BRADYKININ SIGNALING IN REGULATION OF eNOS

描述（由申请人提供）：内皮源性一氧化氮（NO）在调节血管舒张状态并因此调节血压方面具有至关重要的作用。此外，由于 NO 调节这一过程以及许多其他重要的血管过程，因此血管 NO 产生的异常被认为与某些血管疾病（例如动脉粥样硬化、糖尿病和高血压）的发病机制有关。 NO 在内皮细胞中通过内皮一氧化氮合酶 (eNOS) 催化的反应中 L-精氨酸的氧化而合成。最近几个实验室的研究已经确定了 Ser-1179 磷酸化和 Thr-497 去磷酸化在内皮细胞 eNOS 活性的激动剂调节中的作用。该提案的初步研究部分提供的数据为 Ser-617 和 Ser-635 两个额外的 eNOS 磷酸化位点提供了证据。我们的初步数据表明，在响应 eNOS 激活激动剂缓激肽 (BK)、ATP 和血管内皮生长因子的刺激时，这两个位点在培养的内皮细胞中短暂磷酸化。 本提案的主要目的是检验以下假设：血管内皮细胞中的 eNOS 活性部分受到 BK 刺激的 Ser-617 和 Ser-635 酶磷酸化的调节。其他目的是阐明 Ser-617 和 Ser-635 磷酸化改变酶催化活性的分子机制，并测试 Ser-617 或 Ser-635 磷酸化改变 eNOS 与 Caveolin-1、缓激肽 B2 受体或热休克蛋白 90 的相互作用的假设。最后，我们将检查某些病理生理条件（例如高血糖或氧化应激）是否会改变eNOS Ser-617 或 Ser-635 的基础或 BK 刺激磷酸化。

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.