Vasoactive Peptides that Stimulate Smooth Muscles

描述(由申请人提供)：研究计划总结在具体目标1-4下，源于我们对代谢多肽的酶的初步研究，如缓激肽(BK)、血管紧张素(Ang)和其他。血管紧张素转换酶抑制剂(激氨酶II；ACE)目前被用于治疗数以百万计的高血压患者，人们普遍认为它们的治疗作用是由于阻断了高血压肽(Ang II)的激活，同时抑制了降压肽(BK)的失活。我们的研究表明，ACE抑制剂还有其他重要的作用。它们增强BK及其抗肽酶的B2受体类似激动剂，并诱导某种形式的酶-受体串扰。我们将使用带有转染酶和受体的培养细胞，并用各种技术研究人ACE和B2受体之间的异源二聚体的形成，以及ACE抑制剂如何通过增强受体上的BK效应(间接激活)来影响这一复合体。这导致NO和花生四烯酸(前列腺素；特异性目标1)等血管介质的释放增加--在没有ACE和多肽激动剂的情况下，ACE抑制剂还通过与受体胞外区域上的锌结合五聚体序列直接激活desArg BK和desArg-Lysl-BK(特异性目标2和3)的B1受体。B1受体(特异性目标2和3)的激活会导致NO和其他心血管介质从细胞中释放的时间延长。它们对B1受体的长时间作用可能会影响胶原的合成。我们将使用钙通道阻滞剂和蛋白激酶抑制剂来研究B2(特异性靶标1)和B1受体的不同激活模式对不同转导通路的启动作用。我们将继续我们对多肽酶的研究，并了解更多关于人激氨酶I的结构，它通过从BK和Lys-BK上切割Arg来释放B1受体的多肽配体。我们的目标是结晶激氨酸酶I的活性亚基(羧基肽酶N)(特定目标4)。为了获得镁量，我们将使用一种改进的毕赤酵母细胞发酵技术。我们的实验结果将有助于了解广泛使用的ACE抑制剂如何与受体相互作用，从而影响血管反应性。

DESCRIPTION (provided by applicant): The research plans, summarized under Specific Aims 1-4, stem from our initial investigations of enzymes that metabolize peptides such as bradykinin (BK), angiotensins (Ang) and others. Inhibitors of Ang I converting enzyme (kininase II; ACE) are currently used to treat millions of patients for hypertension, and it is widely assumed that their therapeutic action is due to blocking the activation of a hypertensive peptide (Ang II) while inhibiting the inactivation of the hypotensive one (BK). Our research indicates that ACE inhibitors have other important actions as well. They potentiate BK and its peptidase-resistant analogue agonists of the B2 receptor and induce a form of an enzyme-receptor crosstalk. We shall use cultured cells with either transfected or constitutively expressed enzymes and receptors and study, with various techniques, the heterodimer formation between human ACE and the B2 receptor and how ACE inhibitors affect this complex by enhancing BK effects (indirect activation) on the receptor. This leads to an increased release of vascular mediators such as NO and arachidonic acid (prostaglandins; Specific Aim 1) -- ACE inhibitors also directly activate the B1 receptor of desArg BK and desArg-Lysl-BK (Specific Aims 2 and 3) by combining with a Znbinding pentameric sequence on an extracellular domain of the receptor, in the absence of ACE and peptide agonists. Activation of the B1 receptor (Specific Aims 2 and 3) leads to a prolonged release of NO and possibly other cardiovascular mediators from cells. Their prolonged action on the B1 receptor may affect collagen synthesis. We will investigate the initiation of different transduction pathways by the different modes of activation of the B2 (Specific Aim 1) and B1 receptors using calcium channel blockers and protein kinase inhibitors. We shall continue our study of peptidases and learn more about the structure of human kininase I, which releases the peptide ligands of B1 receptors by cleaving Arg from BK and Lys-Bk. We aim to crystallize the active subunit of kininase I (carboxypeptidase N) (Specific Aim 4). To obtain mg quantities, we will use an improved fermentation techniques with Pichia pastoris cells. Results of our experiments will help to understand how widely used ACE inhibitors interact with receptors and consequently influence vascular reactivity.