Vasoactive Peptides that Stimulate Smooth Muscles

描述（由申请人提供）：在特定目标1-4下总结的研究计划源于我们对代谢肽（如缓激肽（BK）、血管紧张素（Ang）等）的酶的初步研究。 血管紧张素I转化酶（激肽酶II; ACE）的抑制剂目前用于治疗数百万高血压患者，并且人们广泛认为它们的治疗作用是由于阻断高血压肽（Ang II）的活化，同时抑制高血压肽（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B2受体的肽酶抗性类似物激动剂，并诱导一种形式的酶-受体串扰。 我们将使用转染或组成型表达的酶和受体的培养细胞，用各种技术研究人ACE和B2受体之间的异源二聚体形成，以及ACE抑制剂如何通过增强BK效应（间接激活）影响该复合物。 这导致血管介质如NO和花生四烯酸（Escherlandins;特异性目的1）的释放增加-- ACE抑制剂也通过在不存在ACE和肽激动剂的情况下与受体的胞外结构域上的Zn结合五聚体序列结合而直接激活desArg BK和desArg-Lysl-BK的B1受体（特异性目的2和3）。 B1受体的激活（特定目的2和3）导致NO和可能的其他心血管介质从细胞中的释放延长。 它们对B1受体的长期作用可能会影响胶原蛋白的合成。 我们将使用钙通道阻滞剂和蛋白激酶抑制剂，通过B2（特异性Aim 1）和B1受体的不同激活模式研究不同转导途径的启动。 我们将继续研究肽酶，并进一步了解人激肽酶I的结构，它通过从BK和Lys-Bk上裂解Arg释放B1受体的肽配体。我们的目标是结晶激肽酶I的活性亚基（羧肽酶N）（具体目标4）。 为了获得mg量，我们将使用改进的巴斯德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.