ENDOTHELIUM-DEPENDENT RELAXATION--G PROTEIN REGULATION

内皮依赖性松弛——G蛋白调节

• 批准号: 3472819
• 负责人: NICHOLAS A FLAVAHAN
• 金额: $ 12.21万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3472819
• 关键词: adrenergic receptor; artery occlusion; atherosclerosis; biological signal transduction; nitric oxide; pertussis toxin; serotonin receptor; swine; tissue /cell culture; vascular endothelium; vasoconstriction; vasodilators; vasospasm; western blottings

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.

关于释放中涉及的信号转导知之甚少 内皮衍生的舒张因子。 多种G蛋白是 已知在神经递质偶联受体中起关键作用， 细胞内效应系统的激素。 百日咳毒素，其中 使某些 G 蛋白失活，抑制内皮依赖性 α2-肾上腺素能和血清素能受体引起的放松 刺激，以及猪体内血小板聚集引起的刺激 冠状动脉。 对一氧化氮和内皮依赖性的放松 对缓激肽、二磷酸腺苷或 A23187 的松弛不受影响 由毒素。 在具有再生内皮的冠​​状动脉中（以下 体内剥脱），内皮依赖性α2-松弛 肾上腺素能和血清素能刺激，以及聚集血小板 减少，而对其他激动剂的反应则不变。 而且，在这些动脉中，百日咳毒素对动脉没有影响。 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 内皮细胞中的敏感G蛋白。 此外，在再生 内皮细胞可能会选择性丧失这种 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 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) 的鉴定 与内皮血清素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 和蛋白质印迹分析）， 2) 表征该 G-激活所产生的亚细胞效应 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- 的功能障碍 增殖内皮细胞中发生的蛋白质依赖性机制 （通过分析 G 蛋白功能的改变，或 内皮受体，或 G 蛋白与内皮受体偶联的能力 受体或激活内皮），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 了解血管功能的正常生理学并希望 提供了对可能启动血管的机制的重要见解 疾病。