Oxidant activity in skeletal muscle fibers is influenced by temperature, CO2 level, and muscle-derived nitric oxide.
Free radicals are produced continuously by skeletal muscle fibers. Extracellular release of reactive oxygen species (ROS) and nitric oxide (NO) derivatives has been demonstrated, but little is known about intracellular oxidant regulation. We used a fluorescent oxidant probe, 2',7'-dichlorofluorescin (DCFH), to assess net oxidant activity in passive muscle fiber bundles isolated from mouse diaphragm and studied in vitro. We tested the following three hypotheses. 1) Net oxidant activity is decreased by muscle cooling. 2) CO(2) exposure depresses intracellular oxidant activity. 3) Muscle-derived ROS and NO both contribute to overall oxidant activity. Our results indicate that DCFH oxidation was diminished by cooling muscle fibers from 37 degrees C to 23 degrees C (P < 0.001). The rate of DCFH oxidation correlated positively with CO(2) exposure (0-10%; P < 0.05) and negatively with concurrent changes in pH (7.0-8.5; P < 0.05). Separate exposures to anti-ROS enzymes (superoxide dismutase, 1 kU/ml; catalase, 1 kU/ml), a glutathione peroxidase mimetic (ebselen, 30 microM), NO synthase inhibitors (N(omega)-nitro-l-arginine methyl ester, 1 mM; N(omega)-monomethyl-l-arginine, 1 mM), or an NO scavenger (hemoglobin, 1 microM) each inhibited DCFH oxidation (P < 0.05). Oxidation was increased by hydrogen peroxide, 100 microM, an NO donor (NOC-22, 400 microM), or the substrate for NO synthase (l-arginine, 5 mM). We conclude that net oxidant activity in resting muscle fibers is 1) decreased at subphysiological temperatures, 2) increased by CO(2) exposure, and 3) influenced by muscle-derived ROS and NO derivatives to similar degrees.
Arbogast
Sandrine
S
Dept. of Physiology, Univ. of Kentucky, 800 Rose St., Rm. MS-509; Lexington, KY 40536-0298, USA.
Reid
Michael B
MB
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001393:Azoles / Q000494:pharmacology; D002245:Carbon Dioxide / Q000494:pharmacology*; D002374:Catalase / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D005456:Fluorescent Dyes; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007091:Image Processing, Computer-Assisted; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D054833:Isoindoles; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D018485:Muscle Fibers, Skeletal / Q000201:enzymology / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D009569:Nitric Oxide / Q000502:physiology*; D016566:Organoselenium Compounds / Q000494:pharmacology; D016877:Oxidants / Q000378:metabolism* / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D013482:Superoxide Dismutase / Q000378:metabolism; D013696:Temperature
D000975:Antioxidants; D001393:Azoles; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D054833:Isoindoles; D016566:Organoselenium Compounds; D016877:Oxidants; D017382:Reactive Oxygen Species; D002245:Carbon Dioxide; D009569:Nitric Oxide; C042986:ebselen; D006861:Hydrogen Peroxide; D002374:Catalase; D013482:Superoxide Dismutase