ENDOTHELIUM-DEPENDENT RELAXATION--G PROTEIN REGULATION

关于释放过程中涉及的信号转导知之甚少 血管内皮源性松弛因子(S)。各种各样的G蛋白是 已知在偶联神经递质受体和 激素对细胞内效应系统的影响。百日咳毒素，它 使某些G蛋白失活，抑制内皮依赖性 α2-肾上腺素能和5-羟色胺能受体引起的松弛 刺激，也包括猪的血小板聚集所引起的刺激 冠状动脉。对一氧化氮的松弛与内皮依赖性 对缓激肽、二磷酸腺苷或A23187的松弛作用不受影响 被毒素毒害。在有再生内皮的冠状动脉中(如下 体内剥脱)，内皮依赖的松弛对α2- 肾上腺素能和5-羟色胺能刺激以及血小板聚集 减少，而对其他激动剂的反应不变。 此外，在这些动脉中，百日咳毒素对血管没有影响。 剩下的放松。这些结果表明，某些内皮细胞- 依赖的松弛是由百日咳毒素的激活所介导的- 内皮细胞中的敏感G蛋白。此外，在再生方面 内皮细胞中可能存在选择性丢失的这种G蛋白依赖性 可能使血管容易发生血管痉挛的机制， 动脉粥样硬化和血管闭塞。本提案的目的是 在离体猪冠状动脉和离体猪冠状动脉中进行测定 内皮细胞(原代、培养和再生)：1)鉴定 与内皮5-羟色胺能偶联的百日咳毒素敏感G蛋白 和α2-肾上腺素能受体(使用SDS-PAGE和Western印迹分析)， 2)表征这种G-激活引起的亚细胞效应 蛋白质(通过分析酶活性、钙通量和 释放内皮细胞介质)，3)确定G- 增殖内皮细胞中的蛋白质依赖机制 (通过分析G蛋白功能的变化，或在 内皮受体，或G蛋白与血管内皮细胞结合的能力 受体或激活内皮)，以及4)分析可能的 功能障碍的原因(通过分析已知的潜在机制 破坏G蛋白功能)。这些实验将进一步推动我们的 了解血管功能的正常生理学，希望 对可能引发血管病变的机制提供重要的见解 疾病。

Little is known concerning the signal transduction involved in the release of endothelium-derived relaxing factor(s). A variety of G-proteins are known to have key roles in coupling receptors for neurotransmitters and hormones to intracellular effector systems. Pertussis toxin, which inactivates certain G-proteins, inhibits the endothelium-dependent relaxations evoked by alpha2-adrenergic and serotonergic receptor stimulation, and also those evoked by aggregating platelets in porcine coronary arteries. Relaxations to nitric oxide and endothelium-dependent relaxations to bradykinin, adenosine diphosphate or A23187 were unaffected by the toxin. In coronary arteries with regenerated endothelium (following in vivo denudation), the endothelium-dependent relaxations to alpha2- adrenergic and serotonergic stimulation, and also to aggregating platelets are reduced, whereas responses to the other agonists are unchanged. Moreover, in these arteries, pertussis toxin is without effect on the remaining relaxations. These results suggest that certain endothelium- dependent relaxations are mediated by activation of a pertussis toxin- sensitive G-protein in the endothelial cells. Furthermore, in regenerating endothelial cells there may be a selective loss of this G-protein-dependent mechanism, which may predispose the blood vessel to vasospasm, atherosclerosis and vascular occlusion. The aim of the present proposal is to determine in isolated porcine coronary arteries, and in isolated porcine endothelial cells (native, cultured and regenerated): 1) the identify of the pertussis toxin-sensitive G-protein coupled to endothelial serotonergic and alpha2-adrenergic receptors (using SDS-PAGE and Western blot analysis), 2) characterize the subcellular effects arisen from activation of this G- protein (by analyzing alterations in enzyme activity, calcium fluxes, and release of endothelial mediators), 3) identify the dysfunction in this G- protein-dependent mechanism that occurs in proliferating endothelial cells (by analyzing alterations in the function of the G-protein, or in the endothelial receptors, or in the ability of the G-protein to couple to the receptors or to activate the endothelium), and 4) analyze the possible cause of the dysfunction (by analyzing potential mechanisms known to disrupt G-protein function). These experiments will further our understanding of the normal physiology of vascular function and hopefully provide important insight into the mechanisms that may initiate vascular disease.