Formation of hydrogen peroxide and nitric oxide in rat skeletal muscle cells during contractions

We examined intra- and extracellular H2O2 and NO formation during contractions in primary rat skeletal muscle cell culture. The fluorescent probes DCFH-DA/DCFH (2,7-dichlorofluorescein-diacetate/2,7-dichlorofluorescein) and DAF-2-DA/DAF-2 (4,5-diaminofluorescein-diacetate/4,5-diaminofluorescein) were used to detect H2O2 and NO, respectively. Intense electrical stimulation of muscle cells increased the intra- and extracellular DCF fluorescence by 171% and 105%, respectively, compared with control nonstimulated cells (p < .05). The addition of glutathione (GSH) or Tiron prior to electrical stimulation inhibited the intracellular DCFH oxidation (p < .05), whereas the addition of GSH-PX + GSH inhibited the extracellular DCFH oxidation (p < .05). Intense electrical stimulation also increased (p < .05) the intra- and extracellular DAF-2 fluorescence signal by 56% and 20%, respectively. The addition of NG-nitro-L-arginine (L-NA) completely removed the intra- and extracellular DAF-2 fluorescent signal. Our results show that H2O2 and NO are formed in skeletal muscle cells during contractions and suggest that a rapid release of H2O2 and NO may constitute an important defense mechanism against the formation of intracellular •OH and •ONOO. Furthermore, our data show that DCFH and DAF-2 are suitable probes for the detection of ROS and NO both intra- and extracellularly in skeletal muscle cell cultures.