Mitochondrial Generation of Oxygen Radicals During Reoxygenation of Ischemic Tissues

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Abstract
Ischemia and reperfusion causes severe mitochondrial damage, including swelling and deposits of hyd-roxyapatite crystals in the mitochondrial matrix. These crystals are indicative of a massive influx of Ca2+ into the mitochondrial matrix occurring during reoxygenation. We have observed that mitochondria isolated from rat hearts after 90 minutes of anoxia followed by reoxygenation, show a specific inhibition in the electron transport chain between NADH dehydrogenase and ubiquinone in addition to becoming uncoupled (unable to generate ATP). This inhibition is associated with an increased H2O2 formation at the NADH dehydrogenase level in the presence of NADH dependent substrates. Control rat mitochondria exposed for 15 minutes to high Ca2+ (200 nmol/mg protein) also become uncoupled and electron transport inhibited between NADH dehydrogenase and ubiquinone. a lesion similar to that observed in post-ischem-ic mitochondria. This Ca2+ -dependent effect is time dependent and may be partially prevented by albumin, suggesting that it may be due to phospholipase A2 activation. releasing fatty acids, leading to both inhibition of electron transport and uncoupling. Addition of arachidonic or linoleic acids to control rat heart mitochondria, inhibits electron transport between Complex I and III. These results are consistent with the following hypothesis: during ischemia, the intracellular energy content drops severely, affecting the cytoplasic concentration of ions such as Na+ and Ca2+. Upon reoxygenation, the mitochondrion is the only organelle capable of eliminating the excess cytoplasmic Ca2+ through an electrogenic process requiring oxygen (the low ATP concentration makes other ATP-dependent Ca?' lransport systems non-operational). Ca2+-overload of mitochondria activates phospholipase A2 releasing free fatty acids, leading to uncoupling and inhibition of the interactions between Complex I and III of the respiratory chain. As a consequence, the NADH-dehydrogenase becomes highly reduced, and transfers electrons directly to oxygen generating O2.