Figure 2.

Alamethicin permeabilization promotes a rapid increase in H₂O₂ production when complex II is inhibited with atpenin A5 or complex III is inhibited with stigmatellin. A, isolated cardiac mitochondria (Mito) were added in concentrations of 1.0, 0.75, or 0.5 mg/ml (traces a–c, respectively) to buffer containing atpenin A5 (AA5, 0.5 μm). After permeabilization with alamethicin (Ala, 20 μg/ml), H₂O₂ production increased and plateaued as endogenous substrates were depleted and then reaccelerated after addition of succinyl-CoA (Suc-CoA) (0.15 mm). B, mitochondria were added at concentrations of 1.0 (trace a) and 0.5 (trace b) mg/ml to buffer containing stigmatellin (Stigm, 1 μm), followed by alamethicin and succinate (50 μm) as indicated. Decreased H₂O₂ production was reactivated with 50 μm succinate and inhibited in trace a with 0.5 and 5 mm succinate (Succ) and in trace b with 1 mm malonate (Malon). In this and other figures, additions to all traces are indicated by black arrows; additions to one trace only are indicated by the arrows of the same color as the trace. C, summary data (median ±95% confidence intervals, *, p < 0.05) for H₂O₂ production in AA5- and stigmatellin-inhibited mitochondria after alamethicin permeabilization and inhibition by malonate.