Reactive Species in Vascular Disease-Injury Mechanisms

描述（由申请人提供）：本申请中的实验将 研究负责细胞调节的分子机制 通过内源性一氧化氮（NO）代谢和抗氧化剂。 已发表的数据表明，NO或直接主要通过可逆的 关键半胱氨酸残基的S-亚硝基化或通过升高cGMP水平 调节适应性反应，使细胞抵抗氧化 应激和凋亡。然而，大多数细胞模型依赖于 通过NO供体或通过诱导性一氧化氮的诱导来递送NO， 一氧化氮合酶（NOS）。为了研究低浓度氮氧化物产生NO的贡献， 输出内皮NOS的细胞保护对氧化剂，我们利用 转染内皮NOS的ECV 3 O 4细胞。转染的细胞产生 足够的NO诱导L-NAME中平滑肌细胞中cGMP的升高 可居住的方式。使用这个定义良好的模型，初步数据显示， NO调节ATP的稳态，葡萄糖的流量， 糖酵解和戊糖磷酸途径和呼吸。而且这 代谢和线粒体生物能量学的动态调节与 具有增加的对H2 O2暴露的抵抗力。暴露于50-100 pM的H2 O2 诱导了近50%的延迟细胞死亡（暴露后18小时）， ECV_3O_4-eNOS细胞的凋亡率低于20%。抑制NO 生产改善了保护效果，恢复了稳态 ATP和葡萄糖流量的水平。使用人肺动脉的初步数据 内皮细胞证实了NO依赖性的抗H2 O2诱导的保护作用。 延迟细胞死亡这些初步数据加上稀少的公开数据 对NO调节代谢能力的研究表明， NO的功能可能与抗氧化应激的适应性有关。 我们认为eNOS产生低水平的NO足以 动态调节细胞葡萄糖代谢和呼吸， 主要的和以前未被认识的分子机制，为NO诱导的 保护免受氧化应激。为了检验这些假设，我们提出了 具体目标如下：（1）确定硝酸盐的分子机理 氧化物介导的细胞代谢调节;（2）调查因果关系 一氧化氮依赖性代谢改变与 适应氧化应激;（3）检查内源性一氧化氮是否 调节线粒体呼吸和线粒体功能， 负责保护免受氧化应激。 第一个目标的实验集中在变构，共价和其他 NO在催化必需和必需的代谢酶中的调节功能， 糖酵解途径和TCA循环中的不可逆步骤。第二个目标将 利用生物化学、药理学和分子方法， NO介导的调节之间潜在因果关系的证据 代谢和抗氧化应激的能力。第三个目标是审查 一氧化氮调节线粒体呼吸重要性及其在保护 细胞从氧化剂暴露和典型的诱导凋亡。总体 拟议的实验将以系统的方式评估的关键作用， 内源性产生的NO作为细胞代谢和呼吸的介质 使细胞能够抵抗氧化应激。

DESCRIPTION (provided by applicant): Experiments in this application will examine the molecular mechanisms responsible for the modulation of cellular metabolism and resistance to oxidants by endogenous nitric oxide (NO). Published data indicated that NO either directly mainly by reversible S-nitrosylation of critical cysteine residues or by elevating cGMP levels modulates the adaptive responses that render cells resistant to oxidative stress and apoptosis. However, the majority of the cellular models rely upon the deliver of NO by NO donors or by the induction of the inducible nitric oxide synthase (NOS). To study the contribution of NO generated by the low output endothelial NOS in the cellular protection against oxidants, we utilized ECV3O4 cells transfected with endothelial NOS. The transfected cells generated sufficient NO to induce elevation of cGMP in smooth muscle cells in an L-NAME inhabitable manner. Using this well-defined model preliminary data revealed that NO regulates the steady state of ATP, the flux of glucose by the glycolytic and pentose phosphate pathways and respiration. Moreover, this dynamic regulation of metabolism and mitochondrial bioenergetics was associated with an increased resistance to H2O2 exposure. Exposure to H2O2 at 50-100 pM induced a delayed cell death (18 hours after exposure) to nearly 50 percent of ECV3O4 but less than 20 percent in the ECV3O4-eNOS cells. Inhibition of NO production ameliorated the protective effect and restored the steady state levels of ATP and glucose fluxes. Preliminary data using human pulmonary artery endothelial cells confirmed the NO-dependent protection against H202 induced delayed cell death. These preliminary data together with scarce published data on the ability of NO to regulate metabolism suggest a previous unrecognized function of NO that may causally relate to adaptation against oxidative stress. We propose that the generation of low levels of NO by eNOS is sufficient to dynamically regulate cellular glucose metabolism and respiration providing a primary and previously unrecognized molecular mechanism for the NO-induced protection against oxidative stress. To examine these hypotheses we propose the following specific aims: (1) define the molecular mechanism(s) of nitric oxide-mediated regulation of cellular metabolism; (2) investigate the causal association between nitric oxide-dependent alterations in metabolism with the adaptation to oxidative stress; and (3) examine if endogenous nitric oxide regulation of mitochondrial respiration and mitochondrial function is responsible for the protection against oxidative stresses. Experiments in the first aim are focused on the allosteric, covalent and other regulatory functions of NO in critical enzymes that catalyze essential and irreversible steps in the glycolytic pathway and TCA cycle. The second aim will utilize biochemical, pharmacological and molecular approaches to provide evidence for the potential causal relationship between NO-mediated regulation of metabolism and resistance to oxidative stress. The third aim examines the importance of NO-regulated mitochondrial respiration and function in protecting cells from oxidant exposures and typical inducers of apoptosis. Overall the proposed experiments will evaluate in a systematic manner the critical role of endogenously generated NO as a mediator of cellular metabolism and respiration that enables cells to resist oxidative stress.