REDUCTION OF GLUTATHIONE DISULFIDE AND THE MAINTENANCE OF REDUCING EQUIVALENTS IN HYPOXIC HEARTS AFTER THE INFUSION OF DIAMIDE

A tissue's response to an oxidative stress is related to its capacity to supply reducing equivalents and may be affected by energy levels. T he ability of intact rat heart tissue to supply NADPH and reduce gluta thione disulfide (GSSG) produced by diamide was determined under normo xic or hypoxic conditions with and without glycolytic energy productio n. Cardiac ATP and phosphocreatine (PCr) levels remained relatively co nstant (approximate to 20 nmol/mg dry weight) during a 60 min perfusio n 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 mu M diamide, after 60 min of normoxia or hypoxia, ox idized 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. T he removal of glucose, or the inhibition of glycolysis with 2-deoxy-D- glucose, did not affect GSSG reduction. Cardiac NADH levels were incre ased from 0.05 to 0.5 nmol/mg dry weight after 60 min hypoxia in heart s perfused with or without glucose. A 5 min infusion of diamide in hyp oxic hearts slightly decreased NADH levels, but there was no further c hange after a subsequent 25 min diamide-free period. Inhibition of glu tathione reductase with 1,3-bis(2-chloroethyl)-1-nitrosourea prevented GSSG reduction, showing NADPH was required. However, NADPH levels wer e not affected by hypoxia or diamide infusion and remained constant at 0.2 nmol/mg dry weight in hearts perfused with or without glucose. In hibition of glycolysis with 2-deoxy-D-glucose also did not affect NADP H levels. These results demonstrate that hypoxia did not affect the ab ility 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 interconversi on pathways.