PREGNANCY EFFECT ON VASCULAR SMOOTH MUSCLE/ENDOTHELIUM

描述：(由申请人提供)心血管适应对 成功怀孕的机制尚不清楚。这项提议继续下去 检验怀孕会降低血管张力和收缩能力的假说 通过加强性激素敏感的血管扩张剂机制。在这里，我们提出一种 新的、统一的机制。虽然雌二醇和黄体酮有无数的 它对调节血液流动的成分通常没有特定的影响，它是通过 改变G蛋白的激动剂-受体偶联以降低GTP酶活性 这种特有的适应就会发生。我们对比了性激素的作用 和妊娠对子宫动脉、肠系膜动脉和主动脉G蛋白的影响 说明响应的特异性。实验是在豚鼠身上进行的 四年了。 目的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/或翻译的方向和 大小与怀孕对血流的影响相一致 动脉。非水解性生物素化底物的光亲和标记 用于从怀孕中获取的动脉的亚细胞部分， 未怀孕，雌激素或黄体酮处理的去势动物之前和 氟化钠活化后。最先进的蛋白质芯片技术将用于 一些功能蛋白质组学研究，以允许微量样本。免疫沉淀 与特异性抗体的结合将确认光标记蛋白的身份。 用核糖核酸酶保护法和细胞特异性方法测定组织mRNA值。 激光捕获法获取的纯细胞样品中mRNA的RT-PCR变化 显微解剖。 目的2：检验怀孕和/或性激素改变G蛋白的假设 以一致的方式偶联到原型血管活性激动剂的受体 已知的怀孕对通过该动脉的血流的影响。方法 上述是在激活特定受体之后进行的。 目标3：检验怀孕和/或性激素下降的假设 动脉中异源三聚体或非异源三聚体GTP酶的活性 具体地说是指具有生殖重要性的器官。光亲和标记后 受体激活将按照目标1中所述执行，除非使用 可水解性生物素化试剂。 目的4：检验怀孕和/或性激素降低GTP酶的假设 通过改变GTP酶激活蛋白的活性(GAP)：RGSS(调节 G蛋白亚基)或小G蛋白的缺口 GTP酶。GTP重叠和蛋白质印迹用于对小的GTP进行量化 结合蛋白质和缺口。血管内皮细胞和血管内皮细胞mRNA的RT-PCR检测 通过激光捕获显微切割获得的细胞将定位这些变化。 总之，这些实验提供了新的理解和途径来阐明 妊娠特有的疾病。

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.