REDOX REGULATION OF eNOS SIGNALING PATHWAYS IN VASCULAR ENDOTHELIUM

项目总结（见说明）： 内皮型一氧化氮合酶（eNOS）是血管稳态的关键决定因素， 内皮细胞代谢eNOS催化涉及几种氧化还原活性辅因子，NO本身可以 与活性氧相互作用。血管内皮源性NO的生物利用度受损， 与氧化应激增加相关的疾病状态。我们假设eNOS的氧化还原调节 途径是一个关键的决定因素，一氧化氮和ROS依赖的信号和代谢调节， 血管内皮细胞所提出的研究与实验研究有重要的交叉点， 本计划中其他项目的计划。eNOS催化涉及几种氧化还原活性辅因子; 细胞内氧化还原状态的变化影响这些关键辅因子的浓度，并导致细胞内氧化还原水平的改变。 eNOS调节的反应。细胞NO加合物的形成受活性氮的影响， 活性氧物种和细胞硫醇氧化还原状态。AMP激活的内皮型一氧化氮合酶的磷酸化 蛋白激酶（AMPK）受活性氧的影响，提供了一个重要的联系， 氧化应激、eNOS信号传导和内皮细胞代谢。他汀类药物还激活AMPK并调节 内皮细胞ROS的产生。靶向信号转导膜微区的eNOS Js被称为 小窝，其中酶与支架/信号蛋白小窝蛋白-1相互作用。我们发现 siRNA介导的小窝蛋白敲低导致内皮细胞ROS产生的显著增加， 发现小窝蛋白-1-/-小鼠的氧化应激显著增加。eNOS-小窝蛋白相互作用 是由他汀类药物调节的，但他汀类药物在调节涉及eNOS的氧化还原途径中的作用仍然存在。 不完全理解。具体目标1中提出的实验将确定 改变的生物蝶呤和巯基代谢影响eNOS翻译后修饰，亚细胞靶向， ROS的产生。《特定目标2》中的实验将确定小窝蛋白的机制， 他汀类药物和活性氧调节AMP激活的蛋白激酶（AMPK）向eNOS的信号传导。

PROJECT SUMMARY (See instructions): The endothelial isoform of nitric oxide synthase (eNOS) is a key determinant of vascular homeostasis and endothelial cell metabolism. eNOS catalysis involves several redox-active cofactors, and NO itself can interact with reactive oxygen species. The bioavailability of endothelium-derived NO is impaired in vascular disease states associated with, increased oxidative stress. We hypothesize that redox regulation ofthe eNOS pathway represents a critical determinant of NO- and ROS-dependent signaling and metabolic regulation in vascular endothelial cells. The proposed studies have important points of intersection with experimental plans proposed by other Projects in this Program. eNOS catalysis involves several redox-active cofactors; changes in intracellular redox state affect the concentrations of these key cofactors, and lead to alterations in eNOS-modulated responses. The formation of cellular NO adducts is influenced by reactive nitrogen and reactive oxygen species and by the cellular thiol redox state. Phosphorylation of eNOS by the AMP-activated protein kinase (AMPK) is influenced by reactive oxygen species, providing an important link between oxidative stress, eNOS signaling, and endothelial cell metabolism. Statins also activate AMPK and modulate endothelial ROS production. eNOSJs targeted to signal-transducing membrane microdomains termed caveolae, where the enzyme interacts with the scaffolding/signaling protein caveolin-1. We discovered that siRNA-mediated knockdown of caveolin leads to a striking increase in ROS production from endothelial cells, and found that caveolin-1-/- mice show dramatic increases in oxidative stress. eNOS-caveolin interactions are modulated by statins, yet the roles of statins in modulation of redox pathways involving eNOS remain incompletely understood. Experiments proposed in Specific Aim 1 will identify the mechanisms whereby altered biopterin and thiol metabolism affects eNOS post-translational modifications, subcellular targeting, and ROS generation. Experiments in Specific Aim 2 will determine the mechanisms whereby caveolin, statins, and reactive oxygen species modulate AMP-activated protein kinase (AMPK) signaling to eNOS.