Membrane potential and delta pH dependency of reverse electron transport-associated hydrogen peroxide production in brain and heart mitochondria

Succinate-driven reverse electron transport (RET) is one of the main sources of mitochondrial reactive oxygen species (mtROS) in ischemia-reperfusion injury. RET is dependent on mitochondrial membrane potential ((m)) and transmembrane pH difference (pH), components of the proton motive force (pmf); a decrease in (m) and/or pH inhibits RET. In this study we aimed to determine which component of the pmf displays the more dominant effect on RET-provoked ROS generation in isolated guinea pig brain and heart mitochondria respiring on succinate or -glycerophosphate (-GP). (m) was detected via safranin fluorescence and a TPP+ electrode, the rate of H2O2 formation was measured by Amplex UltraRed, the intramitochondrial pH (pH(in)) was assessed via BCECF fluorescence. Ionophores were used to dissect the effects of the two components of pmf. The K+/H+ exchanger, nigericin lowered pH(in) and pH, followed by a compensatory increase in (m) that led to an augmented H2O2 production. Valinomycin, a K+ ionophore, at low [K+] increased pH and pH(in), decreased (m), which resulted in a decline in H2O2 formation. It was concluded that (m) is dominant over pH in modulating the succinate- and -GP-evoked RET. The elevation of extramitochondrial pH was accompanied by an enhanced H2O2 release and a decreased pH. This phenomenon reveals that from the pH component not pH, but rather absolute value of pH has higher impact on the rate of mtROS formation. Minor decrease of (m) might be applied as a therapeutic strategy to attenuate RET-driven ROS generation in ischemia-reperfusion injury.