Lipoxygenase-dependent superoxide release in skeletal muscle.
Superoxide anion radical (O(2)(*-)) is released from skeletal muscle at rest and is particularly elevated during conditions of heat stress (42 degrees C). Previous studies have shown that in isolated rat diaphragm O(2)(*-) release is not dependent on mitochondrial electron transport, reduced NADP oxidase activity, or the integrity of membrane anion channels. This study hypothesized that O(2)(*-) release, as measured by cytochrome c reduction, is linked to metabolism of arachidonic acid. Phospholipase A(2) inhibition with manoalide significantly decreased O(2)(*-) release. In downstream pathways, neither the blockage of cyclooxygenase with indomethacin nor the inhibition of cytochrome P-450-dependent monooxygenase with SKF-525A decreased O(2)(*-) release. However, lipoxygenase (LOX) inhibition with general LOX blockers 5,8,11,14-eicosatetraynoic acid and cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate greatly attenuated the signal. Furthermore, the specific 5-LOX inhibitor diethylcarbamazine also significantly decreased O(2)(*-) release. Immunohistochemistry localized 5- and 12-LOX to the cytosol and sarcolemma of muscle cells. Confocal studies, using the O(2)(*-)-sensitive fluorescent indicator hydroethidine, demonstrated that LOX inhibition had no significant influence on intracellular O(2)(*-) formation. When compared with the cytochrome c results, this indicates that intra- and extracellular O(2)(*-) must arise from different sources. These data show for the first time that arachidonic acid metabolism through LOX activity, is a major source of extracellular O(2)(*-) release in skeletal muscle.
Zuo
Li
L
Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Dorothy M. Davis Heart and Lung Research Institute, 473 W 12th Ave., Columbus, OH 43210, USA.
Christofi
Fievos L
FL
Wright
Valerie P
VP
Bao
Shengying
S
Clanton
Thomas L
TL
D015117:5,8,11,14-Eicosatetraynoic Acid / Q000494:pharmacology; D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000378:metabolism*; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D016718:Arachidonic Acid / Q000378:metabolism; D002109:Caffeic Acids / Q000494:pharmacology; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007213:Indomethacin / Q000494:pharmacology; D016859:Lipoxygenase Inhibitors; D008297:Male; D018482:Muscle, Skeletal / Q000201:enzymology*; D011335:Proadifen / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013481:Superoxides / Q000378:metabolism*
D002109:Caffeic Acids; D016861:Cyclooxygenase Inhibitors; D004791:Enzyme Inhibitors; D016859:Lipoxygenase Inhibitors; C099255:cinnamyl-3,4-dihydroxycyanocinnamate; D013481:Superoxides; D015117:5,8,11,14-Eicosatetraynoic Acid; D016718:Arachidonic Acid; D011335:Proadifen; D001092:Arachidonate 12-Lipoxygenase; D001094:Arachidonate 5-Lipoxygenase; D007213:Indomethacin