MITOCHONDRIA AND PULMONARY ENDOTHELIAL CELL DEATH

线粒体和肺内皮细胞死亡

• 批准号: 6537482
• 负责人: JAMES PETERSON
• 金额: $ 20.57万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 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 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6537482
• 关键词: NAD(P)H dehydrogenase; NADPH cytochrome c2 reductase; apoptosis; cell death; circular magnetic dichroism; cytochrome oxidase; electron spin resonance spectroscopy; enzyme activity; enzyme inhibitors; free radical oxygen; hydrogen peroxide; iron compounds; membrane potentials; mitochondria; molecular dynamics; nitric oxide; peroxynitrites; respiratory epithelium; tissue /cell culture

DESCRIPTION (Applicant's abstract): Mitochondria are critical loci of cellular respiration, biosynthesis and metabolism of partially reduced oxygen species and participate in intracellular signaling including calcium homeostasis, oxygen sensing and the initiation of apoptosis. Nitric oxide (NO) is unique amongst signaling molecules in that it affects its target sites by both redox chemistry (S-nitrosylation of thiols) and coordination (especially to Fe2+). Consequently biomolecules affected by NO (and peroxynitrite) tend to have critical metalloregulatory and cysteine-enriched active sites. Accordingly, it has been well known since the discovery of the L-arginine biosynthetic pathway that NO can inhibit mitochondrial respiration. Nonetheless neither the precise targets nor the mechanism by which NO affects mitochondrial function are apparent. We hypothesize that NADH dehydrogenase (complex I) with it's multiple non-heme iron-sulfur (Fe/S) centers and cytochrome c reductase (complex III), an hemoprotein, are targets for NO and peroxynitrite. We propose a series of biophysical and biochemical studies that will reveal the molecular mechanism by which NO, partially reduced oxygen species (PROS) and ONO2 affect mitochondrial respiratory chain function. Aim 1: Investigate the molecular mechanism by which peroxynitrite (ONO2) and other oxidants irreversibly inhibit complex I and complex III. Aim 2: Investigate the mechanism by which complex IV (cytochrome c oxidase) catalyses the detoxification of ONO2 and H2O2. Aim 3: Identify the molecular targets of peroxynitrite and ferrous iron in Keilin-Hartee (K-H, submitochondrial) particles and intact mitochondria. Aim 4: describe the oxidant (NO, superoxide anion, ONO2, etc) induced changes (mitochondrial membrane potential) that occur in mitochondria of intact cultured endothelial cells. These studies, using a combination of low temperature electron paramagnetic resonance spectroscopy and magnetic circular and linear dichroism in isolated bovine heart mitochondria and voltage sensitive dyes in intact bovine pulmonary artery endothelial cells, will provide mechanistic information to support a role for NO induced mitochondrial changes in health and disease.

描述（申请人摘要）：线粒体是细胞增殖的关键位点。 部分还原氧的呼吸、生物合成和代谢 并参与细胞内信号传导包括钙稳态， 氧传感和细胞凋亡的启动。一氧化氮（NO）是一种独特的 在信号分子中，它通过两种氧化还原作用影响其靶位点， 化学（硫醇的S-亚硝基化）和配位（特别是与Fe 2+）。 因此，受NO（和过氧亚硝酸盐）影响的生物分子往往具有 关键的金属调节和富含半胱氨酸的活性位点。因此 自从发现L-精氨酸生物合成途径以来， NO能抑制线粒体呼吸。然而，精确的 NO影响线粒体功能的靶点和机制， 很明显我们假设，NADH脱氢酶（复合物I）与它的多个 非血红素铁-硫（Fe/S）中心和细胞色素c还原酶（复合物III）， 血红素蛋白是NO和过氧亚硝酸盐的目标。我们提出了一系列 生物物理和生物化学研究，将揭示分子机制， NO、部分还原氧（PROS）和ONO2影响线粒体 呼吸链功能目的1：研究其分子机制， 过氧亚硝酸盐（ONO2）和其他氧化剂不可逆地抑制复合物I， 复合体III.目的2：探讨复合物Ⅳ（细胞色素c） 氧化酶）催化ONO2和H2O2的解毒。目标3：确定 Keilin-Hartee（K-H， 亚线粒体）颗粒和完整的线粒体。目标4：描述 氧化剂（NO、超氧阴离子、ONO2等）诱导的变化（线粒体 膜电位）发生在完整培养的内皮细胞的线粒体中 细胞这些研究，使用低温电子 顺磁共振光谱和磁圆和线二色性 在离体牛心脏线粒体和电压敏感染料在完整的 牛肺动脉内皮细胞，将提供机制信息 支持NO诱导的线粒体变化在健康和疾病中的作用。