MITOCHONDRIAL HYPOXIA: PRODUCTION AND REACTION OF ROS

最近的观察已经导致了这样的假设，即在缺血期间由于在缺氧和其他条件下产生活性氧（ROS）而发生显著的线粒体损伤。由于在控制PO 2下研究反应的困难，很少有关于缺氧过程中线粒体ROS产生的研究。这种信息的缺乏留下了一个无法解释的难题：ROS的产生如何随着底物O2的减少而增加。在老年心脏线粒体中发现的缺陷被假设为在缺血期间增加ROS产生，导致老年心脏缺血期间氧化损伤增加。 前两个特定的目标测试两种替代机制，可以单独或组合的帐户在缺血过程中增加的ROS的生产。第一种假设是电子传输链中的载流子被更多地还原，导致泄漏增加到低但非零的O2浓度的位点，以产生。O2.第二 其机制是增强的ROS产生来自作为对缺血和/或缺氧的响应而诱导的细胞环境的变化。这些细胞反应包括Fe的释放、Ca ++的流入、pH的降低和NO的产生。已经开发了一种新的装置，其允许监测ROS的产生，同时在2-15 ℃的受控可变分压下连续呼吸利用存在的O2。使用亚线粒体颗粒，已经显示PO 2的变化改变H2 O2和超氧化物的产生。这些和其他ROS的产生将作为Fe（II）、Ca 2+、降低的pH和NO的存在的函数进行监测，所有这些都在模拟缺血期间观察到的生理变化的浓度下。成年人和老年人的反应， 将比较肌膜下和原纤维间线粒体。在导致最大ROS产生的条件下，ROS产生的位点将通过在特异性抑制剂存在下改变还原底物以及比较亚线粒体颗粒、有丝分裂体和线粒体获得的结果来研究。 在第三个具体目标中，将通过质谱方法研究ROS对蛋白质和心磷脂造成的化学损伤。已经开发了分离用于MS分析的单个电子传递复合物的方法，以便现在可以比较最显著的功能和结构损伤的位置。 在第四个具体目标中，将在我们的系统中模拟由其他三个项目检测或工程化的线粒体中的变化，以便可以表征它们对线粒体ROS直接产生的影响。 此外，还将对PPG研究建议的治疗干预进行测试。

Recent observations have led to the hypothesis that significant mitochondrial damage occurs during ischemia due to the production of reactive oxygen species (ROS) under the hypoxic and other conditions that prevail. Because of the difficulty of studying reactions at controlled PO2, few studies of mitochondrial ROS production during hypoxia exist. This absence of information leaves an unexplained conundrum: how does ROS production increase as the substrate 02, decreases. Defects identified in elderly heart mitochondria are hypothesized to augment ROS production during ischemia, resulting in increased oxidative damage during ischemic periods in the elderly heart. The first two specific aims test two alternative mechanisms that could individually or in combination account for the enhanced production of ROS during ischemia. The first hypothesis is that the carriers in the electron transport chain are more reduced, resulting in sites of increased leakage to a low but nonzero 02 concentration, to generate . O2. The second mechanism is that the enhanced ROS production comes from changes in the cellular environment induced as a response to ischemia and/or hypoxia. Among these cellular responses are the release of Fe, the influx of Ca ++, a decrease in pH, and the production of NO. A novel apparatus has been developed that permits the production of ROS to be monitored, concurrent with continuous respiratory utilization of 02 present at controlled variable partial pressures of 2-15 torr. Using submitochondrial particles, the variation in PO2 has been shown to alter both H202 and superoxide generation. The production of these and other ROS will be monitored as a function of the presence of Fe(II), Ca 2+, decreased pH and NO, all at concentrations mimicking physiological changes observed during ischemia. The response of both adult and elderly and both subsarcolemmal and interfibrillar mitochondria will be compared. The site of ROS production, under conditions that lead to the greatest ROS generation, will be investigated by varying the reducing substrate in the presence of specific inhibitors as well as comparing results obtained with submitochondrial particles, mitoplasts and mitochondria. In the third specific aim the chemical damage inflicted by the ROS on proteins and cardiolipin will be investigated by mass spectrometric methods. Methods of isolating the individual electron transport complexes for MS analyses have been developed so that the location of the most significant functional and structural damage can now be compared. In the fourth specific aim, the variations in mitochondria detected or engineered by the other three projects will be mimicked in our system so that their effects on the direct production of mitochondrial ROS can be characterized. Additionally, therapeutic interventions suggested by the PPG studies will be tested.