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

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We examined intra- and extracellular H(2)O(2) 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 H(2)O(2) 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 N(G)-nitro-L-arginine (L-NA) completely removed the intra- and extracellular DAF-2 fluorescent signal. Our results show that H(2)O(2) and NO are formed in skeletal muscle cells during contractions and suggest that a rapid release of H(2)O(2) 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.
OriginalsprogEngelsk
TidsskriftFree Radical Biology & Medicine
Vol/bind35
Udgave nummer5
Sider (fra-til)455-464
Antal sider10
ISSN0891-5849
DOI
StatusUdgivet - 2003

Bibliografisk note

PUF 2003 5200 219

ID: 17521247