Formation of reactive oxygen species by the contracting diaphragm is PLA₂dependent

Abstract

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 < 0. 001)- and aristolochic acid-treated contracting diaphragms (4.0 +/- 1.0 ng/mg, P < 0.001), and 4) no effect of AACOCF(3) 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, AACOCF(3), 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 PLA(2) and suggest that this enzyme plays a critical role in modulating ROS formation during muscle contraction.

Mitochondrial superoxide production in skeletal muscle fibers of the rat and decreased fiber excitability

“…, which is close to the reported rate values measured extracellularly with different techniques in mammalian skeletal muscles at rest at 37°C (26).…”

A Transverse Tubule NADPH Oxidase Activity Stimulates Calcium Release from Isolated Triads via Ryanodine Receptor Type 1 S -Glutathionylation

“…However, neither the precise cellular location of this NOX activity nor its potential functional effects have been reported. Mitochondrial complexes I and III and cytoplasmic xanthine oxidase are major sources of superoxide anion in skeletal muscle (58,59). Following muscle contraction, superoxide anion has been detected in the extracellular space (58,60,61).…”

The Radical Trap 5,5-Dimethyl-1-PyrrolineN-Oxide Exerts Dose-Dependent Protection against Myocardial Ischemia-Reperfusion Injury through Preservation of Mitochondrial Electron Transport

“…ET is positively charged and has better cellular retention and stability compared with HE. Thus, ET formation was chosen as an indicator of ROS production, which is a common method when using this probe (Nethery et al, 1999;Zuo et al, 2000;Zuo and Clanton, 2002). ROS fluorescence was measured using a tissue fluorometer (C&L Instruments, Inc., Hershey, PA).…”