Mitochondria and Pulmonary Endothelial Cell Death

描述(申请人提供)：肺血管系统，特别是内皮，已知是在分娩和接触内源性NO的过程中对生理和病理变化作出反应的关键部位。线粒体是细胞呼吸、生物合成和活性氧(ROS)和活性氮(RNS)代谢的关键部位。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不可逆抑制的主要部位，而复合体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氧化酶的作用。因此，我们假设在正常生理条件下，络合物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根离子来限制RNS的形成。这种分解反应的效率取决于当时流行的NO/O2比，它也需要消耗O2。在高氧条件下，或在电子传递链受损的病理生理环境中，RNS(和ROS)的形成不能有效地被抑制。这导致了对络合物I和III的不可逆转的抑制，这反过来又通过增加破坏性活性物种的产生而加剧了这种情况，并最终导致细胞死亡。在检验这一广泛的假设时，我们建议确定： 1)络合物IV的NO氧化酶活性限制了肺内皮细胞的亚硝化应激； 2)RNS不可逆抑制络合物I和络合物III的分子位置； 3)亚硝化应激对细胞色素c与复合体Ill和/或复合体IV相互作用的影响程度； 4)RNS依赖的细胞色素c与复合物III和/或IV相互作用的修饰，在促凋亡刺激培养的肺内皮细胞过程中促进细胞色素c从膜间隙丢失的功能作用。

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.