Oxidant activity in skeletal muscle fibers is influenced by temperature, CO2 level, and muscle-derived nitric oxide

Abstract

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) CO2 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°C to 23°C ( P < 0.001). The rate of DCFH oxidation correlated positively with CO2 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 μM), NO synthase inhibitors ( Nω-nitro-l-arginine methyl ester, 1 mM; Nω-monomethyl-l-arginine, 1 mM), or an NO scavenger (hemoglobin, 1 μM) each inhibited DCFH oxidation ( P < 0.05). Oxidation was increased by hydrogen peroxide, 100 μM, an NO donor (NOC-22, 400 μM), 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 CO2 exposure, and 3) influenced by muscle-derived ROS and NO derivatives to similar degrees.