Formation of reactive oxygen species by the contracting diaphragm is PLA(2) dependent

Recent work indicates that respiratory muscles generate superoxide radicals during contraction (M. B. Reid, K. E. Haack, K. NI. Francik, P.A. Volberg, L. Kabzik, and M. S. West. J. Appl. Physiol. 73: 1797-1804, 1992). The int racellular pathways involved in this process are, however, unknown. The pur pose of the present study was to test the hypothesis that contraction-relat ed formation of reactive oxygen species (ROS) by skeletal muscle is linked to activation of the 14-kDa isoform of phospholipase A(2) (PLA(2)). 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 rec ently described fluorescent indicator technique. We examined ROS formation in resting and contracting muscle preparations sind then determined whether contraction-related ROS generation could be altered by administration of v arious PLA(2) inhibitors: manoalide and aristolochic acid, both inhibitors of 14-kDa PLA(2); arachidonyltrifluoromethyl ketone (AACOCF(3)), an inhibit or of 85-kDa PLA(2); and haloenol lactone suicide substrate (HELSS), an inh ibitor of calcium-independent PLA(2). 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 diaphrag ms (trains of 20-Hz stimuli for 10 min, train rate 0.25 s(-1)), 3) near-com plete 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 co ntracting diaphragm. During in vitro studies examining fluorescent measurem ent of ROS formation in response to a hypoxanthine/xanthine oxidase superox ide-generating solution, manoalide, aristolochic acid, AACOCF3, and HELSS h ad no effect on signal intensity. These data indicate that ROS formation by contracting diaphragm muscle can be suppressed by the administration of in hibitors of the 14-kDa isoform of PLA(2) and suggest; that this enzyme play s a critical role in modulating ROS formation during muscle contraction.