The ability of cells and tissues to defend against oxidative stress is determined by the supply of reducing equivalents. Our data demonstrate that cellular energy levels are not directly related to the ability of heart tissue to supply reducing equivalents in response to an oxidative stress. However, the pathways involved in this supply remain unclear, particularly if reactions which complete for reducing equivalents, such as ATP synthesis, are active. Studies in heart are of interest because of the sustained high respiration of this organ, and the potential for oxidative injury during reperfusion subsequent to infarction or surgery. Glutathione (GSH) is a major component of cellular antioxidant systems. GSH is maintained in the reduced form by glutathione reductase. Although this enzyme is specific for NADPH, the ability of intact cells, isolated mitochondria (which are a major source of free radicals and contain antioxidant systems independent of the rest of the cell), and whole tissues to supply reducing equivalents and maintain normal levels of GSH appears to involve NADH. The specific hypotheses to be tested are that: l) reducing equivalents used to reduce exogenous oxidizing agents can be supplied by NADH-linked substrates, 2) the supply of reducing equivalents to defend against oxidative stress is augmented under state 4 and diminished under state 3 respiration, and 3) intact organs can supply reducing equivalents in response to an oxidative stress more effectively than isolated cells. The validity of these hypotheses will be tested by using intact heart tissue, isolated cardiac mitochondria and cardiomyocytes provided with the following energy-linked substrates: glucose, acetate, lactate or pyruvate (intact heart and cardiomyocytes); malate/glutamate, succinate or octanoate (mitochondria). In each of these systems, the contents of ATP, phosphocreatine, NAD, NADH, NADP, and NADPH will be determined and correlated with levels of GSH, GSSH, protein thiols, and protein GSH-mixed disulfides. The effect of oxidatively stressing these systems with diamide (a direct thiol oxidant) or t-butyl hydroperoxide will be determined. The pathways by which reducing equivalents are supplied will be further assessed by providing the different energy-linked substrates in the presence of inhibitors of respiration (rotenone, antimycin A or FCCP [carbonyl cyanide p- trifluoromethoxyphenylhydrazone]). Mitochondrial substrate and inhibitor studies will be performed in both state 4 (without ADP) and state 3 (with ADP) respiration. These data will provide important new information on the pathways used by tissues to supply reducing equivalents and will reveal whether differences, which appear to exist between an intact organ and isolated cells, are real. The results may have important clinical relevance in the context of designing strategies for augmenting the ability of tissues to defend themselves against oxidative stress induced by reperfusion or xenobiotics.
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