Oxidant Stress and Thiol Redox State in Endothelial Cells

正常的氧化代谢导致各种氧化还原形式的分子的产生 氧，称为活性氧物种(ROS)，在一定浓度范围内产生 在细胞内。低水平的ROS产生对于正常的信号机制是重要的，而 较高水平的ROS产生会导致氧化应激，在这种情况下，ROS的通量 超过抗氧化能力。对氧化应激做出反应的一个关键决定因素是细胞的能力 通过自适应增加NADPH的产量来应对增加的ROS生成，主要的 氧化谷胱甘肽还原的还原当量来源。胞浆中的主要酶 合成NADPH所需的是葡萄糖-6-磷酸脱氢酶(G6PD)，这是限速酶 磷酸戊糖途径中的酶。我们以前已经证明了这一点的重要性。 酶在维持正常的内皮功能，包括一氧化氮(NO)生物活性，在面对 氧化应激。在这个提议中，我们假设G6PD及其酶产物NADPH是 内皮细胞硫醇氧化还原状态的关键调节因子对于正常的氧化信号是必不可少的。 和内皮功能，自适应地响应增加的ROS生成，以维持 代偿的氧化应激，以及当氧化应激超过这种适应能力时，是负责的 在内皮细胞中将代偿性氧化应激转化为非补偿性氧化应激 手机。为了验证这一假设，我们将1)评估NADPH和氧化还原状态的关系 血管内皮细胞硫醇库及其酶决定因素；2)评价NADPH对血管内皮功能的影响。 内皮NO及其S亚硝基衍生物的合成、代谢和生物利用度；3)测定 G6PD和NADPH在内皮细胞氧化应激适应性反应中的作用，包括 它们在支持代偿氧化应激状态中的作用；以及4)评估两者之间的关系 在正常氧化信号条件下，硫醇氧化还原状态与内皮NO生物活性之间的关系， 体内代偿性氧化应激和非代偿性氧化应激。这些研究应该摆脱 G6PD和NADPH在调节内皮细胞硫醇氧化还原状态中的关键作用 以及其适应氧化应激的能力，以努力维持正常的内皮功能。

Normal oxidative metabolism leads to the generation of various redox forms of molecular oxygen, termed reactive oxygen species (ROS), that are generated over a range of concentrations within cells. Low levels of ROS production are important for normal signaling mechanisms, while higher levels of ROS production can lead to oxidant stress, a condition under which the flux of ROS exceeds antioxidant capacity. A key determinant of the response to oxidant stress is the cell's capacity to counter increased ROS generation by adaptively increasing the production of NADPH, the principal source of reducing equivalents for the reduction of oxidized glutathione. The primary cytosolic enzyme required for NADPH synthesis is glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway. We have previously demonstrated the importance of this enzyme in maintaining normal endothelial function, including nitric oxide (NO) bioactivity, in the face of oxidant stress. In this proposal, we hypothesize that G6PD and its enzymatic product NADPH are the key regulators of the thiol redox state of the endothelial cell, are essential for normal oxidant signaling and endothelial function, adaptively respond to increased ROS generation to maintain a state of compensated oxidant stress, and when oxidant stress exceeds this adaptive capacity are responsible for the conversion of compensated oxidant stress to uncompensated oxidant stress in the endothelial cell. To test this hypothesis, we will 1) assess the relationship between NADPH and the redox state of endothelial thiol pools and their enzymatic determinants; 2) evaluate the effect of NADPH on the synthesis, metabolism, and bioavailability of endothelial NO and its S-nitroso-derivatives; 3) determine the role of G6PD and NADPH in the adaptive response to oxidant stress in endothelial cells, including their role in supporting the state of compensated oxidant stress; and 4) evaluate the relationship between thiol redox state and endothelial NO bioactivity under conditions of normal oxidant signaling, compensated oxidant stress, and uncompensated oxidant stress in vivo. These studies should shed light on the critical role of G6PD and NADPH in regulating the thiol redox state of the endothelial cell and its ability to adapt to oxidant stress in an effort to maintain normal endothelial function.