Stimulation of H₂O₂ generation by calcium in brain mitochondria respiring on α‐glycerophosphate

It has been reported recently (Tretter et al., 2007b) that in isolated guinea pig brain mitochondria supported by alpha-glycerophosphate (alpha-GP) reactive oxygen species (ROS) are produced through the reverse electron transport (RET) in the respiratory chain and by alpha-glycerophosphate dehydrogenase (alpha-GPDH). We studied the effect of calcium on the generation of H(2)O(2) as measured by the Amplex Red fluorescent assay in this model. H(2)O(2) production in alpha-GP-supported mitochondria was increased significantly in the presence of 100, 250, and 500 nM Ca(2+), respectively. In addition, Ca(2+) enhanced the membrane potential, the rate of oxygen consumption, and the NAD(P)H autofluorescence in these mitochondria. Direct measurement of alpha-GPDH activity showed that Ca(2+) stimulated the enzyme by decreasing the Km for alpha-GP. In those mitochondria where RET was eliminated by the Complex I inhibitor rotenone (2 microM) or due to depolarization by ADP (1 mM), the rate of H(2)O(2) formation was smaller and the stimulation of H(2)O(2) generation by Ca(2+) was prevented partly, but the stimulatory effect of Ca(2+) was still significant. These data indicate that in alpha-GP-supported mitochondria activation of alpha-GPDH by Ca(2+) leads to an accelerated RET-mediated ROS generation as well as to a stimulated ROS production by alpha-GPDH.