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

Pulmonary Division, Department of Medicine, Case Western Reserve University and MetroHealth Medical Center, Cleveland, Ohio 44109 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 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 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 PLA 2 inhibitors: manoalide and aristolochic acid, both inhibitors of 14-kDa PLA 2 ; arachidonyltrifluoromethyl ketone (AACOCF 3 ), an inhibitor of 85-kDa PLA 2 ; and haloenol lactone suicide substrate (HELSS), an inhibitor 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 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