FIGURE 8.
NNT is the principal source of NADPH in depolarized mitochondria resulting from inhibition of electron transport and respiratory activity. As indicated in the representative traces (A–C), the addition of 0.5 mg/ml isolated mitochondria (Mito) was followed by blockage of electron transport induced by the addition of a respiratory complex III inhibitor (2 μm antimycin A, AA), whereas the autofluorescence of reduced NAD(P) and the mitochondrial membrane potential (Δψ, with the use of the dye safranine O) were monitored simultaneously. The NAD(P)H autofluorescence traces are exhibited only after the addition of antimycin A because this reagent causes optical drift. A bolus of t-BOOH was added as indicated before and after the addition of 2 μm rotenone (Rot) to block respiratory complex I (CI) (A and B); alternatively, rotenone was replaced by 20 mm succinate to block forward complex I electron transfer (C). Mitochondria from Nnt−/− were never able to recover the reduced state of NAD(P) when t-BOOH was added in the presence of antimycin A plus rotenone (B) or antimycin A plus succinate (C). Isocitrate (1 mm, Isoc) was added at the end of all traces to fully reduce NAD(P)+. The mean ± S.D. rates of t-BOOH metabolism under these two different experimental conditions are shown in D (n = 4); * indicates different (p < 0.05) from Nnt+/+ in the same condition; # indicates different (p < 0.05) from Nnt+/+ in the presence of antimycin A only; n.d., not detected.