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

Abstract

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 KO 2 produced brain mitochondrial swelling, which was blocked by cyclosporin A (CSA), and which was Ca 2+ 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 Ca 2+ and the fact that ROS were required for Ca 2+-induced mitochondrial swelling. 4. Superoxide anions, but not Ca 2+, 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 Δψm 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.