Formation of reactive oxygen species by the contracting diaphragm is PLA₂ dependent

Recent work indicates that respiratory muscles generate superoxide radicals during contraction (M. B. Reid, K. E. Haack, K. M. Francik, P. A. Volberg, L. Kabzik, and M. S. West. J. Appl. Physiol. 73: 1797-1804, 1992). The intracellular pathways involved in this process are, however, unknown. The purpose of the present study was to test the hypothesis that contraction- related formation of reactive oxygen species (ROS) by skeletal muscle is linked to activation of the 14-kDa isoform of phospholipase A₂ (PLA₂). Studies were performed by using an in vitro hemidiaphragm preparation submerged in an organ bath, and formation of ROS in muscles was assessed by using a recently described fluorescent indicator technique. We examined ROS formation in resting and contracting muscle preparations and then determined whether contraction-related ROS generation could be altered by administration of various PLA₂ inhibitors: manoalide and aristolochic acid, both inhibitors of 14-kDa PLA₂; arachidonyltrifluoromethyl ketone (AACOCF₃), an inhibitor of 85-kDa PLA₂; and haloenol lactone suicide substrate (HELSS), an inhibitor of calcium-independent PLA₂. We found 1) little ROS formation [2.0 ± 0.8 (SE) ng/mg] in noncontracting control diaphragms, 2) a high level of ROS (20.0 ± 2.0 ng/mg) in electrically stimulated contracting diaphragms (trains of 20-Hz stimuli for 10 min, train rate 0.25 s^-1), 3) near-complete suppression of ROS generation in manoalide (3.0 ± 0.5 ng/mg, P < 0.001)- and aristolochic acid-treated contracting diaphragms (4.0 ± 1.0 ng/mg, P < 0.001), and 4) no effect of AACOCF₃ or HELSS on ROS formation in contracting diaphragm. During in vitro studies examining fluorescent measurement of ROS formation in response to a hypoxanthine/xanthine oxidase superoxide- generating solution, manoalide, aristolochic acid, AACOCF₃, and HELSS had no effect on signal intensity. These data indicate that ROS formation by contracting diaphragm muscle can be suppressed by the administration of inhibitors of the 14-kDa isoform of PLA₂ and suggest that this enzyme plays a critical role in modulating ROS formation during muscle contraction.