Cloning of nocistatin receptor and development of new aanalgesics

我们之前从牛脑中分离出一种名为Noc/OFQ的肽，作为孤儿阿片样受体ROR-C的内源性配体。我们证明，鞘内（i.t）给药Noc/OFQ到有意识的小鼠诱导痛觉过敏和触觉痛（异常性疼痛）。我们发现了一种新的七肽，它可以阻断Noc/OFQ引起的疼痛反应，与Noc/OFQ相同的前体，并将其命名为nocistatin。去皮抑素通过假定的去皮抑素受体发挥作用。为了开发新型镇痛药，我们试图表征Noc/OFQ与去皮抑素的疼痛传递，并克隆去皮抑素受体cDNA，得到以下结果。1)根据最近开发的Noc/OFQ拮抗剂的结果，nocistatin仅阻断Noc/OFQ受体介导的应答。2) Nocistatin具有广谱的镇痛药；除Noc/OFQ外，还能减轻PGE2、PGF2α和足底福尔马林所致的疼痛。3) Nocistatin以gtp依赖的方式激活小鼠脑和脊髓膜中的腺苷酸环化酶，并增加cAMP。4)为了开发一种新的镇痛药，我们根据去皮抑素的氨基酸序列合成了多种多肽，并通过镇痛效果和cAMP增加能力对其进行了评价。5)利用哺乳动物细胞系和非洲爪蟾卵母细胞，利用表达克隆方法，从小鼠脑cDNA文库中克隆了去皮抑素受体cDNA。由于nocistatin受体的克隆是开发nocistatin拮抗剂作为新型镇痛药的必要条件，在克隆nocistatin受体cDNA的同时，也尝试了光亲和标记法纯化nocistatin受体。

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.