High Ca2+ load promotes hydrogen peroxide generation via activation of α-glycerophosphate dehydrogenase in brain...

H(2)O(2) generation associated with α-glycerophosphate (α-GP) oxidation was addressed in guinea pig brain mitochondria challenged with high Ca(2+) load (10 μM). Exposure to 10 μM Ca(2+) induced an abrupt 2.5-fold increase in H(2)O(2) release compared to that measured in the presence of a physiological cytosolic Ca(2+) concentration (100 nM) from mitochondria respiring on 5 mM α-GP in the presence of ADP (2 mM). The Ca(2+)-induced stimulation of H(2)O(2) generation was reversible and unaltered by the uniporter blocker Ru 360, indicating that it did not require Ca(2+) uptake into mitochondria. Enhanced H(2)O(2) generation by Ca(2+) was also observed in the absence of ADP when mitochondria exhibited permeability transition pore opening with a decrease in the NAD(P)H level, dissipation of membrane potential, and mitochondrial swelling. Furthermore, mitochondria treated with the pore-forming peptide alamethicin also responded with an elevated H(2)O(2) generation to a challenge with 10 μM Ca(2+). Ca(2+)-induced promotion of H(2)O(2) formation was further enhanced by the complex III inhibitor myxothiazol. With 20 mM α-GP concentration, stimulation of H(2)O(2) formation by Ca(2+) was detected only in the presence, not in the absence, of ADP. It is concluded that α-glycerophosphate dehydrogenase, which is accessible to and could be activated by a rise in the level of cytosolic Ca(2+), makes a major contribution to Ca(2+)-stimulated H(2)O(2) generation. This work highlights a unique high-Ca(2+)-stimulated reactive oxygen species-forming mechanism in association with oxidation of α-GP, which is largely independent of the bioenergetic state and can proceed even in damaged, functionally incompetent mitochondria.