Membrane potential-related effect of calcium on reactive oxygen species generation in isolated brain mitochondria

The effect of Ca2+ applied in high concentrations (50 and 300 µM) was addressed on the generation of reactive oxygen species in isolated mitochondria from guinea-pig brain. The experiments were performed in the presence of ADP, a very effective inhibitor of mitochondrial permeability transition. Moderate increase in H2O2 release from mitochondria was induced by Ca2+ applied in 50 µM, but not in 300 µM concentration as measured with Amplex red fluorescent assay starting with a delay of 100-150 sec after exposure to Ca2+. Parallel measurements of membrane potential (ΔΨm) by safranine fluorescence showed a transient depolarization by Ca2+ followed by the recovery of ΔΨm to a value, which was more negative than that observed before addition of Ca2+ indicating a relative hyperpolarization. NAD(P)H fluorescence was also increased by Ca2+ given in 50 µM concentration. In mitochondria having high ΔΨm in the presence of oligomycin or ATP, the basal rate of release of H2O2 was significantly higher than that observed in a medium containing ADP and Ca2+ no longer increased but rather decreased the rate of H2O2 release. With 300 µM Ca2+ only a loss but no tendency of a recovery of ΔΨm was detected and H2O2 release was unchanged. It is suggested that in the presence of nucleotides the effect of Ca2+ on mitochondrial ROS release is related to changes in ΔΨm; in depolarized mitochondria, in the presence of ADP, moderate increase in H2O2 release is induced by calcium, but only in ≤ 100 µM concentration, when after a transient Ca2+-induced depolarization mitochondria became more polarized. In highly polarized mitochondria, in the presence of ATP or oligomycin, where no hyperpolarization follows the Ca2+-induced depolarization, Ca2+ fails to stimulate mitochondrial ROS generation. These effects of calcium (≤ 300 µM) are unrelated to mitochondrial permeability transition.