MITOCHONDRIA AND PULMONARY ENDOTHELIAL CELL DEATH

描述(申请人摘要)：线粒体是细胞的关键位置 部分还原氧物种的呼吸、生物合成和代谢 并参与细胞内信号传递，包括钙稳态， 氧感应与细胞凋亡的启动。一氧化氮(NO)是唯一的 在信号分子之间，因为它通过两个氧化还原作用影响其目标位置 化学(S-硫醇的亚硝化)和配位(特别是对Fe2+)。 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部位。因此，它 自从发现L-精氨酸生物合成途径以来，人们就已经知道了 NO可以抑制线粒体的呼吸。尽管如此，无论是准确的 NO影响线粒体功能的靶点或机制是 很明显。我们假设NADH脱氢酶(复合体I)与其具有多个 非血红素铁-硫(Fe/S)中心和细胞色素c还原酶(复合体III)， 一种血色素蛋白，是一氧化氮和过氧亚硝酸盐的靶标。我们提出了一系列 生物物理和生化研究将通过以下方式揭示其分子机制 NO、部分还原氧物种(PRO)和ONO2对线粒体的影响 呼吸链功能。目的1：探讨其发病的分子机制。 过氧亚硝酸盐(ONO2)和其他氧化剂不可逆地抑制络合物I和 目标2：研究络合物IV(细胞色素c ONO2和H_2O_2的解毒作用。目标3：确定 过亚硝酸根和亚铁的分子靶标(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)引起的变化(线粒体 膜电位)发生在完整培养内皮细胞的线粒体中 细胞。这些研究结合使用了低温电子 顺磁共振波谱与磁性圆二色性和线性二色性 离体牛心脏线粒体和电压敏感染料的完整 牛肺动脉内皮细胞，将提供机制信息 支持NO诱导的线粒体改变在健康和疾病中的作用。

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.