Mitochondria and Pulmonary Endothelial Cell Death

线粒体和肺内皮细胞死亡

• 批准号: 6776070
• 负责人: JAMES PETERSON
• 金额: $ 28.02万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6776070
• 关键词: NAD(P)H dehydrogenase; NADPH cytochrome c2 reductase; SDS polyacrylamide gel electrophoresis; animal tissue; cell death; cytochrome oxidase; enzyme activity; enzyme inhibitors; enzyme mechanism; enzyme substrate; free radical oxygen; free radicals; hypoxia; in situ hybridization; liquid chromatography mass spectrometry; mitochondria; molecular site; nitric oxide; nitrogen compounds; oxidative phosphorylation; oxidative stress; protein purification; pulmonary circulation; respiratory epithelium; tissue /cell culture; vascular endothelium

DESCRIPTION (provided by applicant): The pulmonary vasculature, particularly the endothelium, is known to be a key site responsive to both physiological and pathological changes in 02 delivery and exposure to endogenous NO. Mitochondria are critical loci of cellular respiration, biosynthesis, and metabolism of reactive oxygen species (ROS) and reactive nitrogen species (RNS). We have established that mitochondrial respiratory complexes I (NADH dehydrogenase) and Ill (cytochrome c reductase) are primary locations of irreversible inhibition by RNS and that complex IV (cytochrome c oxidase), in addition to its known cytochrome c:O2 oxidoreductase activity, may also function as an NO oxidase. Accordingly, we hypothesize that under normal physiological circumstances, the NO oxidase activity of complex IV limits formation of RNS by rapidly converting NO to the relatively innocuous nitrite ion. The efficiency of this catabolic reaction, which also consumes 02, depends upon the prevailing NO/O2 ratio. Under hyperoxic conditions, or in pathophysiological circumstances where the electron-transport chain has been compromised, RNS (and ROS) formation is not effectively suppressed. This results in irreversible inhibition of complexes I and III, which in turn exacerbates the situation by increasing the production of the damaging reactive species and ultimately, leads to cell death. In testing this broad hypothesis, we propose to determine: 1) that the NO oxidase activity of complex IV limits nitrosative stress in pulmonary endothelial cells; 2) the molecular sites at which RNS irreversibly inhibit complex I and complex III; 3) the extent to which nitrosative stress may affect the interaction of cytochrome c with complex Ill and/or complex IV; 4) the functional role of RNS-dependent modification of the interaction between cytochrome c and complexes III and/or IV in promoting the loss of cytochrome c from the intermembrane space during proapoptotic stimulation of cultured pulmonary endothelial cells.

描述（由申请人提供）：众所周知，肺血管，尤其是内皮，是响应02递送和内源性NO暴露的生理和病理变化的关键部位。线粒体是细胞呼吸、生物合成以及活性氧（ROS）和活性氮（RNS）代谢的关键位点。我们已经确定线粒体呼吸复合体I （NADH脱氢酶）和Ill（细胞色素c还原酶）是RNS不可逆抑制的主要位置，复合体IV（细胞色素c氧化酶）除了具有已知的细胞色素c:O2氧化还原酶活性外，还可能具有NO氧化酶的功能。因此，我们假设在正常生理情况下，复合物IV的NO氧化酶活性通过将NO快速转化为相对无害的亚硝酸盐离子来限制RNS的形成。这种分解代谢反应的效率，也消耗02，取决于普遍的NO/O2比率。在高氧条件下，或在电子传递链受损的病理生理环境下，RNS（和ROS）的形成不能有效抑制。这导致复合物I和III的不可逆抑制，这反过来又通过增加破坏性反应物质的产生而加剧了这种情况，并最终导致细胞死亡。为了验证这一宽泛的假设，我们建议确定：