Oxypurinol and indomethacin effects on superoxide release by the contracting isolated diaphragm

Several reports have indicated that free radicals are generated within the diaphragm during strenuous contraction, but the mechanism of this free radical generation remains unclear. In theory, a number of metabolic pathways may contribute to this process, including the generation of Superoxide as a byproduct of xanthine oxidase-catalyzed reactions and as a byproduct of prostaglandin metabolism. The purpose of the present study was to examine the effects of oxypurinol (a xanthine oxidase inhibitor) and indomethacin (an inhibitor of prostaglandin metabolism) on Superoxide release by the contracting diaphragm. Studies were performed using isolated, arterially perfused rat hemidiaphragms for which Superoxide release was assessed by measuring arterio-venous cytocbrome c reduction gradients across this muscle and calculating cytochrome c reduction rates (CCRR). CCRR was measured at rest and during electrically stimulated diaphragmatic contractions (15 contractions/min at 20 Hz) in 6 control, 5 oxypurinol treated (50 mg/1) and 6 indomethacin treated (10 mg/1) diaphragms. We observed a small CCRR in the resting control diaphragm (78 ±_ 10 pmole/min) that was reduced by indomethacin (32 ±_ 11 pmol/min, p<.02) and was unaffected by oxypurinol (87 ±_ 9 pmol/min). During contraction, CCRR rose to 127 +_ 12 pmol/min above resting levels for controls and by 136 ±20 pmol/min for the indomethacin group, but only increased by 36 ±_ 10 pmqlnin in the oxypurinol group (p< .01). In additional experiments, Superoxide dismutase (4000 unit/1) also suppressed contraction-related CCRR. These data suggest that superoxideproduction and release by the contracting in vitro diaphragm is unrelated to prostaglandin metabolism and is primarily due to xanthine oxidase catalyzed reactions.