Reactive oxygen species induce swelling and cytochrome c release but not transmembrane depolarization in isolated rat brain mitochondria

1 In this study, we have used isolated brain mitochondria to investigate the effects of superoxide anions (O-2(-)) on mitochondrial parameters related to apoptosis, such as swelling, potential, enzymatic activity, NAD(P) H, cytochrome c release, and caspase activity. 2 Addition of the reactive oxygen species (ROS) generator KO2 produced brain mitochondrial swelling, which was blocked by cyclosporin A (CSA), and which was Ca2+ independent. 3 Calcium induced mitochondrial swelling only at high concentrations and in the presence of succinate. This correlated with the increase in O-2(-) production detected with hydroethidine in mitochondrial preparations exposed to Ca2+ and the fact that ROS were required for Ca2+-induced mitochondrial swelling. 4 Superoxide anions, but not Ca2+, decreased citrate synthase and dehydrogenase enzymatic activities and dropped total mitochondrial NAD( P) H levels. 5 Calcium, but not O-2(-), triggered a rapid loss of mitochondrial potential. Calcium-induced Deltapsi(m) dissipation was inhibited by Ruthenium Red, but not by CSA. 6 Calcium- and superoxide-induced mitochondrial swelling released cytochrome c and increased caspase activity from isolated mitochondria in a CS A-sensitive manner. 7 In summary, superoxide potently triggers mitochondrial swelling and the release of proteins involved in activation of postmitochondrial apoptotic pathways in the absence of mitochondrial depolarization.