H2O2 generation is decreased by calcium in isolated brain mitochondria

Release of H2O2 in response to Ca2+ loads (1-100 μM) was investigated using Amplex red fluorescent assay in isolated guinea-pig brain mitochondria respiring on glutamate plus malate or succinate. In mitochondria challenged with Ca2+ (10 μM), in the absence of adenine nucleotides and inhibitors of the respiratory chain, the rate of H2O2 release, taken as an indication of H2O2 production, was decreased by 21.8 ± 1.6% in the presence of NADH-linked substrates and by 86.5 ± 1.8% with succinate. Parallel with this, a Ca2+-induced loss in NAD(P)H fluorescence, sustained depolarization, decrease in fluorescent light scattering signal and in calcein fluorescence were detected indicating an increased permeability and swelling of mitochondria, which were prevented by ADP (2 mM). In the presence of ADP H2O2 release from mitochondria was decreased, but Ca2+ no longer influenced the generation of H2O2. We suggest that the decreased H2O2 generation induced by Ca2+ is related to depolarization and NAD(P)H loss resulting from a non-specific permeability increase of the mitochondrial inner membrane.