ENDOTHELIUM-DEPENDENT RELAXATION--G PROTEIN REGULATION

关于参与释放的信号转导知之甚少 内皮源性舒张因子。 多种G蛋白 已知在神经递质受体偶联中起关键作用， 激素到细胞内效应系统。 百日咳毒素， 使某些G蛋白失活，抑制内皮依赖性 α_2-肾上腺素能和肾上腺素能受体引起的舒张 刺激，以及由猪血小板聚集引起的刺激 冠状动脉 一氧化氮和内皮依赖性舒张 缓激肽、二磷酸腺苷或A23187的舒张作用不受影响 毒素的影响 在具有再生内皮的冠状动脉中（以下 在体内剥脱），内皮依赖性舒张α 2- 肾上腺素能和肾上腺素能刺激，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- 依赖性松弛是由百日咳毒素的激活介导的， 敏感的G蛋白。 此外，在再生 内皮细胞中可能存在这种G蛋白依赖性的选择性丧失， 机制，这可能使血管易于发生血管痉挛， 动脉粥样硬化和血管闭塞。 本建议的目的是 以测定离体猪冠状动脉和离体猪 内皮细胞（天然的、培养的和再生的）：1）鉴定 百日咳毒素敏感性G蛋白偶联内皮细胞粘附素 和α 2-肾上腺素能受体（使用SDS-PAGE和Western印迹分析）， 2)表征由这种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 内皮介质的释放），3）确定该G- 在增殖的内皮细胞中发生的蛋白质依赖性机制 (by分析G蛋白功能的改变，或 内皮受体，或在G蛋白的能力，以耦合到 受体或激活内皮），和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）。 这些实验将进一步促进我们的 了解血管功能的正常生理学， 提供了重要的洞察机制，可能启动血管 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.