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

Abstract

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 ∼60% of total glutathione. In both the 500× g (with mitochondria) and 10 000× g (without mitochondria) rat heart supernatants, isocitrate supported reduction of ∼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 10 000× g supernatant and 47% in the 500× 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× g and reconstituted 500× g supernatants whereby G6-P-supported GSSG reduction is attenuated.