脊髄における神経可塑性モデル、アロディニアの発症機構

• Project/Area Number: 12053263
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Kansai Medical University
• Principal Investigator: 伊藤 誠二 関西医科大学, 医学部, 教授 (80201325)
• Co-Investigator(Kenkyū-buntansha): 西澤 幹雄 関西医科大学, 医学部, 講師 (40192687) ; 芦高 恵美子 関西医科大学, 医学部, 講師 (50291802) ; 松村 伸治 関西医科大学, 医学部, 助手 (70276393)
• Project Period (FY): 2000 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥60,200,000 (Direct Cost: ¥60,200,000); Fiscal Year 2004: ¥11,600,000 (Direct Cost: ¥11,600,000); Fiscal Year 2003: ¥11,900,000 (Direct Cost: ¥11,900,000); Fiscal Year 2002: ¥11,700,000 (Direct Cost: ¥11,700,000); Fiscal Year 2001: ¥12,800,000 (Direct Cost: ¥12,800,000); Fiscal Year 2000: ¥12,200,000 (Direct Cost: ¥12,200,000)
• Keywords: PACAP / NMDA受容体 / 神経因性疼痛 / 中枢性感作 / ノックアウトマウス / 一酸化窒素合成酵素 / NADPHジアホラーゼ / トランスロケーション / 神経可塑性 / ノシセプチン / ノシスタチン / 脊髄 / 痛覚 / アロディニア / プロスタグランジン / JTC-801 / グルタミン酸 / 一酸化窒素 / BRET / 脊髄スライス / アクロメリン酸

Research Abstract

最近のDNAマイクロアレイの実験では、神経損傷に伴い100以上の遺伝子発現が変化することが報告されているが、どのように疼痛反応に関与するかは不明であった。今年度はPACAP(pituitary adenylate cyclase-activating polypeptide)のノックアウト(PACAP^<-/->マウスを用いて検討を行った。神経損傷に伴いPACAPの発現がDRGの中型・大型細胞、脊髄後角の浅層で増加するが、PACAP^<-/->マウスでは見られなかった。痛覚伝達にはグルタミン酸NMDA受容体が重要であり、その活性化に伴い一酸化窒素(NO)の産生が増加する。神経型一酸化窒素合成酵素(nNOS)活性は組織を固定後、NADPHジアホラーゼ活性で組織染色して測定できる。神経損傷後、NADPHジアホラーゼ活性がPACAPの発現誘導部位に一致して増加していたが、PACAP^<-/->マウスでみられなかった。NADPHジアホラーゼがnNOSの活性化を反映しているかどうか確認するために、蛍光NO指示薬DAF-FMを用いてNO産生を検討した。脊髄スライスにNMDAあるいはPACAPを単独投与した場合にはNO産生がみられなかったが、NMDA存在下にPACAPは濃度依存的にNO産生を増加させた。PACAP^<-/->マウスでNMDAとPACAPが相乗的に作用してアロディニアを誘発することから、疼痛行動とNO産生との関連が確認された。さらに、培養細胞を用いてNMDAとPACAPでnNOSの細胞質から細胞膜へのトランスロケーションが引き起こされ、NO産生が上昇することが示された。nNOSは後シナプス膜肥厚(PSD)においてPSD-95を介してNMDA受容体と会合することが知られている。現在、神経因性疼痛に伴うNMDA受容体複合体の構成分子の変化をプロテオミクスで解析を進めている。

Research Products

• [Journal Article] Involvement of Rho-Kinase in inflammatory and neuropathic pain through phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS) (2005)
  • Author(s): Tatsumi, Shinichi
  • Journal Title: Neuroscience 131 Pages: 491-498
• [Journal Article] Pituitary adenylate cyclase-activating polypeptide is required for the development of spinal sensitization and induction of neuropathic pain (2004)
  • Author(s): Mabuchi, Tamaki
  • Journal Title: J.Neurosci. 24 Pages: 7283-7291
• [Journal Article] Monitoring for dynamic biological processing by intra-molecular bioluminescence resonance energy transfer system using secreted luciferase (2004)
  • Author(s): Otsuji, Tomomi
  • Journal Title: Anal.Biochem. 329 Pages: 230-237
• [Journal Article] Acute and effects on induction of allodynia by acromelic acid, a mushroom poison related structurally to kainic acid (2004)
  • Author(s): Minami, Toshiaki
  • Journal Title: Br.J.Pharmacol. 142 Pages: 679-688
• [Journal Article] Membrane-associated prostaglandin E synthase-I is required for neuropathic pain (2004)
  • Author(s): Mabuchi, Tamaki
  • Journal Title: Neuroreport 15 Pages: 1395-1398
• [Journal Article] Synthesis of prostaglandin F ethanolamide by prostaglandin F synthase and identification of Bimatoprost as a potent inhibitor of the enzyme : new enzyme assay method using LC/ESI/MS (2004)
  • Author(s): Koda, Noriko
  • Journal Title: Arch.Biochem.Biophys. 424 Pages: 128-136
• [Book] 痛みの基礎と臨床(緒方 宣邦, 柿木 竜介編) (2003)
  • Author(s): 伊藤 誠二(分担)
  • Total Pages: 310
  • Publisher: 真興貿易(株)医書出版部
• [Patent(Industrial Property Rights)] 蛋白質のプロセッシングを測定するためのモニター蛋白質 (2007)
  • Inventor(s): 伊藤 誠二(他2名)
  • Industrial Property Rights Holder: 科学技術振興財団
  • Filing Date: 2007
• [Publications] Mabuchi, T.: "Attenuation of neuropathic pain by the nociceptin/orphanin FQ antagonist JTC-801 is mediated by inhibition of nitric oxide production."Eur.J.Neurosci.. 17. 1384-1392 (2003)
• [Publications] Muratani, T.: "Functional characterization of prostaglandin F_<2α> receptor in the spinal cord for tactile pain (allodynia)."J.Neurochem.. 86. 374-382 (2003)
• [Publications] Minami, T.: "Functional evidence for interaction between prostaglandin EP3 and κ-opioid receptor pathways in tactile pain induced by human immunodeficiency virus type-1 (HIV-1) glycoprotein gp120."Neurophamacology. 45. 96-105 (2003)
• [Publications] Muratani, T.: "Preemptive analgesia by Zaltoprofen in a mouse model of post-operative pain through blockade of the bradykinin B_2 pathway."J.Pharmacol.Exp.Ther.. (in press). (2004)
• [Publications] Koda, N.: "Synthesis of prostaglandin F ethanolamide by prostaglandin F synthase and identification of bimatoprost as a potent inhibitor of the enzyme-new enzyme assay method by LC/ESI/MS."Arch.Biochem.Biophys.. (in press). (2004)
• [Publications] Otsuji, T.: "Monitoring for dynamic biological processing by intra-molecular bioluminescence resonance energy transfer system using secreted luciferase."Anal.Biochem.. (in press). (2004)
• [Publications] 伊藤 誠二: "痛み-基礎・診断・治療-"朝倉書店. 407 (2003)
• [Publications] Doi, Y.: "Central nociceptive role of prostacyclin (IP) receptor induced by peripheral inflammation"Neuroreport. 13. 93-96 (2002)
• [Publications] Okuda-Ashitaka, B.: "Pain transmission regulated by novel neuropeptides nocistatin and nociceptin/orphanin FQ"J. Biol. Macromol.. 2. 3-10 (2002)
• [Publications] Tanikawa, N.: "Identification and characterization of a novel type of membrane -associated prostaglandin E synthase"Biochem. Biophys. Res. Commun.. 291. 884-889 (2002)
• [Publications] Muratani, T.: "Characterization of nociceptin/orphanin FQ-induced pain responses by the novel receptor antagonist N-(4-amino-2-methylquinolin-6-yl) -2-(4-ethylphenoxymethyl) benzamide monohydro chloride"J. Pharmacol. Exp. Ther.. 303. 424-430 (2002)
• [Publications] Mabuchi, T.: "Attenuation of neuropatic pain by nociceptin/orphanin FQ antagonist is mediated by inhibition of nitric oxide production"Eur. J. Neurosci.. (in press). (2003)
• [Publications] Minami, T.: "Characterization of the glutamatergic system for induction and maintenance of allodynia"Brain Res.. 895. 178-185 (2001)
• [Publications] Minami, T.: "Effects of Capsaicin Cream on Prostaglandin-induced Allodynia"Anesth.Analg.. 93. 419-423 (2001)
• [Publications] Minami, T.: "Characterization of EP receptor subtypes responsible for prostaglandin E_2-induced gain responses by use of EP_1 and EP_3 receptor knockout mice"Br.J.Pharmacol.. 133. 438-444 (2001)
• [Publications] Maruyama, R.: "Effects of amino acids on the amidation of polyaromatic carboxylic acids by bacillus cereus"Biosci.Biotechnol.Biochem.. 65. 1761-1765 (2001)
• [Publications] Ito, S.: "Central and peripheral roles of prostaglandins in pain and their interactions with novel neuropeptides nociceptin and nocistatin"Neuroscience Research. 41. 299-332 (2001)
• [Publications] Doi, Y.: "Central nociceptive role of prostacyclin (IP) receptor induced by peripheral inflammation"Neuro Report. 13. 93-96 (2002)
• [Publications] 伊藤誠二: "オピオイド治療 課題と新潮流 V. 痛みの最前線ノシセプチン関連(1)ノシスタチンの鎮痛作用"エルゼビア・サイエンス株式会社ミクス. 309 (2001)
• [Publications] Nakano,H.: "Effect of intrathecal nocistatin on the formalin-induced pain in mice versus that of nociceptin/orphanin FQ."J.Pharmacol.Exp.Ther.. 292. 331-336 (2000)
• [Publications] Minami,T.: "Characterization of nociceptin/orphanin FQ-induced pain in responses conscious mice : neonatal capsaicin treatment and NMDA receptor GluR ε subunit knockout mice."Neuroscience. 97. 133-142 (2000)
• [Publications] Ito,S.: "Central roles of nociceptin/orphanin FQ and nocistatin : allodynia as a model of neural plasticity."Progress Brain Res.-Nervous system plasticity and chronic pain. 129. 205-218 (2000)
• [Publications] Okuda-Ashitaka,E.: "Nocistatin : a novel neuropeptide encoded by the gene for the nociceptin/orphanin FQ precursor."Peptides. 21. 1101-1109 (2000)
• [Publications] Nishizawa,M.: "Close kinship of human 20a-hydroxysterold dehydrogenase gene with three aldo-keto reductase genes."Genes to Cells. 5. 111-125 (2000)
• [Publications] Minami,T.: "Characterization of the glutamatergic system for induction and maintenance of allodynia."Brain Research. (in press). (2001)