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2002 Fiscal Year Final Research Report Summary

Analysis of receptor and processing enzyme of pain regulated peptide, nocistatin using bioluminescence.

Research Project

Project/Area Number 13670132
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field General medical chemistry
Research InstitutionKansai Medical University

Principal Investigator

OKUDA-ASHITAKA Emiko  Kansai Medical University, Factory of Medicine, Lecturer, 医学部, 講師 (50291802)

Co-Investigator(Kenkyū-buntansha) ITO Seiji  Kansai Medical University, Factory of Medicine, Professor, 医学部, 教授 (80201325)
Project Period (FY) 2001 – 2002
KeywordsNocistatin / Nociceptin / orphanin FQ / Pain transmission / BRET / Processing / Propotein covertase
Research Abstract

We identified a neuropeptide and named it "nocistatin (NST)". NST is derived from the same precursor protein as nociceptin/orphanin FQ (N/OFQ), while NST exhibits antagonism against Noc/OFQ-actions. Both NST and N/OFQ are co-localized in the superficial laminae of mouse spinal dorsal horn, but these peptides induce several functions via the respective receptors. We carried out a photaffiniiy labeling approach for identification of the NST receptor, and we attempt an intra-molecular BRET system for monitoring dynamic biological process of the production of NST and N/OFQ in the living cells.
1. NST receptor: Mouse spinal cord membranes were incubated with a ^<125>I-labeled photoaffinity NST analogue, and this analogue bound approximately 33-kDa protein. The 33-kDa protein labeling was decreased by NST but not by N/OFQ. The photoaffinity-labeled protein disappeared after GTPγS treatment, suggesting that the NST-specific photolabeled protein may be associated with a GTP-binding protein.
2. P … More eptide-production: We constructed a fusion protein (Rluc-GFP) covalently linking luciferase (Renilla luciferase; Rluc) to Aequorea GFP as an intra-molecular BRET partner, and we inserted constructs of NST and Noc/OFQ containing a proteolytic cleavage motif (Lys-Arg) within Rluc-GFP. The bioluminescence spectrum of the fusion protein with DeepBlueC as a substrate was bimodal (λmax= 400 nm (Rluc) and 510 nm (GFP)), indicating that the excited-state energy of Rluc transfers to GFP (in short, BRET). Furthermore, we developed a novel BRET system using a secreated luciferase, which enabled us to follow a real-time and continuous BRET in culture medium. The BRET had the desired profile for monitoring the protein processing of NST and N/OFQ in intact cells, and the change in BRET signals quantified the processing of the protein. Co-transfection of the BRET probe with proprotein convertases showed that PC1 was involved in the processing of Lys-Arg between NST and N/OFQ and furin in that of Lys-Arg upstream of NST. Less

  • Research Products

    (12 results)

All Other

All Publications (12 results)

  • [Publications] Minami, T.: "Characterization of the glutamatergic system for induction and maintenance allodynia"Brain.Res.. 895. 178-185 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Ito, S.: "Central and peripheral roles of prostaglandins in pain and their interactions with novel neuropeptides nociceptin and nocistatin"Neuroscience Res.. 41. 299-322 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Okuda-Ashitaka, E.: "Pain transmission regulated by novel neuropeptides nocistatin and nociceptin/orphanin FQ"J.Biol.Macromol.. 2. 3-10 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Muratani, T.: "Characterization of nociceptin / orphanin FQ-induced pain responses by the novel receptor antagonist JTC-801"J.Pharmacol.Exp.Ther.. 303. 424-430 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Mabuchi, T.: "Attenuation of neuropathic pain by nociceptin/orphanin FQ antagonist is mediated by inhibition of nitric oxide production"Eur.J.Neurosci.. 17. 1384-1392 (2003)

    • Description
      「研究成果報告書概要(和文)」より
  • [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"エルセビア・サイエンス株式会社ミクス. 309 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Zeilhofofer, H.U.: "Proceeding of the 10^<th> World Congress on Pain Nociceptin, Nocistatin and Pain"International Association for the Study of Pain Press (In press). (2003)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Minami, T.: "Characterization of the glutamatergic system for induction and maintenance allodynia."Brain. Res.. 895. 178-185 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Ito, S.: "Central and peripheral roles of prostaglandins in pain and their interactions with novel neuropeptides nociceptin and nocistatin."Neuroscience Res.. 41. 299-322 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Okuda-Ashitaka, E.: "Pain transmission regulated by novel neuropeptides nocistatin and nociceptin/orphanin FQ."J. Biol. Macromol.. 2. 3-10 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Muratani, T.: "Characterization of nociceptin / orphanin FQ-induced pain responses by the novel receptor antagonist JTC-801."J. Pharmacol. Exp. Ther.. 303. 424-430 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Mabuchi, T.: "Attenuation of neuropathic pain by nociceptin/orphanin FQ antagonist is mediated by inhibition of nitric oxide production."Eur. J. Neurosci.. 17. 1384-1392 (2003)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 2004-04-14  

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