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

Abstract

H2O2 generation associated with α-glycerophosphate (α-GP) oxidation was addressed in guinea pig brain mitochondria challenged with high Ca2+ load (10μM). Exposure to 10μM Ca2+ induced an abrupt 2.5-fold increase in H2O2 release compared to that measured in the presence of a physiological cytosolic Ca2+ concentration (100nM) from mitochondria respiring on 5mM α-GP in the presence of ADP (2mM). The Ca2+-induced stimulation of H2O2 generation was reversible and unaltered by the uniporter blocker Ru 360, indicating that it did not require Ca2+ uptake into mitochondria. Enhanced H2O2 generation by Ca2+ 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 H2O2 generation to a challenge with 10μM Ca2+. Ca2+-induced promotion of H2O2 formation was further enhanced by the complex III inhibitor myxothiazol. With 20mM α-GP concentration, stimulation of H2O2 formation by Ca2+ 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 Ca2+, makes a major contribution to Ca2+-stimulated H2O2 generation. This work highlights a unique high-Ca2+-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.