PREGNANCY EFFECT ON VASCULAR SMOOTH MUSCLE/ENDOTHELIUM

描述：（由申请人提供）心血管适应对于 成功怀孕的机制尚不清楚。这项建议继续 验证怀孕会降低血管张力和收缩力的假设 通过增强对性敏感的血管扩张机制。在这里，我们提出一个 新的统一机制。虽然雌二醇和孕酮有无数的 通常对调节血流的成分产生非特异性作用， 改变激动剂-受体与G蛋白的偶联，以降低GT3活性， 发生了特征性的适应。我们将性激素的作用 和妊娠对子宫和肠系膜动脉以及主动脉中G蛋白的影响， 说明反应特异性。实验是在豚鼠身上进行的， 4年 目的1：验证妊娠和/或性激素改变G蛋白的假设 激活和它们的mRNA表达和/或翻译， 这与怀孕对血液流动的影响一致， 动脉用不可水解的生物素化底物进行光亲和标记 用于从孕妇获得的动脉的亚细胞部分， 非妊娠动物和雌激素或孕酮治疗的去势动物， NaF活化后。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，以允许微量样品。免疫沉淀 用特异性抗体将确认光标记的蛋白质的身份。 组织mRNA通过核糖核酸酶保护测定和细胞特异性免疫组织化学测定来测量。 使用激光捕获获得的纯细胞样品中mRNA的RT-PCR变化 显微解剖 目的2：验证妊娠和/或性激素改变G蛋白的假设 与原型血管活性激动剂的受体以一致的方式偶联 怀孕对动脉血流的影响方法 在特定受体活化后进行上述的方法。 目的3：检验怀孕和/或性激素减少的假设 动脉中的异源三聚体或非异源三聚体GT3活性 特别是生殖器官的损伤光亲和标记后 受体活化将如目的1所述进行，除了使用 可水解生物素化试剂。 目的4：验证妊娠和/或性激素降低GT3的假设 通过改变GT3活化蛋白（GAP）：RGSs（调节 对于异源三聚体G-蛋白，GAPs可以是G-蛋白亚基），或者对于小G-蛋白，GAPs可以是GAPs。 GTP酶。GTP覆盖和蛋白质印迹用于定量小GTP 结合蛋白和GAP。内皮和平滑肌mRNA的RT-PCR 通过激光捕获显微切割获得的细胞将定位变化。 总之，这些实验提供了新的理解和途径来阐明 妊娠特殊疾病

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.