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

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• 作者：Silveira ; Leonardo R. ; Pereira-Da-Silva ; Lucia ; Juel ; Carsten ; Hellsten ; Ylva
• 关键词：Reactive oxygen species ; Cell culture ; Oxidative stress ; Free radicals
• 刊名：Free Radical Biology and Medicine
• 出版年：2003
• 期刊代码：105_08915849
• 类别：med
• 出版时间：September 1, 2003
• 卷：35
• 期：5
• 页码：455-464
• 文件大小：189 K

摘要

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 N^G-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.