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Cloning of nocistatin receptor and development of new aanalgesics

诺西他汀受体的克隆及新型镇痛药的开发

基本信息

批准号：
11558093
负责人：
ITO Seiji
金额：
$ 6.34万
依托单位：
Kansai Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

We previously isolated a peptide termed as nociceptin/orphanin FQ (Noc/OFQ) from bovine brain as an endogenous ligand for the orphan opioid-like receptor ROR-C.We demonstrated that intrathecal (i.t.) administration of Noc/OFQ into conscious mice induced hyperalgesia and tactile pain (allodynia). We found a novel heptadecapeptide which blocks the Noc/OFQ-evoked pain responses, on the same precursor as Noc/OFQ and named it nocistatin. Nocistatin exerts actions through the postulated nocistatin receptor. To develop novel analgesics, we tried to characterize pain transmission involving Noc/OFQ and nocistatin and clone cDNA of nocistatin receptor and obtained following results.1) Based on the results obtained by a Noc/OFQ antagonist developed recently, nocistatin only blocked the responses mediated by the Noc/OFQ receptor. 2) Nocistatin has a broad spectrum of analgesics ; it alleviated the pain induced by i.t. PGE2, i.t. PGF2α and intraplantar formalin in addition to i.t. Noc/OFQ.3) Nocistatin activated adenylate cyclase in the membrane prepared from mouse brain and spinal cord in a GTP-dependent manner and increased cAMP.4) In order to develop a new analgesic, we synthesized various peptides on the basis of amino acid sequence of nocistatin and evaluated them by analgesic effects and capability of cAMP increase. 5) We have tried to clone a nocistatin receptor cDNA from a cDNA library prepared from mouse brain with expression-cloning methods by use of mammalian cell lines and Xenopus oocytes. Since the cloning of nocistatin receptor is a requisite for the development of nocistatin antagonists as new analgesics, purification of the nocistatin receptor has been also tried by photoaffinity labeling, in parallel with the cloning of the nocistatin receptor cDNA.

我们先前从牛脑中分离出一种称为伤害素/孤啡肽FQ(Noc/OFQ)的多肽，作为孤儿阿片样受体ROR-C的内源性配体。清醒小鼠注射Noc/OFQ可引起痛觉过敏和触觉痛(痛觉异常)。我们在与Noc/OFQ相同的前体上发现了一种新的七肽，它可以阻断Noc/OFQ引起的疼痛反应，并将其命名为nocistatin。Nocistatin通过假设的nocistatin受体发挥作用。为了开发新的止痛药，我们试图研究涉及Noc/OFQ和nocistatin的痛觉传递，并克隆了nocistatin受体的cDNA，得到了以下结果：1)根据新近开发的Noc/OFQ拮抗剂的结果，nocistatin只阻断了Noc/OFQ受体介导的反应。2)诺西他汀具有广谱的止痛药作用；它能减轻I.T.引起的疼痛。PGE2，I.T.前列腺素F2α和足底福尔马林及I.T.NOC/OFQ。3)Nocistatin以GTP依赖的方式激活小鼠脑和脊髓细胞膜上的腺苷环化酶，并使cAMP升高。4)为了开发新的止痛药，我们根据nocistatin的氨基酸序列合成了各种多肽，并从镇痛效果和cAMP升高能力两个方面进行了评价。5)利用哺乳动物细胞系和非洲爪哇卵母细胞，采用表达克隆的方法，从小鼠脑组织的cDNA文库中克隆了nocistatin受体基因。由于克隆nocistatin受体是开发nocistatin拮抗剂作为新的止痛药的必要条件，因此在克隆nocistatin受体基因的同时，还尝试了光亲和标记法纯化nocistatin受体。