Cosubstrates involved in the reduction of cytosolic glutathione disulfide in rat heart

The functionality of glutathione (GSH), which is present in separate mitochondrial and cytosolic pools, hinges on a steady supply of reducing equivalents, provided by NADPH, to convert glutathione disulfide (GSSG) to GSH. It is believed traditionally that glucose 6-phosphate (G6-P) via the pentose phosphate pathway is the main cellular source of NADPH. The current study examined the ability of NADH-and NADPH-linked cosubstrates to support cardiac cytosolic GSSG reduction. Exogenous NADP(+) was added to the incubation mixtures because of the loss of this nucleotide during homogenization. Exogenous GSSG was added to all samples to levels that were similar to 60% of total glutathione. In both the 500 x g (with mitochondria) and 10 000 x g (without mitochondria) rat heart supernatants, isocitrate supported reduction of similar to 90% of available GSSG within 10 min. Malate, pyruvate and palmitoyl carnitine did not support GSSG reduction in either supernatant. G6-P yielded GSH levels within 10 min equal to 77% of total glutathione in the 10000 x g supernatant and 47% in the 500 x g supernatant. The current data indicate: (1) The pentose phosphate pathway, alone, is less efficient than isocitrate at supplying reducing equivalents for cytosolic GSSG reduction; and (2) some confounding factor(s) occur in the 500 x g and reconstituted 500 x g supernatants whereby G6-P-supported GSSG reduction is attenuated. (C) 1997 Elsevier Science Ireland Ltd.