Fig. 2.
Peroxide-induced permeability pore transition (PTP) opening and pyridine nucleotide oxidation in rat liver mitochondria. A. Representative tracings of respiration-dependent mitochondrial Ca2+ uptake and subsequent PTP-dependent release. Extramitochondrial free Ca2+ was monitored with the Calcium Green 5N fluorescent dye in medium containing succinate as a respiratory substrate and the respiratory chain complex I inhibitor, rotenone. At time 0 mitochondria (0.5 mg/ml) from vehicle-treated or sulforaphane (SFP)-treated rats were loaded with 25 μM calcium, which is a level that was retained almost completely during the duration of the measurements (Line 1 & 4). At 250 sec, tert-butyl hydroperoxide (tBOOH) was added at either 5 μM (Line 2) or 250 μM (Line 3), resulting in an almost immediate increase in calcium green 5N fluorescence, representing mitochondrial Ca2+ release. Ca2+ efflux from control mitochondria was completely inhibited by the presence of 1 μM cyclosporin A (CsA) (Line 7) and was substantially greater than that observed with mitochondria from sulforaphane-treated rats (Lines 5 & 6). At 450 sec, ionomycin, a non-specific calcium ionophore, was added to completely release the small, residual mitochondrial Ca2+ (Line 3). B. Representative tracings of NAD(P)H autofluorescence in the absence and presence of tBOOH. Pyridine nucleotide redox state for mitochondria from either vehicle- or sulforaphane-treated rats remained highly reduced and stable after the addition of Ca2+ alone (Lines 1 & 4). The addition of 5 μM (Line 2) or 250 μM (Line 3) tBOOH caused immediate and complete oxidation of NAD(P)H within vehicle control mitochondria. Treatment of rats with SFP inhibited NAD(P)H oxidation at 5 μM tBOOH (Line 5), but did not inhibit oxidation at 250 μM tBOOH (Line 6).