Résumé :
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Communication n° 309 Under physiological conditions, muscle fibers continually produce reactive oxygen species (ROS) and nitric oxide (NO). Intracellular oxidant activity is increased by fatiguing exercise, inflammatory mediators, and heat stress. Other physiological factors that affect oxidant regulation remain poorly defined. We hypothesized that 1/ detectable NO and ROS activities could be measured in the cytosol of intact fibers. 2/ stretch, CO2 exposure, and cooling would alter oxidant activity in muscle fibers. The fluorescent oxidant probe, 2',7'-dichlorofluorescein (DCFH), was used to measure oxidant activity in muscle fibers from mouse diaphragm, extensor digitorus longus (EDL), and soleus. Muscle preparations were isolated in vitro, loaded with DCFH, and incubated under control (Lo, 37oC, CO2/O2 5%/95%) or experimental conditions using paired comparisons. We found that: 1) In DCFH loaded muscle, fluorescence was diminished by the NO synthase inhibitor L-NAME (43.6% control; P<0.05) and by hemoglobin (55%; P < 0.05). D-NAME had no effect and the NO donor increased fluorescence (284%; P<0.01). Anti-ROS enzymes diminished fluorescence (catalase 69.3%, SOD 33%; P<0.01) and H2O2 increased the signal (344%; P<0.05). These data indicate that muscle-derived NO and ROS are detectable within the cytosolic compartment of skeletal muscle fibers and contribute importantly to intracellular oxidant activity under resting conditions. 2.) Stretch from 90% to 110% optimal length increased oxidant activity in muscle fibers of soleus (123%; p<0.001) and EDL (171%; p<0.01). 3.) In diaphragm fibers, oxidant activity correlated positively with CO2 exposure (0-10%; P<0.05) and negatively with concurrent changes in pH (7.0-8.5; P<0.05). 4.) Cooling from 37oC to 23oC decreased oxidant activity in fibers of diaphragm (-55%, P<0.001) and EDL (-69%, P<0.001). We conclude that passive stretch and CO2 exposure increase cytosolic oxidant activity in skeletal muscle fibers whereas cooling decreases activity. Supported by the National Space Biomedical Research Institute, the Muscular Dystrophy Association, and NIH grant HL45721.
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