Abstract
It has been reported recently (Tretter et al., 2007b) that in isolated guinea pig brain mitochondria supported by α‐glycerophosphate (α‐GP) reactive oxygen species (ROS) are produced through the reverse electron transport (RET) in the respiratory chain and by α‐glycerophosphate dehydrogenase (α‐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 α‐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 α‐GPDH activity showed that Ca^2+^ stimulated the enzyme by decreasing the Km for α‐GP. In those mitochondria where RET was eliminated by the Complex I inhibitor rotenone (2 μM) 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 α‐GP‐supported mitochondria activation of α‐GPDH by Ca^2+^ leads to an accelerated RET‐mediated ROS generation as well as to a stimulated ROS production by α‐GPDH. © 2007 Wiley‐Liss, Inc.