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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 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.
free radicals; skeletal muscle; respiratory muscles
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