Depolarization of In Situ Mitochondria Due to Hydrogen Peroxide‐Induced Oxidative Stress in Nerve Terminals

Abstract

Mitochondrial membrane potential (Δ?_m) was determined in intact isolated nerve terminals using the membrane potential-sensitive probe JC-1. Oxidative stress induced by H₂O₂ (0.1-1 mM) caused only a minor decrease in Δ?_m. When complex I of the respiratory chain was inhibited by rotenone (2 μM), Δ?_m was unaltered, but on subsequent addition of H₂O₂, Δ?_m started to decrease and collapsed during incubation with 0.5 mM H₂O₂ for 12 min. The ATP level and [ATP]/[ADP] ratio were greatly reduced in the simultaneous presence of rotenone and H₂O₂. H₂O₂ also induced a marked reduction in Δ?_m when added after oligomycin (10 μM), an inhibitor of F₀F₁-ATPase. H₂O₂ (0.1 or 0.5 mM) inhibited α-ketoglutarate dehydrogenase and decreased the steady-state NAD(P)H level in nerve terminals. It is concluded that there are at least two factors that determine Δ?_m in the presence of H₂O₂: (a) The NADH level reduced owing to inhibition of α-ketoglutarate dehydrogenase is insufficient to ensure an optimal rate of respiration, which is reflected in a fall of Δ?_m when the F₀F₁-ATPase is not functional. (b) The greatly reduced ATP level in the presence of rotenone and H₂O₂ prevents maintenance of Δ?_m by F₀F₁-ATPase. The results indicate that to maintain Δ?_m in the nerve terminal during H₂O₂-induced oxidative stress, both complex I and F₀F₁-ATPase must be functional. Collapse of Δ?_m could be a critical event in neuronal injury in ischemia or Parkinson’s disease when H₂O₂ is generated in excess and complex I of the respiratory chain is simultaneously impaired.