Researchers Database

IMAIZUMI Yuji

    Executives Trustee/Vice President
Contact: yimaizumphar.nagoya-cu.ac.jp
Last Updated :2024/03/19

Researcher Information

J-Global ID

Research Interests

  • calcium signaling   バイオイメージング   イオントランスポーター   平滑筋   イオンチャネル   

Research Areas

  • Life sciences / Pharmacology

Academic & Professional Experience

  • 1978-1981 Nagoya1981-1996 Nagoya

Education

  •        - 1976  The University of Tokyo  Faculty of Pharmaceutical Science  生命薬学

Association Memberships

  • 日本神経科学会   日本生理学会   日本平滑筋学会   日本薬理会   日本薬学会   

Published Papers

Books etc

  • 血管平滑節カルシウムチャネルの機能制御とその役割。
    循環器科 1998
  • 「平滑節」
    薬学必携シリーズ 薬理学、第11章 朝倉書店 1997
  • 「オータコイド・抗アレルギー薬」
    薬学必携シリーズ 薬理学、第8章 朝倉書店 1997
  • Kチャネルと薬。
    ファルマシア(セミナー) 1997
  • Regulation of Ca-dependent K current and action potential shape by intracellular Ca storage sites in some types of smooth muscle cells.
    1996
  • Noradrenaline-induced Ca-channel current modulation in smooth muscles.
    1996
  • 心血管以外の臓器に対するKチャネル開口薬の作用。
    治療学 1996
  • 「下剤と止瀉剤」
    病気とくすり 南山堂 1994
  • Effects of 9-methyl-7-bromoeudistomin D(MBED), a powerful Ca
    1993
  • Ca拮抗薬の平滑節Ca電流に対する選択的抑制作用について
    1993
  • 各種平滑節細胞の興奮性の多様性とその機序について
    日本薬理学会誌(日本薬理学会奨励賞受賞総説) 1993
  • Electrical properties of iris sphincter.
    1992
  • 「平滑節組織及び単離細胞からの電気現象誘導法」
    生物薬科学実験講座 臓器機能測定法(]G0003[)広川書店 1992
  • Measurement of noninactivating calcium current in smooth muscle cells.
    1991
  • A comparative study about voltage-dependent Ca currents in smooth muscle cells isolated from several tissues.
    1989

MISC

Awards & Honors

  • 2018/03 日本薬学会 日本薬学会賞
     疾患治療標的および創薬標的としてのイオンチャネル分子機能解明 
    受賞者: 今泉 祐治
  • 1992/03 日本薬理学会 日本薬理学会学術奨励賞
     各種平滑筋細胞の興奮性の多様性とその機序について

Research Grants & Projects

  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Fund for the Promotion of Joint International Research (Fostering Joint International Research (B))
    Date (from‐to) : 2018/10 -2022/03 
    Author : 今泉 祐治; 大矢 進; 山村 寿男; 鈴木 良明; 鬼頭 宏彰
     
    炎症慢性化過程の細胞機能変動において、免疫担当細胞に機能発現するイオンチャネルがどのような病態生理学的意義を果たしているかは明らかにされていない。本研究の目的は、貪食機能を有する免疫担当細胞の活性化と、炎症慢性化による組織リモデリングにおける細胞内Ca2+濃度制御に関わるイオンチャネル群とその分子機構を解明し、炎症慢性化・組織リモデリングにおける新規治療標的イオンチャネルを探索・同定することである。 これまでの研究において、炎症性腸疾患モデルマウスの病態発症・悪化にtwo-pore型K+チャネルK2P5.1の発現・機能亢進による炎症性サイトカイン産生増加が関与すること、および炎症誘発性の低酸素環境によるHypoxia-Inducible Factor-1αシグナルの活性化がTリンパ球K2P5.1の発現亢進に関与することを明らかにした (Endo et al., 2020)。 また、炎症性腸疾患の回復期においてCa2+活性化K+チャネルKCa3.1の阻害がCD4+CD25+制御性Tリンパ球におけるIL-10発現・分泌を亢進させ、症状の改善に寄与することを明らかにした(Ohya et al., 2021)。 変形性関節症(OA)に対するCa2+シグナルの関係を明らかにするため、マウス初代軟骨細胞を用い、OA病変とイオンチャネルの関連を調べた。IL-1βがCa2+遊離活性化Ca2+(CRAC)チャネルを介した細胞内Ca2+濃度上昇によりNFATの核移行を促して、OAマーカー発現を担うことが示唆された。また、IL-1βの持続的処置により、電位依存性K+チャネルKv1.6の発現が低下し、膜脱分極・細胞内Ca2+濃度上昇が引き起こされ、OAマーカー誘導が増強されることを見出した。ヒト軟骨細胞株でClC3が浸透圧刺激による容量制御に関わることを見出した(Yamada et a., 2020)。 これまでの軟骨細胞に関する研究の総説を国際誌に発表した(Suzuki et al., 2020)。
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
    Date (from‐to) : 2016/04 -2018/03 
    Author : Imaizumi Yuji
     
    To develop a new ion channel-targeted drug screening system for high throughput screening (HTS) assay, we have established HEK293-based “test cell” expressing a mutated Na+ channel lacking inactivation and a K+ channel (Kir2.1) that hyperpolarizes the membrane potential (PCT/JP2011/064967). We found that only treatment of the test cells with Ba2+ is enough to induce action potential and cell death. Then two-pore domain potassium (K2P) channels were additionally expressed in the test cells because K2P channels are involved in progression of various disease and thought to be druggable targets. In this system, both activating and inhibitory effects of drugs on K2P channels can be easily and accurately estimated using simple cell death assay. IC50 values of these blockers acquired by both manual and automated process were close to those obtained using patch-clamp recordings. Now drug screenings using compound library are in progress.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2014/04 -2018/03 
    Author : Imaizumi Yuji; Higuchi Tsunehiko; Asai Kiyofumi; Hirono Syuichi
     
    The present study revealed that in non-excitable cells such as chondrocytes andendothelial cells, Ca2+-activated K+ (KCa) channels and store-operated Ca2+ (SOC) channels play significant roles in the positive feedback mechanism for the regulation of intracellular Ca2+ concentration ([Ca2+]i). This [Ca2+]i elevation forms cellular responses to various types of stimulations in physiological conditions or gets involved in pathological process. In chondrocytes, it is demonstrated that ion channels, such as large-conductance Ca2+-activated K+ (BK) channels, Ca2+-release-activated Ca2+ (CRAC) channels, ClC-3 and ClC-7, are involved in progression of osteoarthritis. In brain capillary endothelial cells, hypoxic stress induces the up-regulation of Kir2.1, augment of positive-feedback mechanisms and promotion of cell proliferation. These events may play a role in disruption of blood-brain-barrier after cerebral hypoxia.
  • 日本学術振興会:科学研究費助成事業 新学術領域研究(研究領域提案型)
    Date (from‐to) : 2015/04 -2017/03 
    Author : 今泉 祐治
     
    ①低酸素ストレスによる脳血管内皮細胞の異常な細胞増殖は、血液脳関門を破綻させ、低酸素脳症の悪化に関与することが知られているが、その分子機構については不明な点が多い。これまでの研究で、脳血管内皮細胞株であるt-BBEC117細胞は、低酸素ストレスによりストア作動性Ca2+流入を増大させるが、その分子実体であるOrai1/Orai2のタンパク質発現量は変化させなかった。本研究では、脳血管内皮細胞の膜電位制御に関与する内向き整流性K+チャネルであるKir2.1チャネルとその発現を調節するダイナミン2に注目した。低酸素ストレスによって、ダイナミン2の発現は増加した。ダイナミン阻害薬は、低酸素ストレス誘発性Kir2.1チャネルの発現増加と活性上昇を抑制した。低酸素ストレスによるダイナミン2を介したKir2.1チャネルの活性増大が、脳血管内皮細胞の異常な細胞増殖を起こすことから、この機構が低酸素脳症における血液脳関門の破綻に関連することが示唆された。
    ②平滑筋Ca2+マイクロドメインはタンパク質複合体のプラットフォームであり、効率的なシグナル伝達に重要であると認識されている。本研究では、筋小胞体とミトコンドリアを近接させるミトフュージン2の細胞内Ca2+動態における役割の解明を目指した。ミトフュージン2のノックダウンによって、アゴニスト誘発性Ca2+増加に伴う細胞質Ca2+緩衝能は緩徐になり、ミトコンドリアCa2+取り込み能は低下した。ミトフュージン2のmRNA発現は酸化ストレスにより増加し、その発現亢進は活性酸素のスカベンジャーによって抑制された。活性酸素誘発性のミトフュージン2発現亢進はミトコンドリアの融合を促進した。平滑筋Ca2+マイクロドメインにおいて、筋小胞体とミトコンドリアの機能連関を促進するミトフュージン2は、細胞内Ca2+制御におけて重要な機能を果たしていると考えられる。
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
    Date (from‐to) : 2014/04 -2016/03 
    Author : Imaizumi Yuji; YAMAMURA Hisao; SUZUKI Yoshiaki
     
    In the present study, we developed a novel screening system for drugs acting on ion channels. The achievements of results in this study are as follows; (1) We established a recombinant cell line co-expressing mutant Nav1.5 and Kir2.1 (IFM/Q3+Kir). Electrical stimulation (ES) of the cell line induced prolonged action potentials and subsequent cell death. Furthermore "third" ion channel (Kv,K2P or α7-nicotinic receptor) that are candidates for therapeutic targets was expressed in IFM/Q3+Kir. In these cells, drugs that modulate the third ion channel activity can change the mortality of the cells after ES. MTT assay using already-known drugs acting on the "third" ion channels demonstrated that dose-response curves obtained from our new system are as accurate as those obtained from patch-clamp recordings. (2) We developed a new device that enables us to stimulate the recombinant cell lines cultured on 96 well plates with ES. Thus, our new system is available for high throughput screening.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2013/04 -2016/03 
    Author : YAMAMURA Hisao; IMAIZUMI Yuji
     
    The calcium-activated chloride (ClCa) channel plays substantial roles in the regulation of membrane excitability in vascular smooth muscles. Recently, TMEM16A-coding protein has been identified as the molecular entity responsible for ClCa channel in several types of vascular smooth muscles. In this study, the functional expression of TMEM16A and its regulatory factors was examined in murine portal vein smooth muscle cells. TMEM16A was abundantly expressed in portal vein myocytes and formed a dimeric ClCa channel. The activity of TMEM16A ClCa channel was modified by the interaction with actin cytoskeleton. TMEM16A was also interacted with TMEM16B to form a heteromeric ClCa channel. Finally, TMEM16A was downregulated in portal vein smooth muscle cells from hepatic cirrhosis-induced portal hypertensive mice. These results provide useful information for elucidating physiological and pathological significances of TMEM16A ClCa channels in vascular smooth muscles.
  • 日本学術振興会:科学研究費助成事業 新学術領域研究(研究領域提案型)
    Date (from‐to) : 2013/04 -2015/03 
    Author : 今泉 祐治
     
    (1)Ca2+クロックとイオンチャネルの機能連関によるペースメーカーモデル構築 3型リアノジン受容体(RyR3)を介したCa2+遊離がペースメーカー電位発生・調律・伝播に果たす役割を明らかにするため、Ca2+活性化Cl-チャネル(TMEM16A)を定常発現させたHEK293細胞に、RyR3受容体を発現させて検討を行った。巨大タンパクであるRyR3を安定的に発現させるためにバキュロウイルス系による遺伝子導入法を用いた。その結果、細胞内Ca2+濃度上昇に同期した細胞膜電位の脱分極が観測され、ペースメーカー細胞のCa2+クロックによるペースメーキングをHEK293細胞で再現することができた。 (2)Ca2+クロックによるペースメーキング電位発生機構の普遍性検討比較 Ca2+活性化Cl-チャネル(TMEM16 類)の分子機能として、Ca2+振動の電位変化へのシグナル変換素子としての機能を想定し、概日リズムの形成に関与する松果体でのTMEM16の発現及び機能の解明を目指した。これまで、松果体細胞における短周期のCa2+クロック機構が存在すること、またTMEM16A,TMEM16Bが高発現し、ヘテロ二量体を形成することを見出した。
  • 日本学術振興会:科学研究費助成事業 特別研究員奨励費
    Date (from‐to) : 2012/04 -2015/03 
    Author : 今泉 祐治; AZIZIEH Regis
     
    性ホルモンによるカリウムチャネル発現調節機構の解明 本研究の目的は男性生殖器系組織での男性ホルモン受容体刺激による情報伝達系が、BKチャネル機能発現を制御している可能性と疾患との関連を明らかにすることである。男性ホルモンの影響を調べるため、雄性Wistar/STラットに対して去勢手術を行ったCAST群、擬似手術を施したSHAM群、CAST群にテストステロンを経口投与したTES群、溶媒のみを投与したVehicle群を用いた。ユビキチン系がBKαの分解に寄与し、アンドロゲン受容体刺激により、強く抑制される可能性を示唆する予備的結果を得ていため、ユビキチン連結酵素(E3)のサブタイプの同定を試みた。その結果、Nedd4 mRNAが精管平滑筋において、高発現していた。更に、CAST及びVehicle群で発現が上昇する傾向にあった。共免疫沈降の結果、CASTにおいて、BKチャネルと結合するNedd4のタンパク量がSHAMと比較して有意に多かった。前立腺のアンドロゲン受容体発現に対して、ユビキチンプロテアソーム系の活性化を介した負帰還機構として働く因子に、PMEPA1(Prostate Transmembrane Protein, Androgen Induced 1)が知られている。ラット輸精管においてこのPMEPA1が発現するか、またテストステロンによってその発現量が影響を受けるか否かについて検討した。その結果、CAST、Vehicle群において、PMEPA1 mRNAレベルが有意に上昇していた。この現象はテストステロンの投与によって消失した。これら結果は、去勢によるテストステロンの減少がPMEPA1の発現量の上昇と、ユビキチンプロテアソーム系の活性化を介して、BKチャネル発現量を減少させることを示唆している。ヒト由来の初代培養前立腺平滑筋細胞に対して遺伝子導入あるいはsiRNA等を用いてより詳細な検討を試みた。しかし、この培養細胞ではBKチャネルのタンパク質発現が低かったため、実験を進めることができなかった。
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2011/11 -2014/03 
    Author : IMAIZUMI Yuji; OHYA Susumu; YAMAMURA Hisao; HIGUCHI Tsunehiko; ASAI Kiyofumi; HIRONO Syuichi
     
    The present study revealed that, in non-excitable cells such as endothelial cells, lymphocytes, chondrocytes and airway cilliated cell, Ca2+-activated K+ (KCa) channels and store-operated Ca2+ (SOC) channels play significant roles in the positive feedback mechanism for the regulation of intracellular Ca2+ concentration ([Ca2+]i). This [Ca2+]i elevation elicites cellular responses to various types of stimula under physiological conditions or is involved in pathological processes. In brain capillary endothelial cells and chondrocytes, Ca2+-release activated Ca2+ channel is a major component of SOC channels and regulates cell proliferation. In T lymphocytes isolated from inflammatory disease model mice, the change in expression level of intermediate-conductance KCa channel is related to pathological processes. In ciliated cells, ATP-sensitive K+ channel contribute to the positive feedback mechanism for [Ca2+]i, and facilitate ciliary movement and consequently promote airway clearance.
  • 日本学術振興会:科学研究費助成事業 新学術領域研究(研究領域提案型)
    Date (from‐to) : 2011/04 -2013/03 
    Author : 今泉 祐治
     
    本研究の目的は幾種類かの平滑筋組織においてペースメーカー電位発生源として認識されているカハール間質系細胞でのペースメーカー電位発生機構を再構築し、シミュレーションすることにより、ペースメーカー発生機構を解明することである。ミトコンドリア呼吸リズムは近傍の小胞体へのATP供給、小胞体Ca2+ポンプによるCa2+取り込み、そしてCa2+遊離を制御し、さらに小胞体から遊離されたCa2+の一部はミトコンドリアへ取り込まれ、呼吸を促進す可能性がある。この連鎖で、ミトコンドリアと小胞体間の機能的一体性が生じ、Ca2+オシレーションを安定的に発生させると推測している。細胞内Ca2+オシレーション発生装置としてCa2+遊離チャネルのリアノジン3型受容体をHEK293細胞に強制発現させると、一部の細胞で自発Ca2+遊離により、Ca2+オシレーションが発生する。特定のミトコンドリア呼吸周期と近傍小胞体からCa2+遊離を画像解析するため、1つの細胞からミトコンドリア膜電位と細胞内Ca2+濃度変化を僅かな時間差で画像解析する方法を開発した。さらにこのようなCa2+オシレーション発生装置として機能する特定のオルガネラ連関が、どのような分子機構で生じるのかを明らかにするため、細胞膜直下の小胞体と近傍のミトコンドリアの表面が、エバネッセント光領域で観察できることを全反射顕微を用いた1分子可視化法により確立した。一方、Ca2+活性化K+(SK2)チャネルを定常発現した細胞に、3型リアノジン受容体を一過性に発現させると、シグナル変換されCa2+オシレーションに同期した過分極オシレーションが生じた。Ca2+活性化Cl-チャネルであるヒトTMEM16AのcDNAを独自に単離し、hTMEM16A定常発現HEK細胞を作製脱分極性のペースペーカー電位の再構築を行う準備が完了した。
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
    Date (from‐to) : 2011 -2012 
    Author : IMAIZUMI Yuji; OHYA Susumu; YAMAMURA Toshio
     
    To provide a simple but high throughput screening method for compounds acting on ion channels, a new recombinant cell line, in which single action potential (AP) induced cell death, was produced by gene transfection. Mutated human cardiac Na^+channel Nav1.5 (IFM/Q3), which shows extremely slow inactivation, and wild type inward rectifier K^+channel, Kir2.1 were stably co-expressed in HEK293 cells (IFM/Q3+K_2.1). In IFM/Q3+K_2.1, application of single electrical stimulation (ES) elicited a long AP lasting over 30 s and led cells to die by over 70 %, while HEK293 co-transfected with wild type Nav1.5 and K_2.1 fully survived. The additional expression of hERG K+channels in IFM/Q3+K_2.1 shortened the duration of evoked AP and, thereby, markedly reduced the cell death. The treatment of the cells with nifekalant, E-4031, cisapride, terfenadine and verapamil, hERG channel inhibitors, recovered the prolonged AP and dose-dependently facilitated cell death upon ES. Results indicate the high utility of this cell system for hERG K+channel safety assay. Moreover, to develop a screening system for blockers of voltage-gated Kv1.3 and Kv1.5 channels, new cell lines co-expressing IMF/Q3, K_2.1 and Kv1.3 or Kv1.5 were introduced as IFM/Q3+K_+Kv1.3 and IFM/Q3+K_+Kv1.5, respectively. Co-expression of Kv1.3 or Kv1.5 to IFM/Q3+K_ shortened the evoked APs and prevented the cell death. In the presence of margatoxin, a selective Kv1.3 blocker, ES induced the cell death in IFM/Q3+K_+Kv1.3, but not in IFM/Q3+K_+Kv1.5. In the presence of 4-aminopyridine, a non-selective Kv channel blocker, ES application elicited cell death in both cell lines. The IC50s of acacetin, a Kv1.5 blocker, and citalopram, a 5-HT uptake-inhibitor, in IFM/Q3+K_+Kv1.3 were almost identical to those in IFM/Q3+K_+Kv1.5. It was, thereby, found that acacetin and citalopram block both Kv1.3 and Kv1.5 without significant selectivity. The new cell lines for hERG, Kv1.3 or Kv1.5 channel inhibition assay fit to the high-throughput screening because of its simplicity, accuracy and high cost-performance.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)
    Date (from‐to) : 2011 -2012 
    Author : YAMAMURA Hisao; IMAIZUMI Yuji
     
    To clarify the molecular dynamics of large conductancecalcium-activated potassium channels in the functional calcium microdomain, we examined the single-molecule imaging using total internal reflection fluorescence microscopy. These results indicate that the molecular dynamics are strongly restricted by cytoskeleton and direct interaction with caveolin in vascular smooth muscle cells.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)
    Date (from‐to) : 2008 -2011 
    Author : HIGUCHI Tsunehiko; UMEZAWA Naoki; KATO Nobuki; IMAIZUMI Yuji
     
    We have succeeded in the development of 2, 4, 6-triethyl-1, 3, 5-di(alkylamino) benzene dynamic combinatorial library having affinity to hemin by utilizing aldehyde/amine-imine equilibrium reaction between 2, 4, 6-triethyl-1, 3, 5-benzenetricarbaldehyde(1) scaffold and amines. Increased products in the presence of hemin had higher affinity with hemin than not increased products. Porphyrin having four aldehyde precursors as a scaffold was synthesized. Equilibrium reaction between scaffold 1 and boomerang-form diamines converged to a cage-type supramolecule mainly.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2008 -2010 
    Author : IMAIZUMI Yuji; OHYA Susumu; YAMAMURA Hisao; ASAI Kiyofumi; HIGUCHI Tunehiko
     
    The present study revealed that functional expression of Ca^<2+>-activated K+ (BK, IK, SK) channels in non-excitable cells, such as chondrocytes, vascular endothelial cells and T-lymphocytes, substantially contributes to sustained increase in intracellular Ca^<2+> concentration in response to various types of stimuli. We proved that Ca^<2+>-activated K+ channels play the central roles in the positive feedback mechanism for the regulation of intracellular Ca^<2+> concentration via the membrane hyperpolarization, which increases the driving force of store-operated Ca^<2+> influx through non-selective cation channels.
  • 日本学術振興会:科学研究費助成事業 特定領域研究
    Date (from‐to) : 2008 -2009 
    Author : 今泉 祐治; 大矢 進; 山村 寿男
     
    細胞膜上でイオンチャネルは他分子と特定の分子複合体(トランスポートソーム)を形成して、初めて正常な生体内機能を果たしている場合の多いことが明らかになりつつある。本研究は、平滑筋、およびペースメーカー細胞としての間質系カハール細胞、非興奮性細胞としての軟骨細胞におけるCa^<2+>活性化K^+チャネル(BKチャネルなど)とその他の細胞膜上のイオンチャネルやトランスポーターやカベオリン、更には小胞体膜上のリアノジン受容体との分子間連関の可能性とトランスポートソームの実体を一分子可視化法により明らかにすることを、目的としている。蛍光タンパクでラベルされたこれら分子の遺伝子を上記細胞に導入・発現させ、全反射顕微鏡で可視化するとともに、その機能を電気生理学的に解析した。 (1) リアノジン受容体とBKチャネルの機能連関を可視化するとともに、男性ホルモンによる発現調節機構を明らかにした(J Pharmacol Sci, 2009)。 (2) 軟骨細胞由来の培養細胞において、細胞内Ca2+濃度制御機構において、非選択的陽イオンチャネルとの機能連関により、BKチャネルなどのCa^<2+>活性化K^+チャネルが重要な役割を果たしていることを明らかにした(Am J Physiol, 2010)。またCl^-チャネルとの機能連関を明らかにした(J Pharmacol Sci, 2010) (3) Na^+-Ca^<2+>交換体を高発現したマウス膀胱平滑筋において、その生理機能を明らかにした。(J Pharmacol Sci, 2010)
  • 日本学術振興会:科学研究費助成事業 特定領域研究
    Date (from‐to) : 2006 -2007 
    Author : 今泉 祐治; 大矢 進; 山村 寿男; 村木 克彦
     
    <細胞内CAa^<2+>信号の電気信号への変換機構におけるトランスポートソーム機能と構造実体の解析>2型リアノジン受容体(RyR2)異型接合性欠損マウス膀胱平滑筋を用いてRyR2の寄与を明らかにした。膀胱平滑筋においてRyR2を介した自発Ca^<2+>遊離(Ca^<2+>spark)の発生とそのCa^<2+>信号をSTOCsという電気信号に変換するトランスポートソーム機能は,尿貯留・排泄調節という膀胱機能発現において根源的な果たす役割を果たすことが示され,当該トランスポートソームの実体はカベオラ構造内に存在することが強く示唆された。さらにこのような信号機構がカハール間質細胞において生じ,ペースメーカー電位発生の根源となっている可能性を再構築系を用いて明らかにした。 <大コンダクタンスCa^<2+>活性化K^+(BK)チャネルのβサブユニット特異的開口物質の発見>電位感受性蛍光色素のDiBAC_4(3)および関連オキソノール色素にBKチャネルβ1およびβ4サブユニット選択性(β2には無効)を有するBKチャネル開口作用があることを発見し,創薬の可能性を示した。 <一分子可視化によるCa^<2+>信号から電気信号への変換トランスポートソームの機能解析>トランスポートソームにおけるCa^<2+>信号から電気信号への変換に関する分子機構解明における新たな手法として一分子レベルでの可視化技術を導入した。全反射蛍光顕微鏡とホールセルクランプ法の併用により,電位固定化で細胞膜直下200nm以内でのナノスケールの蛍光分子動態が測定可能となった。また記録電極からCa^<2+>蛍光色素Fluo4を細胞内に導入し,脱分極刺激時の膜直下の局所Ca^<2+>濃度変化をナノスケールで計測することが可能となった。この方法を用いて電位固定化でのチャネルを中心としたトランスポートソーム機能の定量的ナノイメージング解析を行った。トランスポートソームの信号変換分子機構を解明する上で一分子可視化技術は画期的な技術となる可能性を示した.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2005 -2007 
    Author : IMAIZUMI Yuji; OHYA Susumu; YAMAMURA Hisao; TOGARI Akihumi
     
    In excitable cells, such as CNS neurons, negative feedback regulation systems for intracellular Ca^<2+> homeostasis work to prevent Ca^<2+> overlord, when excess excitability and resulting excess Ca^<2+> influx occurs. Activation of Ca^<2+> activated K+ (Kca) channels is know to be one ` of the most important components responsible for the negative feedback regulation of [Ca2^+]_i in excitable cells. This project was undertaken to elucidate the molecular mechanism for the regulation of Kca channel activity and to search changes in the regulation diseases. The goal of this project is to find out molecular seeds targeting on K_ca channels in some diseases. The following development was obtained during the research period. (1) It was found that small conductance K_ca (SK) channel in vascular endothelial cell lines originally derived from bovine blood-brain barrier has significant functional role in endothelial; proliferation stimulated by ATP presumably released from astrocytes in CNS. (JBC,2006; J Pharmacol Sci, 104, 2007). (2) The deep impact of ryanodine receptor type2 (RyR2) to the mechanism for negative feedback regulation of [Ca^<2+>], via spontaneous Ca^<2+> release(Ca^<2+> spark) from sarcoplasmic reticulum and subsequent activation of large conductance Kca (BK) channel was found using urinary bladder smooth muscle cells from wild type and RyR2 heterozygous KO mice. A line of evidence indicates that RyR2 contributes to the bladder continent as 'a key molecule regulating resting membrane potential and muscular tonus as well as excitation-contraction coupling (J Physiol, 2007, J Pharmacol Sci, 103, 2007). (3) It was found that potential sensitive oxonol dyes act as potent BK channel openers. It is the first synthesized compound which shows opening property selective to BK81 and 84 subunits over BK62. The oxonol compounds may be a seed of 8 subunit selective BK channel opener (Mol Pharmacol, 2007). (4) It has been known that the contractile responses of isolated large arteries from spontaneously hypertensive rats (SHR) to agonists are markedly potentiated in low pH bathing solution. It was found that the enhanced expression of BK channels in arterial smooth muscles of SHR and its sensitivity to extracellular pH are responsible for the acid pH induced potentiation of contraction (Am J Physiol, 2007).
  • 日本学術振興会:科学研究費助成事業 萌芽研究
    Date (from‐to) : 2005 -2006 
    Author : 今泉 祐治; 大矢 進; 山村 寿男
     
    全反射蛍光顕微鏡を利用して、生きた細胞内で機能している細胞内小器官、特に小胞体の膜上に存在する特定の蛋白質(特にはリアノジンCa^<2+>遊離チャネル)のリアルタイムでの機能を一分子可視化法により、解明することを試みた。さらにその技術を一般化し、その他の小胞体上の蛋白質、あるいはその他のオルガネラ(ミトコンドリアなど)の膜表面蛋白を一分子可視化し、その機能解析に新分野を切り開くための端緒とするべく検討した。 (1)膜電位固定下で平滑筋細胞膜上の1分子のまたは凝集した分子群のCa^<2+>チャネルが脱分極で開口することにより、細胞膜直下の筋小胞体からのリアノジン受容体を介したCa^<2+>遊離を可視化することに成功した(07年日本薬理学会年会発表;論文投稿準備中)。 (2)YFPでラベルされた2および3型リアノジン受容体をHEK293細胞に発現させ、リアノジン受容体開口による自発的Ca^<2+>遊離現象を一分子可視化することを試行している。 (3)CFPラベルされた大コンダクタンスCa^<2+>活性化K^+チャネルをHEK293細胞に発現させ、機能的クラスター形成の過程を一分子可視化法により明らかにした。また(2)と同時に発現させることにより両分子の機能連関の可視化を検討している。
  • 日本学術振興会:科学研究費助成事業 萌芽研究
    Date (from‐to) : 2003 -2004 
    Author : 今泉 祐治; 大矢 進
     
    現在、汎用されている代表的なオキソノール系蛍光色素としてDiBAC_4(n)やDisBAC_4(n)などが挙げられる。陰イオンであるDiBACは細胞膜を良く透過するため、細胞外液中に存在させると膜電位差が減少した時には細胞内へより多く分布し、不特定の細胞質蛋白と結合して、細胞内からの蛍光強度を増す。細胞外のfreeのDiBACは微弱な蛍光しか発しない。アーチファクトが生じやすい理由は、本来は極めて短い色素の蛍光寿命が、不特定の細胞質蛋白との結合により延長されて測定が可能となっているという根本的な測定原理に由来する。蛋白と色素の結合に影響を与える化合物は、イオンチャネルに作用しなくても蛍光強度を変化させるからである。上記の欠点を解消するため、次のような系を考案した。DiBAC系膜電位感受性蛍光色素に標識化学構造を化学合成により付加した。一方、その標識部位を特異的に認識する蛋白を遺伝子導入により細胞に高発現させることを試みた。付加的化学構造を持つ色素は特異的結合蛋白に優先的に結合するため、不特定の細胞内蛋白との結合は防がれ、かつより高効率の蛍光を発することが可能性となると考えた。構造が比較的単純な精巣型アンジオテンシン転換酵素タンパクの一部とその阻害薬カプトプリル類縁化合物の利用を検討した。特異的結合蛋白を低分子化することにより色素との結合・解離速度を上昇させることができると想定した。以上から、当方法によりアーチファクトの減少と反応速度の上昇が得られると推測し検討を加えたものの、現在のところ従来と比較して明らかな反応速度の変化は観察されていない。DiBACが既に細胞質のタンパクのうちでも比較的低分子なものに良く結合していた可能性、まだ低分子化が不足している可能性、細胞内では何らかの理由で結合能が低下しているなどの理由が考えられるのでさらに検討が必要である。
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2002 -2004 
    Author : IMAIZUMI Yuji; MURAKI Katsuhiko; OHYA Susumu; OHWADA Tomohiko
     
    Although the increase in intracellular Ca^<2+> concentration ([Ca^<2+>]i) is commonly observed in responses to various types of stimuli, the excess increase in [Ca^<2+>]i, or in other words, overload of cells with calcium is one of the key steps and very popular in the process of accumulation in cellular damages under pathophysiological settings. To minimize calcium overload, cells have various systems to extrude Ca^<2+> to and/or prevent Ca^<2+> entry from outside. The Ca^<2+> entry is usually due to opening of two separate types of Ca^<2+> entry channels; voltage-dependent Ca^<2+> channels (VDCCs) and non selective cation channels. Ion channels, whose activities are directly modulated by [Ca^<2+>]i, strongly contributes to the regulation of Ca^<2+> entry via the changes in membrane potential. Large conductance Ca^<2+> activated K^+ (BK) channels are ubiquitously expressed in excitable cells except cardiac myocytes and also expressed in some non-excitable cells. The activation of BK channel induces membrane hyperpolarization, reduces VDCC activity and minimizes Ca^<2+> overload in excitable cells. We surveyed low molecular natural products mainly from plants to find out new prototype of BK channel opener, since this type of agents may reduce the hyper contractility of smooth muscle tissues or Ca^<2+> over load in neurons under pathophysiological conditions. Among over 60 natural products and their synthesized derivatives, we found pimaric acid (PiMA)and related compounds as potent openers of BK channel. Effects of PiMA and other compounds on BK channels were examined using HEK293 cells, in which either the a-subunit of BK channel (HEKBKα) or both α and 01 (HEKBKαβ1) subunits was heterologously expressed. Effects of these compounds (10μM) on the membrane potential of HEKBKαβ1 were monitored by use of DiBAC_4(3), a voltage-sensitive dye. PiMA, isopimaric acid, sandaracoisopimaric acid, dihydropimaric acid, dihydroisopimaric acid and dihydroisopimarinol induced substantial membrane hyperpolarization. The direct measurement of BKαβ1 opening under whole cell voltage-clamp showed that these six compounds activated BKαβ1 in a very similar concentration range (1-10 μM), in contrast abietic acid, sclareol and methyl pimarate had no effect. PIMA did not affect the charybdotoxin-induced block of macroscopic BKaβ1 current. Single channel recordings of BKαβ1 in inside-out patches showed that 10 μM PiMA did not change channel conductance, but significantly increased its open probability due to increase in sensitivity to Ca^<2+> and voltage. Since co-expression of β1 subunit did not affect PiMA-induced potentiation, the site of action for PiMA is suggested to be BKα subunit. PiMA was selective to BK over cloned small and intermediate Ca^<2+> activated K^+ channels. It can be concluded that PiMA (>1μM) increases Ca^<2+> and voltage-sensitivity of BKα when applied from either side of the cell membrane. The marked difference in potency as BK channel openers between PiMA and abietic acid, despite only very small differences in their chemical structures, may provide insight into the fundamental structure-activity relationship governing BKα activation. Moreover, we found that BK channel-like K^+ channels, which may be expressed in mitochondria of cardiac myocytes, are also activated PiMA. The protective effects of PiMA to reduced cell injury in ischemic conditions were also detected in rat cardiac myocytes. Taken together, we found a useful compound, PiMA, as a prototype of BK channel opener and obtained basic information about the activity-structure relationships for BK channel opener.
  • 日本学術振興会:科学研究費助成事業
    Date (from‐to) : 1999 -2000 
    Author : 渡辺 稔; 大矢 進; 村木 克彦; 今泉 祐治
     
    単離平滑筋細胞に電位固定法を適用し、膜電流を記録するとともに、記録電極内からfluo-3を細胞に負荷しレーザー共焦点顕微鏡を用いて細胞内Ca^<2+>分布画像を同時に取得する手法を用いて、精管・膀胱・門脈等の興奮性の高い平滑筋細胞における興奮収縮連関の画像解析とCa^<2+>依存性K^+チャネル活性化の解析を行い、次の知見を得た。(1)細胞膜直下に局在する特定の筋小胞体(の一部分)は、活動電位などの脱分極時に、電位依存性Ca^<2+>チャネルを介したCa^<2+>流入によるCa^<2+>遊離機構の起点となり、数百ミリ秒持続するCa^<2+>ホットスポットを形成する。(2)膜直下筋小胞体からの局所遊離Ca^<2+>によるK^+チャネル活性化は、活動電位波形を制御し活動電位発生頻度を調節する。(3)この局所Ca^<2+>遊離は他の筋小胞体へCa^<2+>遊離を伝播した場合にのみ、収縮を誘発する可能性が高い。(4)このようなCa^<2+>ホットスポット形成という特定機能を持つ膜直下筋小胞体の数は、1細胞当り比較的少数(<20)と考えられる。 一方、保持電位-40mV付近での観察から、膜直下の特定の筋小胞体(数箇所)から、自発的な一過性局所Ca^<2+>遊離(Ca^<2+>スパーク)が繰り返し生じ、近傍の細胞膜上のCa^<2+>依存性K^+チャネル(10-100個)を活性化することにより自発性・一過性の外向き電流(STOC)が生じることが示唆されている。Ca^<2+>スパークとSTOCの持続時間は半値幅50ミリ秒程度で、その発生に外液Ca^<2+>の流入は直接必要ではなく、特定の筋小胞体からのリアノジン受容体を介する自発性Ca^<2+>遊離によると考えられる。今回、上記の活動電位発生初期に重要な働きをする特定の筋小胞体のさらに一部で、Ca^<2+>スパークがほぼ定期的に生じるていること、それがSTOCと完全に同期していることを画像解析と膜電流の同時記録により明らかにした。Ca^<2+>スパークは心筋や骨格筋において興奮収縮連関の最少ユニットとして解析されている。一方、横行小管と筋小胞体の発達が悪い平滑筋においては、細胞膜直下の筋小胞体で生じるCa^<2+>スパークが細胞膜上のCa^<2+>依存性イオンチャネルを活性化して静止膜電位や興奮性、さらに筋緊張度の調節に関与していることが明らかとなった。静止時にSTOCを活性化するCa^<2+>スパーク部位は、脱分極時にはより大きく増幅され持続するCa^<2+>ホットスポット部位となる。
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 1998 -1999 
    Author : WATANABE Minoru; OHYA Susumu; IMAIZUMI Yuji
     
    The mechanisms underlying nonspecific supersensitivity induced by parasympathetic denervation were examined using RT-PCR, semi-quantitative PCR and skinned fiber techniques. It was found that microinjection of AF64A, a novel choline uptake inhibitor, into anterior eye chamber of the rat resulted in changes in iris sphincter smooth muscle similar to those elicited by surgical parasympathetic denervation by removal of ciliary gangrionectomy. The changes were characterized in vitro by reduced contractile response to nerve stimulation presumably via the decrease in acetylcholine (ACh) release from parasympathetic nerve endings and by enhanced contractile responses both specific to exogenously applied ACh and nonspecific to serotonin and high KィイD1+ィエD1. Semi-quantitative PCR analyses of muscarinic receptors in rat iris indicate that mRNAs of m2, m3 and m4 subtypes are expressed in iris and that, unexpectedly, the expression level of m4 is much higher than that of m2 and m3. The sequence homology of m2, m3 and m4 in rat iris to those reported in genome or cDNA in other tissues of the rat was over 99%, while several differences in amino acid sequence in intracellular loops were found. Following the injection of AF64A, mRNA levels of m2 and m3 increased and that of m4 decreased, respectively. The CaィイD12+ィエD1 sensitivity of the contractile machinery measured in skinned fiber was slightly but significantly increased by AF64A injection. The increase in CaィイD12+ィエD1 sensitivity was abolished by addition of H7, protein kinase C inhibitor. AF64A injection resulted in marked increase in the maximum contractile response of iris sphincter muscle. A slight increase in amount of actin and no change in those of myosin and calmodulin in iris can not explain the increase in the maximum contractile response. A novel mechanism distinctive from the changes in major contractile and related regulatory proteins may underlie the nonspecific supersensitivity induced by AF64A.
  • 心血管系病態でのイオンチャネル発現変化とその機構
    Date (from‐to) : 1998 -1999
  • 電位依存性一過性Kチャネルのアラキドン酸感受性と遺伝子解析
    Date (from‐to) : 1998 -1999
  • 日本学術振興会:科学研究費助成事業
    Date (from‐to) : 1997 -1997 
    Author : 渡辺 稔; 村木 克彦; 今泉 祐治
     
    心室筋や平滑筋の細胞内Ca^<2+>濃度は、特に外的刺激のない状態においても均一あるいは一定ではない。筋小胞体から、Ca遊離チャネルであるリアノジン受容体を介して自発的なCa遊離が局所的に生じる現象を、Ca蛍光色素と共焦点蛍光顕微鏡などを用いることにより数十ミリ秒間のCaスパークとして可視化することができる。我々は高速共焦点蛍光顕微鏡を用い平滑筋細胞においてCaスパークの2次元画像解析を行うとともに、自発性一過性外向き電流を室温で同時記録した。Caスパークが細胞内数箇所で見られる場合にも、スパークの中心が細胞膜から1μm以上離れている場合は明確に同期した外向き電流は観察されないことから、細胞膜と非常に緊密な位置関係にある筋小胞体だけが自発性一過性外向き電流を生じさせ得ると考えられる。興奮性の高い膀胱や精管の平滑筋細胞では、膜電位固定下での脱分極によりまずCa電流が活性化され、その後にCa依存性K電流(1_)が活性化される。1_の活性化は極めて速やかで、+10mVでは20ミリ秒以内にピークに達する。Ca画像解析によるとCa濃度の上昇は細胞膜に沿って均一に生じるのではなく、細胞膜直下に数個から20個程度の直径1μm以下のCaホットスポットが1_の発生と同時に生じ、その後、徐々に広がり百ミリ秒以上続いて細胞全体のCa濃度が上昇することがわかった。Caホットスポットは電位依存性Caチャネルを介して流入したCa細胞膜直下の筋小胞体からCaを遊離させることによって生じると考えられる。活動電位の発生によっても同様のCaホットスポットが観察された。平滑筋において細胞膜直下の筋小胞体の一部は、リアノジン受容体を介するCa遊離により局所Ca動態に重要な生理的役割を持ち、BKチャネル活性を制御することにより、活動電位の波形や発生頻度および静止膜電位の調整を行うとともに、興奮収縮連関でのCa遊離連鎖の起点となる可能性が示唆された。
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 1996 -1997 
    Author : WATANABE Minoru; OHYA Susumu; IMAIZUMI Yuji
     
    The amount of mRNAs of muscarinic acetylcholine receptor subtypes, m1, m2, m3, m4 and m5, in the rat iris was determined quasi-quantitatively using RT-PCR methods. The mRNA expression levels of m2 and m3 were higher than those of m1 and m5 in the rat iris as in other smooth muscle tissues. It was characteristic in the rat iris that the m4 mRNA expression level was high. In addition, the cloning of cDNAs encoding m2, m3 and m4 subtypes in the rat iris was performed using RT-PCR methods. Amino acids sequence of m2 subtype in the rat iris included nine differences in comparison with that in the heart, whereas it was 100% identical to genomic one. To examine the mechanisms underlying the parasympathectomy-induced effects in the rat iris smooth muscles, the mRNA expression levels of muscarinic receptor subtypes were determined in iris from ciliaryganglionectomized rats. The results suggested that the parasympathectomy does not markedly affect the mRNA levels. It was also clarified that the intracellular Ca^<2+> concentration ([Ca^<2+>]_i) in the rat iris dilator under the resting conditions is higher than those in the iris sphincter and many other smooth muscles which do not have an inherent tone. Since the muscarinic receptor stimulation resulted in the decrease in [Ca^<2+>]_i in the rat iris dilator, the muscarinic relaxation appeared to be due to the decrease in [Ca^<2+>]_i. In denervated iris dilators, muscarinic receptor stimulation induced neither the decrease in [Ca^<2+>]_i nor the relaxation.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 1996 -1997 
    Author : WATANABE Minoru; OHYA Susumu; MURAKI Katsuhiko; IMAIZUMI Yuji
     
    Early inactivating K^+ (A type) current can be recorded widely in neuron, cardiac myocytes and smooth muscle cells. the current plays significant roles for the regulation of action potential firing and formation of the repolarizing phase of an action potential. Although several distinct genes encoding the K^+ channels responsible for A type K^+ current (I_A) in cardiac myocytes and brain have been reported, nothing is known in smooth muscle cells. The electrophysiological characteristics of I_A in smooth muscle cells (vas deferens, colon and ureter) and similar to those in cardiac myocytes. Nevertheless, it was found that arachidonic acid selectively blocks the I_A in smooth muscle cells (Am.J.Physiol.1997). The possibility that the K^+ channel gene for I_A in smooth muscle is deferent from that in cardiac myocytes was examined using RT-PCR techniques. In addition, the related K^+ channels in smooth muscle cells in the rat was cloned. Kv1.4,4.2 and 4.3 have been reported as K^+ channel genes in brain and cardiac myocytes. Based on the expression levels of mRNAs of these genes, it was found taht Kv4.3 is predominant among them in smooth muscle cells. Moreover, Kv4.3 in smooth muscle cells was a new spliced variant of the original Kv4.3 (Kv.4.3M) in brain and has additional 19 amino acids in cytosolic domain close to the C terminus (Kv4.3L) (FEBS Letters, 1997). In the rat heart, the mRNA level of Kv4.3L was higher than that of Kv4.3M.Electrophysiological characteristics of Kv1.4,4.2,4.3M and 4.3L K^+ channels were determined in HEK 293 cells in which one of these channel types was highly expressed. The voltage-dependence of activation and inactivation, the recovery time course from inactivation and the sensitivity to arachidonic acid of Kv4.3L were not significantly different from those of Kv4.3M.Further research is required to elucidate the functional roles of additional 19 amino acids in Kv4.3L.It was also found that the different expression levels of mRNAs encoding the large conductance Ca^<2+> -dependent K^+ channels and the voltage-dependent Ca^<2+> channels in various type of smooth muscle cells, at least in part, explains the difference in membrane excitability in different types of smooth muscle cells.
  • 気道平滑筋細胞のカルシウム依存性クロライドチャネルと気道過敏症
    Date (from‐to) : 1996 -1997
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 1994 -1995 
    Author : WATANABE Minoru; OHYA Susumu; IMAIZUMI Yuji
     
    The activation of muscarinic receptors by exogenously applied acetylcholine (ACh) elicits unique responses in rat iris dilator smooth muscle ; relaxation at low doses and contraction at high doses (1). The pA_2 values of muscarinic antagonists for antagonism to relaxation in dilator muscles were most similar to those for M_3-type muscarinic receptors and contraction might be mediated by M_3-like receptor. The effects of Pertussis toxin (PTX) on contraction and/or relaxation induced by agonists were examined in the rat iris dilator. Only the ACh-induced relaxation was affected by injection of PTX into the anterior eye chamber. Relaxation had completely disappeared after injection of PTX.However, Preteatment with PTX did not significantly affect contraction induced by norepinephrine or 5-hydroxytryptamine or the relaxation induced by isoprenaline in dilator muscles (2). Therefore, only relaxation of the biphasic response to ACh was mediated by pertussis toxin sensitive GTP binding protein in rat iris dilator. To isolate the gene coding muscarinic receptor expressed in rat iris, RT-PCR was carried out using total RNA of the rat eye as the template and specific primers which were designed based on the sequence of cDNA for the rat cardiac m2 receptor and cDNA for the rat brain m3 receptor.Consequently, PCR products amplified were 1.4k bps for m2 receptor specific primers (3) and 1.8k bps m3 receptor specific primers (4). Each PCR product was subcloned into plasmid vector, and each nucleotide sequence was abalyzed. The protein sequence of m2 receptor cDNA obtained from rat eye differed from that reported for rat cardiac m2 receptor cDNA by 9 amino acid residues. The protein sequence of m3 receptor cDAN obtained from rat eye differed from that reported for the rat brain m3 receptor cDNA by 5 amino acid residues. To confirm the expression of the m2 and m3 receptor molecular subtype around the tissue of rat iris, RT-PCR_Swere performed using specific PCR primers targeted short fragment (350-450 bps) of third intracellular loop. From the results of RT-PCR,it was comfirmed that m2 and m3 receptor mRNAs expressed around the tissue of rat iris. Furthermore, m1 and , m5 receptor mRNA were not expressed substantialy.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 1994 -1995 
    Author : WATANABE Minoru; WALSH Michael; GILES Wayne; UYAMA Yoshiaki; OHYA Susumu; MURAKI Katsuhiko; IMAIZUMI Yuji
     
    The protein responsible for slowly activating outward current upon depolarization (IsK or mini K) in several smooth muscle tissues were cloned using RT-PCR and its distribution was examined using RNase protection assay. The techniques of RT-PCR method were taught in The University of Calgary. The cDNA encoding IsK protein cloned in rat aorta, duodenum, iris, ileum, stomach, trachea and urinary bladder and rabbit aorta, clon, stomach and urinary bladder was 100% identical to that has been reported in the rat kidney. The RNA protection assay showed that IsK is expressed in a similar extent in all these tissues. IsK was recorded upon depolarization in Xenopus oocytes injected with IsK cRNA. Pharmacological experiments using perfusion preparations of the rabbit mesenteric vascular bed suggested that Ba^<2+>-sensitive inward rectifier type K current is partly responsible for the resting membrane potential and tone of mesenteric arterial smooth muscle. In isolated mesenteric arterial myocytes, small K current which is sensitive to Ba^<2+> was observed under whole-cell voltage clamp. Inward rectifier type K channel (IRK1) was cloned from the rabbit mesenteric artery using RT-PCR.Total RNA extracted from mesenteric artery was used as a template. The primers were designed based on the sequence of IRK1 cDNA of the rabbit heart. A PCR product of 1392 bps (RBMAIK1) was obtained from arteries both with or without endothelium. RBMAIK1 was divided into three fragments by restriction endonucleases and subcloned into a plasmid vector. All fragments were, then, sequenced. RBMAIK1 showed 100% identity with rabbit heart IRK1. The injection of cRNA from the RBMAIK1 cDNA into Xenopus oocytes resulted in expression of Ba^<2+> sensitive inward rectifier K current. The techniques of gene transfection to mammalian cell lines will be transferred to Watanabe's group from that in the University of Calgary. Existence of A-type K currents has been reported in several types of smooth muscle cells including portal vein. Kv4.2 or Kv1.4 is the major K channel responsible for A-type K current in the rat heart. To identify the K channels responsible for those in smooth muscle, RT-PCR was performed using the total RNA of several types of smooth muscle tissues as templates and the primers designed from Kv4.2 and 1.4 of the rat brain. The PCR products of about 1.7Kbps were obtained. The subcloning of the PCR products and sequencing will be carried out.
  • ラット胃セロトニン受容体に関連した低分子量タンパクの遺伝子クロ-ニングと発現
    Date (from‐to) : 1994 -1995
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 1992 -1993 
    Author : WATANABE Minoru; IMAIZUNI Yuji
     
    This project was undertaken to investigate molecular mechanisms of transmission between autonomic nerve and smooth muscle cell by means of electrophysiological technics and measuring intracellular Ca concentration if preparations where neuro-muscular interaction is reconstituted by co-culture of autonomic neurons and smooth muscle. Singles neuron and smooth muscle cells isolated fron superior cervical ganglia and vas deferens or iris of young or infant rats were co-cultured for up to 7 days. Cardiac myocytes isolated from infant rats were also used as the target cedds of the sympathetic innervation.Membrane currents recorded by whole-cell clamp were compared in freshly isolated cells and from cells cultured for 3-4 days. Major currents resolved by using specific blockers were not changed significantly during the culture. Since the success rate of synapse formation during co-culture was low, synaptic current could not be measured in innervated smooth muscle cells. Morphological changes in interaction between nerve endings and smooth muscle cells were observed under scanning electron microscopy. Although cardiac myocytes were co-cultured with sympathetic neurons to increase the success rate, electrical recordings form innervated myocytes were not succedful either. To investigate the functional changes in intracellular Ca storage setes during the primary clture, effects of cyclopiazonic acid. a novel inhibitor of Ca-pump in endo-or sarco-plasmic reticulum(ER/SR), were examined. The decrease in stored Ca in ER/SR resulted in the decrease in Ca activated membrane currents, especially Ca activated K current (I_), in both sympathetic neurons and vas deferens smooth muscle cells. Since I_ is the major current responsible for action potential afterhyperpalarization if both cell types, the inhibition of ER/SR Ca-pump by CPA resulted in the potentiation of membrane excitability. Intracellular Ca mobikization was investigated using Ca-fluorescent indicator, Fluo 3-AM, and laser confocal fluorescent microscopy in these cells. although the reconstifution of synatic interaction by co-culture of freshly isolated sympathetic neurons and smooth muscle cells was not very successful, it is suggested that the ionic channels in co-cultured cells remained unchanged.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 1992 -1993 
    Author : WATANABE Minoru; MICHAEL walsh; WAYNE giles; MURAKI Katsuhiko; IMAIZUMI Yuji
     
    1. Differences in regulation of early transient K (A-type) currents by arachidonic acid (AA) in cardiac and smooth muscle cells A-type currents have been identified in smooth muscle cells isolated from portal vein, seminal vesicle, vas deferens, colon and stomach fundus. The activation and inactivation kinetics and the sensitivity to 4-aminopyridine of A-type current sin smooth muscle cells are similar to those in cardiac muscle cells, suggesting the same family of the channels in these muscles. The functional roles of the current in smooth muscle cells are suppressing AP or making a delay for AP firing during the early stage of depolarization and are, therefore, different from those in cardiac myocytes. We found that the application of 1muM AA reduced A-type current by about 50 % in these smooth muscle cells. In contract, substantial reduction of A-type current was observed in rabbit atrial myocytes only when much higher concentration of AA (>30muM) was applied, implying that physiological significance of AA-induced reduction of A-type current is larger in smooth muscle cells. The AA-induced reduction of A-type current was not affected by inhibitors of cyclooxy-genase, lipoxygenase or superoxide dismutase. The reduction was partly decreased by proteinkinase C inhibitior. 2. Differences in modulation of delayed rectifier K current by Class III antiarrythmic drugs Class III antiarrythmic drug prolongs AP duration and refractory period in cardiac myocytes. Although delayed rectifier K current is selectively suppressed by Class III anitiarrythmic drugs in cardiac myocytes, effects of the drugs on delayed rectifier K current in smooth muscle have not been clarified yet. The delayed rectifier K current in smooth muscle cells of the porcine coronary artery was much less sensitive to Class III antiarrythmic drugs, such as E-4031 and MS-551 but was equally sensitive to quinidine. The selectivity of Class III antiarrythmic drugs to cardiac delayed rectifier K current over that in vascular smooth muscle, especially that of coronary artery is quite important because suppression of delayed rectifier K current increases membrane excitability and contractility of the smooth muscle. 3. Increase in Ca sensitivity in smooth muscle by quaternary ammonium salt. To examine the difference in Ca mobilization in cardiac and smooth muscle cells by inositol 1,4,5 trisphosphate (IP_3), effects of a putative blocker of K channels which couple with Ca release from sarcoplasmic reticulum by IP_3 were examined. Unexpectedly, tetrahexylammonium bromide, which has been reported as a potent K channel blocker in endoplasmic reticulum of central nervous system, markedly increased Ca sensitivity of smooth muscle strip skinned by beta-escin by separate mechanism. The mechanism underlying the increase in Ca sensitivity includes neither an increase in myosin light chain kinase activity nor a decrease in myosin dephosphatase activity.
  • 各種平滑筋細胞におけるCaチャネル活性調節機構の多様性とそのメカニズム
    Date (from‐to) : 1992 -1993
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 1990 -1991 
    Author : WATANABE Minoru; KAWAI Tomoyuki; IMAIZUMI Yuji
     
    The present study was undertaken to reconstitute neuromuscular junction by co-culture of autonomic ganglia or ganglion cells with smooth muscle cells and to elucidate the synaptic transmission mechanisms using electrophysiological techniques and measurement of intracellular Ca ion concentration. As preparations for co-culture, the combination of superior cervical ganglia and vas deferens smooth muscle cells were used. Superior cervical ganglia was removed from infant rat, sliced into several pieces and organ-cultured. Occasionally, single ganglion cells were isolated by enzymatical dispersion from the slices. Single smooth muscle cells were also obtained with coliagenase from vas deferentia of matured rats and guinea-pig. Before establishing co-culture methods, effects of norepinephrine and ATP, chemical transmitters released from sympathetic nerve endings, on membrane ionic currents-were examined in single smooth muscle cells freshly isolated from vas deferens or primary cultured for a few days. NE decreased both Ca current(1_)and Ca-dependent K current(1_). The decrease in the latter current was more prominent, NE increased the membrane excitability. It is suaaested that the mechanisms for 1_ decrease by NE includes Ca channel ind('Ftivation via an increase in intracellukar Ca concentration and, in addition, more direct inhibition of Ca channel activity by activation of GTP binding protein. On the other hand, it is suggested that the decrease in 1_ by Ne is mediated by changes in intracellular Ca mobilization by NE. The co-culture was performed by seeding single smooth muscle cells after neurocladism had been found in ganglion cells sprouting from ganglia slices. The probability of success of co-culture was, however, low since contamination with bacteria often happened in the later part of the procedure. The formation of synaps between sympathetic neuron and smooth muscle cells had been found in morphological survey with scanning electronmicroscope. Exact lines of evidence that indicate formation of functionally available synaps, such as contraction or post synaptic potentials in smooth muscle cells following electrical stimulation of the nerve, have not been obtained yet.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 1988 -1989 
    Author : WATANABE Minoru; TAKEI Satomi; KAWAI Tomoyuki; IMAIZUMI Yuji
     
    The aim of this project is to elucidate the Ca mobilizing mechanism underlying changes in muscle tension when transmitters, autacoids and drugs are applied to iris dilator muscle of the rat and some other species. Special efforts were made to investigate the mechanisms of relaxation induced by muscarinic stimulation. First, it is found that the muscarinic relaxation is mediated by M_2-like receptor, whereas the conventional classification of muscarinic receptors into M_1 and M_2 does not fit well to characteristics of the receptor in the rat and pig iris dilator (J. Eye, 1988). Second, a striking finding was obtained that the relaxation induced not only by nerve stimulation but also by exogenously applied muscarinic agonist disappeared after parasympathectomy by ciliary ganglionectomy (E.J.P.,1988). This suggests that the muscarinic relaxation may not occur directly but mediate a release of relaxing substance from cells nearby. Therefore, substances which induce relaxation in dilator muscle were widely surveyed. It was finally found that high K^+ solution induced marked relaxation when Ca, alpha- and beta- adrenergic, and muscarinic antagonists were simultaneously present (B.J.P., 1990). Since the high K^+-induced relaxation was also abolished after parasympathectomy (manuscript in preparation), an additional indirect evidence to the assumption was obtained. The relaxing substance is neither the endothelium derived relaxing factor in vascularture and probably nor a peptide. On the other hand, sympathectomy by superior ganglionectomy did not alter the muscarinic relaxation, whereas a specific increase in sensitivity to noradrenaline was observed. The supersensitivity is due to the abolishment of noradrenaline-uptake after degeneration of sympathetic nerve endings (J.J.P., 1989). Beta-adrenergic and some other relaxations were not affected by sympathectomy either (manuscript in preparation). The measurement of intracellular Ca concentration by fluorescent indicator, fura2, was considered to be one of the most effective approaches to clarify the cellular Ca mobilization, the success rate of concomitant measurements of [Ca]_i and tension was very low in the rat dilator tissue because of its small size. Sufficient results for publication have not been obtained. From the results mentioned above, it is highly possible that muscarinic agonists and high K^+ solution induce relaxation of dilator muscle via release of a relaxing substance somewhat relating to parasympathetic but not sympathetic innervation. Further study is required to determine the relaxing substance.
  • 日本学術振興会:科学研究費助成事業
    Date (from‐to) : 1988 -1989 
    Author : 渡辺 稔; 武井 智美; 河合 智之; 今泉 祐治
     
    ラット虹彩散瞳筋において副交感神経刺激あるいはムスカリン様作用薬により生じる弛緩の機序の解明が研究課題である。 まず副交感神経切除により、ムスカリン様作用薬による弛緩反応も消失することを報告した(Eur.J.Pharmacol.,1988)。 またムスカリン様作用がM_2タイプのリセプターを介する可能性が高いものの従来のM_1,M_2,という分類では充分説明できない可能性を示唆した(眼薬理,1988)。 さらに弛緩を引き起こす物質として多くの生理活性物質を検索した。その結果、highK^+により生じる弛緩に副交感神経終末からのアセチルコリンの遊離を介さない成分がかなり存在することをつきとめ、報告した(あたらしい眼科,1989)。 これについては、highK^+のNa^+ーK^+pumpに対する作用、あるいは外液中のNa^+の減少による直接作用ではなく、未知の弛緩物質の遊離が関与している可能性の高いこと、副交感神経除去により、highK^+の弛緩も消失することなどを加え、外国雑誌に投稿中である。 アセチルコリンによる弛緩のセカンドメッセンジャーに関する検索では直接にcyclic AMP,GMPを定量した結果、これらがメッセンジャーである可能性は殆んど無いことがわかった。 蛍光色素による細胞内Ca^<2+>濃度変化の測定は大動脈等の大きな平滑筋標本では測定可能なものの、ラット虹彩散瞳筋ではいまのところ充分な蛍光変化が得られておらず、何等かの新たな工夫の必要を要すると思われる。
  • 日本学術振興会:科学研究費助成事業
    Date (from‐to) : 1983 -1983 
    Author : 今泉 祐治
  • 日本学術振興会:科学研究費助成事業
    Date (from‐to) : 1980 -1980 
    Author : 今泉 祐治
  • 日本学術振興会:科学研究費助成事業
    Date (from‐to) : 1979 -1979 
    Author : 今泉 祐治

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