Examination of the downstream mediators responsible for inhibition of mitochondrial respiration by dopamine (DA) was investigated. Consistent with findings reported by others, exposure of rat brain mitochondria to 0.5 mM DA for 15 min at 30°C inhibited pyruvate/glutamate/malate-supported state-3 respiration by 20%. Inhibition was prevented in the presence of pargyline and clorgyline demonstrating that mitochondrial inhibition arose from products formed following MAO metabolism and could include hydrogen peroxide (H2O2), hydroxyl radical, oxidized glutathione (GSSG) or glutathione-protein mixed disulfides (PrSSG). As with DA, direct incubation of intact mitochondria with H2O2 (100 μm) significantly inhibited state-3 respiration. In contrast, incubation with GSSG (1 mM) had no effect on O2 consumption. Exposure of mitochondria to 1 mM GSSG resulted in a 3.3-fold increase in PrSSG formation compared with 1.4- and 1.5-fold increases in the presence of 100 μm H2O2 or 0.5 mM DA, respectively, suggesting a dissociation between PrSSG formation and effects on respiration. The lack of inhibition of respiration by GSSG could not be accounted for by inadequate delivery of GSSG into mitochondria as increases in PrSSG levels in both membrane-bound (2-fold) and intramatrix (3.5-fold) protein compartments were observed. Furthermore, GSSG was without effect on electron transport chain activities in freeze-thawed brain mitochondria or in pig heart electron transport particles (ETP). In contrast, H2O2 showed differential effects on inhibition of respiration supported by different substrates with a sensitivity of succinate > pyruvate/malate > glutamate/malate. NADH oxidase and succinate oxidase activities in freeze-thawed mitochondria were inhibited with IC50 approximately 2-3-fold higher than in intact mitochondria. ETPs, however, were relatively insensitive to H2O2. Co-administration of desferrioxamine with H2O2 had no effect on complex I-associated inhibition in intact mitochondria, but attenuated inhibition of rotenone-sensitive NADH oxidase activity by 70% in freeze- thawed mitochondria. The results show that DA-associated inhibition of respiration is dependent on MAO and that H2O2 and its downstream hydroxyl radical rather than increased GSSG and subsequent PrSSG formation mediate the effects.
- Glutathione-protein-mixed disulfides
- Parkinson's disease