ACTIVATION OF PROTEIN KINASE C IN VASCULAR SMOOTH MUSCLE

蛋白激酶C是一种磷脂和钙依赖的酶，是 被认为在信号转导过程中起重要作用 激活磷脂酶C并释放二酰甘油的激素。 最近的研究表明，蛋白激酶C的激活 可能在维持补药(持续)状态方面发挥核心作用 血管平滑肌中可见的收缩。初步研究 已经证明血管扩张剂、缓激肽和 血管收缩药，血管紧张素II，都会增加细胞内 钙和二酰甘油的形成与酶的激活一致 磷脂酶C对膜结合有不同的影响 培养的血管平滑肌细胞中蛋白激酶C的活性 (Cvsmc)。这表明，蛋白激酶C的调节 活动可能是荷尔蒙调节的一个基本机制 血管反应性。本提案的目标是 进一步探讨解释这些差异的机制 在血管紧张素II和缓激肽之间。最有可能的解释是 这些激素作用于不同的磷脂池 释放二酰甘油(或其他因素)，这些因素具有不同的 激活蛋白激酶C的能力。 这项提议将使用定量酶分析以及 内源性磷脂的放射性标记研究 血管紧张素II和缓激肽对不同磷脂的影响 泳池。此外，气相色谱、镀银薄层 层析和高压液相色谱会 用于测定释放的二甘油的分子种类 在接触了这些荷尔蒙之后。最后，对荷尔蒙敏感的 将制备质膜制剂，以允许进一步 对调节酶活性的因素的表征 胞浆和膜结合蛋白激酶C的结果 这些研究将提供重要的新信息 缓激肽的跨膜信号系统，以及 激素调节蛋白激酶C活性的机制(S) 在血管平滑肌细胞中。最终，这些研究将 更好地理解荷尔蒙的细胞基础-- 诱导血管收缩和血管扩张，并将提供新的 对血管反应性的生理学和 高血压的病理生理学。

Protein kinase C, a phospholipid- and calcium-dependent enzyme, is thought to be important in the signal transduction process of hormones which activate phospholipase C and release diacylglycerol. Recent studies have suggested that activation of protein kinase C may play a central role in maintaining the tonic (sustained) state of contraction seen in vascular smooth muscle. Preliminary studies have demonstrated that the vasodilator, bradykinin and the vasoconstrictor, angiotensin II, both increase intracellular calcium and diacylglycerol formation consistent with activation of phospholipase C, yet have different effects on membrane-bound protein kinase C activity in cultured vascular smooth muscle cells (cvsmc). This suggests that regulation of protein kinase C activity may be a fundamental mechanism whereby hormones modulate vascular reactivity. The goal of the present proposal is to further explore the mechanisms which account for these differences between angiotensin II and bradykinin. The most likely explanation is that these hormones act on different phospholipid pools to release diacylglycerols (or other factors) which possess different capacities to activate protein kinase C. The studies outlined in this proposal will use quantitative enzyme assays as well as radiolabelling of endogenous phospholipids to characterize the effects of angiotensin II and bradykinin on different phospholipid pools. In addition, gas chromatography, argentation thin layer chromatography, and high pressure liquid chromatography will be used to determine the molecular species of diacyglycerol released after exposure to these hormones. Finally, a hormone-sensitive plasma membrane preparation will be prepared to allow further characterization of the factors which regulate the activity of cytosolic and membrane-bound protein kinase C. The results of these studies will provide significant new information on the transmembrane signalling system for bradykinin, and the mechanism(s) by which hormones regulate protein kinase C activity in vascular smooth muscle cells. Ultimately, these studies will provide a better understanding of the cellular basis of hormone- induced vasoconstriction and vasodilitation and will provide new insights into the physiology of vascular reactivity and the pathophysiology of hypertension.