  • 研究・臨床セミナー
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 獣医学部
  • 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

大学運営

委員歴

  • 2019年12月 - 現在   日本薬理学会認定   薬理学エデュケーター
  • 2018年10月 - 現在   日本薬理学会   代議員
  • 2018年 - 現在   農林水産省   獣医事審議会専門委員
  • 2015年 - 現在   獣医系大学間獣医学教育支援機構   獣医学共用試験センター委員
  • 2010年 - 現在   日本獣医学会   評議員   日本獣医学会
  • 2009年 - 現在   日本薬理学会   学術評議員   日本薬理学会

社会貢献活動

  • モンゴル獣医・畜産分野人材育成能力強化プロジェクト
    期間 : 2018年
    役割 : 講師
    主催者・発行元 : 国際協力機構・モンゴル生命科学大学 獣医学部
  • スーパーサイエンスハイスクール
    期間 : 2012年 - 2016年
    役割 : 講師
    主催者・発行元 : 北海道釧路湖陵高等学校
    イベント・番組・新聞雑誌名 : KCS基礎


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