Reactive Species in Vascular Disease-Injury Mechanisms

血管疾病损伤机制中的反应物种

• 批准号: 6726925
• 负责人: HARRY ISCHIROPOULOS
• 金额: $ 34万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-05 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6726925
• 关键词: Krebs' cycle; apoptosis; biological signal transduction; cardiovascular injury; cell line; cellular respiration; cytoprotection; cytotoxicity; free radicals; gene induction /repression; glycolysis; mitochondria; molecular pathology; nitric oxide; nitric oxide synthase; oxidative stress; peroxidation; transfection; vascular endothelium; western blottings

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.

描述（由申请人提供）：本申请中的实验将 检查负责调节细胞的分子机制 内源性一氧化氮对氧化剂的代谢和抗性（NO）。 已发布的数据表明，这两个直接不是直接是由可逆的 临界半胱氨酸残基的S-亚硝基化或通过升高CGMP水平 调节适应性反应，使细胞具有抗氧化的抗性细胞 压力和凋亡。但是，大多数细胞模型都依赖 NO捐赠者或通过诱导一氮的诱导来交付NO 氧化物合酶（NOS）。研究低下的NO产生的贡献 在细胞保护剂中，输出内皮NOS抗氧化剂，我们使用了 用内皮NOS转染的ECV3O4细胞。转染的细胞生成 足以诱导L-NAME平滑肌细胞中CGMP的升高 居住的方式。使用这个明确定义的模型初步数据显示 没有调节ATP的稳态，葡萄糖的通量 糖酵解和戊糖磷酸盐途径和呼吸。而且，这 新陈代谢和线粒体生物能的动态调节是相关的 对H2O2暴露的阻力增加。 50-100 pm暴露于H2O2 诱导延迟的细胞死亡（暴露于18小时）至近50％ ECV3O4，但在ECV3O4-ENOS细胞中不到20％。抑制否 生产改善了保护效果并恢复了稳态 ATP和葡萄糖通量水平。使用人肺动脉的初步数据 内皮细胞证实了针对H202诱导的无依赖性保护 延迟细胞死亡。这些初步数据以及稀缺的数据 关于NO调节新陈代谢的能力，表明先前的未识别 NO的功能可能与氧化应激的适应性有关。 我们建议，eNOS的低水平NO生成足以 动态调节细胞葡萄糖代谢和呼吸，提供 无诱导的主要和以前未识别的分子机制 防止氧化应激。为了审查这些假设，我们提出了 以下特定目的：（1）定义硝酸的分子机制 氧化物介导的细胞代谢调节； （2）研究因果 一氧化氮依赖性的代谢改变与 适应氧化应激； （3）检查内源性一氧化氮是否 线粒体呼吸和线粒体功能的调节是 负责保护氧化应激。 第一个目标的实验集中于变构，共价和其他实验 关键酶中NO的调节功能，该酶催化了必不可少的和 糖酵解途径和TCA周期中的不可逆步骤。第二个目标 利用生化，药理学和分子方法提供 无介导的调节之间潜在因果关系的证据 代谢和对氧化应激的抗性。第三目检查 无调线粒体呼吸和保护功能的重要性 来自氧化剂暴露和典型凋亡诱导剂的细胞。总体而言 提出的实验将以系统的方式评估 内生产生的NO作为细胞代谢和呼吸的介体 这使细胞能够抵抗氧化应激。