ENDOTHELIUM-DEPENDENT RELAXATION--G PROTEIN REGULATION

内皮依赖性松弛——G蛋白调节

基本信息

批准号：
2221017
负责人：
NICHOLAS A FLAVAHAN
金额：
$ 12.8万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
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/2221017
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

项目摘要

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.

关于释放涉及的信号转导的知之甚少内皮衍生的放松因子（S）。多种G蛋白是已知在神经递质和激素到细胞内效应系统。百日咳毒素，这灭活某些G蛋白，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，也是通过在猪中聚集血小板引起的刺激冠状动脉。一氧化氮和内皮依赖性的放松对心动激肽，腺苷二磷酸或A23187的放松不受影响由毒素。在具有再生内皮的冠状动脉中在体内剥夺），依赖于α2-的内皮依赖性松弛肾上腺素和血清素能刺激，也聚集了血小板减少了，而对其他激动剂的反应不变。此外，在这些动脉中，百日咳毒素对剩余的放松。这些结果表明某些内皮依赖的松弛是通过激活百日咳毒素的激活来介导的内皮细胞中敏感的G蛋白。此外，再生内皮细胞可能会选择性丧失该G蛋白依赖性机制，可能使血管易于血管痉挛，动脉粥样硬化和血管阻塞。本提案的目的是在孤立的猪冠状动脉和孤立的猪中确定内皮细胞（天然，培养和再生）：1）百日咳毒素敏感的G蛋白与内皮血清素能的耦合和α2-肾上腺素能受体（使用SDS-PAGE和Western印迹分析）， 2）表征由于该G-激活而产生的亚细胞效应蛋白质（通过分析酶活性的改变，钙通量和释放内皮介体），3）确定该G-中的功能障碍蛋白质依赖性机制发生在增殖的内皮细胞中（通过分析G蛋白功能的变化或内皮受体，或G蛋白夫妇搭配的能力受体或激活内皮），4）分析可能功能障碍的原因（通过分析已知的潜在机制破坏G蛋白功能）。这些实验将进一步了解血管功能的正常生理学，并希望提供有关可能引发血管的机制的重要见解疾病。