BRADYKININ SIGNALING IN REGULATION OF eNOS

说明书(申请人提供)：内皮源性一氧化氮(NO)在调节血管扩张状态中起关键作用，因此在调节血压中起重要作用。此外，由于NO调节这一过程和许多其他重要的血管过程，血管中NO产生的异常被认为与某些血管疾病的发病有关，如动脉粥样硬化、糖尿病和高血压。在内皮细胞中，一氧化氮是在内皮型一氧化氮合酶(ENOS)的催化下，通过氧化L精氨酸而合成的。最近几个实验室的研究已经确定了Ser-1179磷酸化和Thr-497去磷酸化在激动剂调节内皮细胞eNOS活性中的作用。本提案的初步研究部分提供的数据为eNOS在Ser-617和Ser-635处的另外两个磷酸化位点提供了证据。我们的初步数据显示，在培养的内皮细胞中，这两个位点在eNOS激活激动剂缓激肽(BK)、ATP和血管内皮生长因子的刺激下被瞬时磷酸化。 本提案的主要目的是验证一种假设，即血管内皮细胞中eNOS的活性部分受BK刺激的Ser-617和Ser-635处的酶的磷酸化调节。另一个目的是阐明Ser-617和Ser-635的磷酸化改变酶催化活性的分子机制(S)，并检验Ser-617或Ser-635的磷酸化改变eNOS蛋白与小窝蛋白-1、缓激素B2受体或热休克蛋白90的相互作用的假说。最后，我们将检查某些病理生理条件，如高血糖或氧化应激，是否改变基础或BK刺激的eNOS在Ser-617或Ser-635的磷酸化。

DESCRIPTION (provided by applicant): Endothelium-derived nitric oxide (NO) has a crucial role in regulation of the state of vasodilation of blood vessels and hence in regulation of blood pressure. Furthermore, because NO regulates this and a number of other important vascular processes, abnormalities in vascular NO production are thought to contribute to the pathogenesis of certain vascular disorders such as those of atherosclerosis, diabetes, and hypertension. NO is synthesized in endothelial cells by oxidation of L-arginine in a reaction catalyzed by the enzyme, endothelial nitric oxide synthase (eNOS). Recent investigations in several laboratories have established a role for Ser-1179 phosphorylation and Thr-497 dephosphorylation in agonist regulation of eNOS activity in endothelial cells. Data presented in the Preliminary Studies section of this proposal provide evidence for two additional sites of eNOS phosphorylation at Ser-617 and Ser-635. Our preliminary data shows that these two sites are transiently phosphorylated in cultured endothelial cells in response to stimulation with the eNOS-activating agonists bradykinin (BK), ATP, and vascular endothelial growth factor. The principal aim of the present proposal is to examine the hypothesis that eNOS activity in vascular endothelial cells is regulated in part by BK-stimulated phosphorylation of the enzyme at Ser-617 and Ser-635. Additional aims are to elucidate the molecular mechanism(s) by which phosphorylation at Ser-617 and Ser-635 alters enzyme catalytic activity and to test the hypothesis that phosphorylation at Ser-617 or Ser-635 alters eNOS protein-protein interactions with caveolin-1, the bradykinin B2 receptor, or heat shock protein 90. Finally, we will examine whether certain pathophysiological conditions, such as hyperglycemia or oxidative stress, alter either basal or BK-stimulated phosphorylation of eNOS at Ser-617 or Ser-635.