PREGNANCY EFFECT ON VASCULAR SMOOTH MUSCLE/ENDOTHELIUM

妊娠对血管平滑肌/内皮的影响

• 批准号: 6472568
• 负责人: CARL P WEINER
• 金额: $ 33.41万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-01 至 2006-03-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6472568
• 关键词: G protein; RNase protection assay; affinity labeling; aorta; biological signal transduction; enzyme activity; estradiol; guanosinetriphosphatase activating protein; guanosinetriphosphatases; guinea pigs; hormone regulation /control mechanism; immunocytochemistry; immunoprecipitation; laser capture microdissection; mesenteric artery; microarray technology; nuclear runoff assay; polymerase chain reaction; pregnancy circulation; progesterone; proteomics; receptor coupling; uterus; vascular endothelium; vascular smooth muscle; vasodilators

DESCRIPTION: (Provided By Applicant) Cardiovascular adaptation essential for a successful pregnancy occurs by unclear mechanisms. This proposal continues testing the hypothesis that pregnancy decreases vascular tone and contractility by enhancing sex hormone-sensitive vasodilator mechanisms. Here, we propose a new, unifying mechanism. Though estradiol and progesterone have a myriad of often nonspecific effects on components regulating blood flow, it is by altering agonist-receptor coupling to G-proteins to reduce GTPase activity that the characteristic adaptations occur. We contrast the effects of sex hormones and pregnancy on G-proteins in uterine and mesenteric arteries and the aorta to illustrate response specificity. Experiments are performed in guinea pigs over 4 years. AIM 1: Test the hypothesis that pregnancy and/or sex hormones alter G-protein activation and their mRNA expression and/or translation in a direction and magnitude consistent with the effect of pregnancy on blood flow through that artery. Photoaffinity labeling with a nonhydrolyzable, biotinylated substrate is used in subcellular fractions of arteries obtained from pregnant, nonpregnant, and estrogen or progesterone-treated castrate animals before and after NaF activation. State of the art protein chip technology will be used for some functional proteomic studies to allow micro samples. lmmunoprecipitation with specific antibody will confirm the identity of the photolabeled protein. Tissue mRNA is measured by ribonuclease protection assays and cell specific changes by RT-PCR of mRNA from pure cell samples obtained using laser capture microdissection. AIM 2: Test the hypothesis that pregnancy and/or sex hormones alters G-protein coupling to receptors of prototypic vasoactive agonists in a manner consistent with the known effect of pregnancy on blood flow through that artery. Methods described above are performed after activation of the specific receptor. AIM 3: Test the hypothesis that pregnancy and/or sex hormones decrease heterotrimeric or non-heterotrimeric GTPase activity in the arteries specifically of reproductively important organs. Photoaffinity labeling after receptor activation will be performed as described in Aim 1 except using a hydrolysable biotinylated agent. AIM 4: Test the hypothesis that pregnancy and or sex hormones decrease GTPase activity by altering GTPase activating proteins (GAPs): RGSs (regulatory G-protein subunits) for heterotrimeric G-proteins or the GAPs for small GTPases. GTP overlay and western blots are used to quantify the small GTP binding proteins and GAPs. RT-PCR of mRNA from endothelial and smooth muscle cells obtained by laser capture microdissection will localize the changes. Together, these experiments provide new understanding and avenues to elucidate pregnancy specific diseases.

描述：（由申请人提供）心血管适应 成功的怀孕是通过不清楚的机制发生的。该提议仍在继续 测试怀孕降低血管张力和收缩力的假设 通过增强性激素敏感的血管扩张剂机制。在这里，我们建议 新的，统一的机制。虽然雌二醇和孕酮有无数 通常对调节血流的成分的非特异性影响，这是 改变激动剂受体偶联与G蛋白，以减少GTPase活性 发生了特征的适应。我们对比性激素的影响 以及子宫和肠系膜动脉中G蛋白的怀孕以及主动脉 说明响应特异性。实验是在豚鼠上进行的 4年。 目标1：检验怀孕和/或性激素改变G蛋白的假设 激活及其mRNA表达和/或翻译在方向上， 幅度与怀孕对血液流动的影响一致 动脉。具有不可水解的生物素化底物的光性标记 用于从怀孕获得的动脉的亚细胞级分中 之前和 NAF激活后。最先进的蛋白质芯片技术将用于 一些功能性蛋白质组学研究以允许微样品。 lmmunoprecipitation 使用特异性抗体将确认光标记蛋白的身份。 通过核糖核酸酶保护测定和细胞特异性测量组织mRNA 使用激光捕获获得的纯细胞样品的RT-PCR变化mRNA的变化 显微解剖。 目标2：检验怀孕和/或性激素改变G蛋白的假设 以一致的方式与原型血管活性激动剂的受体耦合 妊娠对血液流过的动脉的已知影响。方法 在激活特定受体后进行上述。 目标3：检验怀孕和/或性激素降低的假设 动脉中的异三聚体或非异三聚体GTPase活性 特别是生殖重要的器官。光性标签之后 受体激活将如AIM 1所述进行，除了使用 可水解生物素化剂。 目标4：检验怀孕和性激素降低GTPase的假设 通过改变GTPase激活蛋白（GAP）的活性：RGSS（调节性 g蛋白亚基）用于异三聚体G蛋白或小的差距 GTPases。 GTP叠加层和蛋白质印迹用于量化小GTP 结合蛋白质和间隙。来自内皮和平滑肌的mRNA的RT-PCR 通过激光捕获微分解获得的细胞将定位变化。 这些实验共同提供了新的理解和途径来阐明 特定疾病。