Our overriding hypothesis is that oxygen radicals are novel mediators of normal diaphragm physiology, modulating contractile properties and accelerating fatigue. We will test this hypothesis using selective antioxidants to identify the participation of specific oxygen radicals and measuring tissue glutathione as an endogenous indicator of oxidative stress. Observations in vitro and in situ will be compared to determine the site(s) and physiologic importance of oxidant effects. We have three Specific Aims: 1. To evaluate selected oxygen radical species as endogenous mediators of diaphragm contractile properties, discriminating effects on neuromuscular excitation from effects on intracellular excitation-contraction coupling. We hypothesize that endogenous oxygen radicals influence twitch characteristics and depress submaximal tetanic force by altering excitation-contraction coupling. Expt. 1 will measure contractile properties of curarized diaphragm bundles in vitro, with or without antioxidant treatment. We further hypothesize that oxidant stress may influence neuromuscular excitation. This will be tested by comparing antioxidant effects on curarized bundles with similar data from indirectly- stimulated, phrenic nerve-diaphragm preparations. 2. To determine whether endogenous oxygen radicals act intracellularly to accelerate fatigue of diaphragm myocytes and, if so, to identify the molecular species involved. Oxidant stress contributes to acute fatigue of skeletal muscles, including the diaphragm, by an unknown mechanism. We hypothesize that endogenous oxygen radicals act directly on the myocyte to depress function. Expt. 2 will test intracellular effects by screening selective antioxidant probes for protection against acute fatigue of skeletal muscles, including the diaphragm, by an unknown mechanism. We hypothesize that endogenous oxygen radicals act directly on the myocyte to depress function. Expt. 2 will test intracellular effects by screening selective antioxidant probes for protection against acute fatigue of directly-stimulated fiber bundles. We also hypothesize that the ratio of reduced-to-oxidized glutathione (GSH:GSSG; a nonspecific measure of oxidant stress) will be less in fatigued than unfatigued bundles. We propose to measure tissue GSG:GSSG under both conditions. 3. To determine the physiological importance of oxygen radicals in diaphragm fatigue. Because in vitro conditions may profoundly and unpredictably influence oxidant kinetics, results of Expt. 2 will be clarified in Expt. 3 by testing the hypothesis that oxygen radicals accelerate fatigue of the normoxic diaphragm when perfused with blood and stimulated neurally. We also will test the hypothesis that the arteriovenous difference in blood GSH:GSSG increases as mechanical failure develops, providing humoral evidence of fatigue.
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