Formation of hydrogen peroxide and nitric oxide in rat skeletal muscle cells during contractions.
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.
Silveira
Leonardo R
LR
Departamento de Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas, São Paulo, Brazil. yhellsten@aki.ku.dk
Pereira-Da-Silva
Lucia
L
Juel
Carsten
C
Hellsten
Ylva
Y
D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D004558:Electric Stimulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D019793:Fluorescein; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005978:Glutathione / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism*; D017665:Hydroxyl Radical / Q000378:metabolism; D007202:Indicators and Reagents / Q000494:pharmacology; D009119:Muscle Contraction / Q000502:physiology*; D018482:Muscle, Skeletal / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis*; D019335:Nitroarginine / Q000494:pharmacology; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000502:physiology*; D030421:Peroxynitrous Acid / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
C414689:4,5-diaminofluorescein diacetate; D004791:Enzyme Inhibitors; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D030421:Peroxynitrous Acid; C029569:diacetyldichlorofluorescein; D019335:Nitroarginine; D009569:Nitric Oxide; D017665:Hydroxyl Radical; D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; D006861:Hydrogen Peroxide; D005978:Glutathione; D019793:Fluorescein