MITOCHONDRIAL HYPOXIA: PRODUCTION AND REACTION OF ROS

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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.