Reduction of glutathione disulfide and the maintenance of reducing equivalents in hypoxic hearts after the infusion of diamide

Abstract

A tissue's response to an oxidative stress is related to its capacity to supply reducing equivalents and may be affected by energy levels. The ability of intact rat heart tissue to supply NADPH and reduce glutathione disulfide (GSSG) produced by diamide was determined under normoxic or hypoxic conditions with and without glycolytic energy production. Cardiac ATP and phosphocreatine (PCr) levels remained relatively constant (∼ 20 nmol/mg dry weight) during a 60 min perfusion with oxygenated Krebs-Henseleit buffer containing glucose. Levels of ATP and PCr were depleted 85–92% following 60 min of hypoxia. A 5 min infusion of 800 μM diamide, after 60 min of normoxia or hypoxia, oxidized 70–80% of cardiac glutathione (GSH), but had no effect on total glutathione. After a subsequent 25 min diamide-free perfusion, 75–85% of the GSSG formed was reduced in both normoxic and hypoxic hearts. The removal of glucose, or the inhibition of glycolysis with2-deoxy- D-glucose, did not effect GSSG reduction. Cardiac NADH levels were increased from 0.05 to 0.5 nmol/mg dry weight after 60 min hypoxia in hearts perfused with or without glucose. A 5 min infusion of diamide in hypoxic hearts slightly decreased NADH levels, but there was no further change after a subsequent 25 min diamide-free period. Inhibition of glutathione reductase with 1,3-bis(2-chloroethyl)-1-nitrosourea p prevented GSSG reduction, showing NADPH was required. However, NADPH levels were not affected by hypoxia or diamide infusion and remained constant at 0.2 nmol/mg dry weight in hearts perfused with or without glucose. Inhibition of glycolysis with 2-deoxy- D-glucose also did not affect NADPH levels. These results demonstrate that hypoxia did not affect the ability of oxidatively stressed, intact heart tissue to supply NADPH for the reduction of GSSG. In addition, GSSG reduction was independent of energy levels and appeared to be unaffected by glucose availability. NADH may be involved in maintaining NADPH levels through interconversion pathways.