Reactive Species in Vascular Disease-Injury Mechanisms

描述(由申请人提供)：本申请中的实验将 探讨细胞内信号转导的分子机制 内源性一氧化氮(NO)的代谢和对氧化剂的抵抗。 已发表的数据表明，NO既不是直接的，主要是可逆的 S亚硝化关键半胱氨酸残基或提高cGMP水平 调节使细胞抵抗氧化的适应性反应 应激与细胞凋亡。然而，大多数细胞模型依赖于 NO由无供体或通过诱导性硝酸的诱导传递 氧化物合成酶(NOS)。研究低氧对大气中NO的贡献 输出内皮一氧化氮合酶在细胞抗氧化保护中的应用 转染内皮型一氧化氮合酶的ECV3O4细胞。转基因细胞产生了 足够的NO诱导血管平滑肌细胞cGMP升高--L 适合居住的方式。使用这个定义明确的模型，初步数据显示 NO通过调节ATP的稳态，葡萄糖的流量， 糖酵解和戊糖磷酸途径与呼吸作用。此外，这一点 代谢的动态调节与线粒体生物能量学有关 增加了对过氧化氢暴露的抵抗力。暴露在50-100 PM的过氧化氢中 导致延迟性细胞死亡(暴露18小时后)，接近50% ECV3O4-eNOS细胞中，ECV3O4-eNOS细胞中的DNA含量低于20%。一氧化氮的抑制作用 生产改善了保护效果，恢复了稳定状态 三磷酸腺苷和葡萄糖通量水平。使用人体肺动脉的初步数据 内皮细胞证实对H202诱导的NO依赖保护作用 延迟性细胞死亡。这些初步数据与稀缺的已公布数据一起 关于NO调节新陈代谢的能力提出了一种先前未被认识的建议 NO的功能可能与对氧化应激的适应有关。 我们认为，eNOS产生的低水平NO足以 动态调节细胞葡萄糖代谢和呼吸，提供 NO诱导的主要和以前未知的分子机制 防止氧化应激。为了检验这些假设，我们提出了 具体目标如下：(1)明确硝酸根的分子机制(S) 氧化物介导的细胞代谢调节；(2)调查原因 一氧化氮依赖的代谢改变与高血压的关系 对氧化应激的适应；以及(3)检查内源性一氧化氮 线粒体呼吸和线粒体功能的调节 负责防止氧化应激。 第一个目标中的实验主要集中在变构、共价和其他 NO在关键酶中的调节功能，这些酶催化必需的和 糖酵解途径和TCA循环中不可逆的步骤。第二个目标是 利用生化、药理学和分子方法提供 NO介导的调控之间潜在因果关系的证据 新陈代谢和抵抗氧化应激的能力。第三个目标是检查 一氧化氮调节的线粒体呼吸及其在保护中的作用 细胞免受氧化剂和典型的细胞凋亡诱导剂的影响。总体而言 拟议的实验将以系统的方式评估 内源性产生的NO作为细胞代谢和呼吸的介质 这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.