FUNCTION OF GUT PEPTIDES IN ISOLATED GUT MUSCLE CELLS

肠肽在分离的肠肌细胞中的功能

• 批准号: 2138120
• 负责人: GABRIEL M. MAKHLOUF
• 金额: $ 14.94万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-04-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2138120
• 关键词: amine oxidoreductase; calcium flux; cholinergic receptors; enzyme activity; enzyme inhibitors; gastrointestinal function; guinea pigs; human tissue; inositol phosphates; laboratory rabbit; laboratory rat; muscle relaxation; myenteric plexus; neuropeptide receptor; neurotransmitter receptor; nitric oxide; opioid receptor; protein kinase; receptor coupling; second messengers; serotonin receptor; smooth muscle; vasoactive intestinal peptide

The objective of this proposal is to characterize the receptors, signalling pathways and cellular mechanisms involved in relaxation of gastric smooth muscle by transmitters of the myenteric plexus. The focus is on peptide receptors coupled to multiple interactive pathways that mediate or modulate relaxation. The experimental approach exploits a technique for selective, site-directed receptor protection that allows each pathway to be examined separately and is based on the discovery of an important link between the myenteric neuropeptide, VIP release; in turn, VIP stimulates (i.e., re-generates) NO in target muscle cells. The link to VIP confers receptor specificity on NO signalling and engages both limbs of the cyclic nucleotide system that sustains relaxation. Accordingly, the Specific Aims of the proposal are: (1) to characterize receptors for NPY, opioid peptides, acetylcholine and 5-HT, which are coupled to IP, generation and IP-dependent Ca release via one receptor subtype, and to increase (5-HT and muscarinic M) or decrease (NPY Y, opioid mu, delta, kappa, and muscarinic M) in cAMP via a distinct receptor subtype; (2) to characterize receptors for VIP and the myenteric homologous peptide, PACAP, in isolated gastric muscle cells which are coupled to stimulation of adenylate cyclase and NO-dependent stimulation of guanylate cyclase; our continuing studies in plasma membranes derived from isolated muscle cells have demonstrated the presence of a membrane- bound, constitutive, G protein-coupled, Ca2+/calmodulin-sensitive NO synthase which is specifically activated by VIP and PACAP; and (3) to identify the loci of regulatory phosphorylation by cAMP- and cGMP- dependent protein kinase (cA-kinase and cG-kinase) which mediate relaxation. In comparative studies using cA-kinase and cG-kinase inhibitors including PKI (6-22) amide, a highly specific inhibitor of cA-kinase, we have identified concentrations of inhibitors that selectively block cA-kinase activity (H-89, Rp-cAMPS) and cG-kinase activity (the indole carbazole KT5823). This development set the stage for identification of events in the signalling cascade that, based on preliminary results, are preferentially regulated by cA-kinase (IP3 generation, IP3-dependent Ca2+ release) and cG-kinase (sarcoplasmic Ca uptake) or equally regulated by both kinases (Ca/calmodulin sensitivity of MLC kinase and MLC phosphorylation). The combined approach embodied the three aims should advance our understanding of the cellular mechanisms mediating smooth muscle relaxation.

该建议的目的是表征受体， 信号通路和蜂窝机制与放松 通过肌丛的发射器的胃平滑肌。 重点 在肽受体上，与多种互动途径相连 介导或调节放松。 实验方法利用了 选择性，定向的受体保护技术，允许 每个要分开检查的途径，并基于发现 肌植物神经肽，VIP释放之间的重要联系；在 转弯，VIP刺激目标肌肉细胞中的NO（即重新生成）。 这 链接到VIP的链接在没有信号和参与的情况下赋予受体特异性 循环核苷酸系统的两个肢体都持续了松弛。 因此，该提案的具体目的是：（1）表征 NPY，阿片类肽，乙酰胆碱和5-HT的受体，它们是 与IP，发电和IP依赖性CA释放相结合 亚型，并增加（5-HT和毒蕈碱M）或减少（npy y， 阿片类Mu，Delta，Kappa和Muscarinic M）在营地中通过一个独特的 受体亚型； （2）表征VIP和肌室的受体 在孤立的胃肌细胞中的同源肽，PACAP 与刺激腺苷酸环化酶和无依赖性刺激相结合 鸟甘酸环化酶的;我们在衍生的质膜中的持续研究 从孤立的肌肉细胞中表明存在膜 结合，组成型，G蛋白耦合，Ca2+/对钙调蛋白敏感的NO 合成酶特异性地通过VIP和PACAP激活； （3）到 确定cAMP和CGMP-调节磷酸化的基因座 依赖性蛋白激酶（CA-激酶和CG-激酶）介导 松弛。在使用CA-激酶和CG激酶的比较研究中 包括PKI（6-22）酰胺在内的抑制剂，这是一种高度特异性的抑制剂 CA-激酶，我们已经确定了抑制剂的浓度 有选择地阻止CA-激酶活性（H-89，RP训练营）和CG-激酶 活性（吲哚甲甲苯KT5823）。 这一发展奠定了舞台 为了识别信号级联中的事件，该事件基于 初步结果优先通过CA-激酶调节（IP3 产生，依赖IP3的Ca2+释放）和CG-激酶（肌质Ca 摄取）或通过两种激酶（CA/钙调蛋白敏感性）平均调节 MLC激酶和MLC磷酸化）。 组合方法体现 这三个目标应该提高我们对细胞的理解 介导平滑肌松弛的机制。