Silveira Leonardo R, Pereira-Da-Silva Lucia, Juel Carsten, Hellsten Ylva
Departamento de Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas, São Paulo, Brazil.
Free Radic Biol Med. 2003 Sep 1;35(5):455-64. doi: 10.1016/s0891-5849(03)00271-5.
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.
我们检测了原代大鼠骨骼肌细胞培养物收缩过程中细胞内和细胞外过氧化氢（H₂O₂）及一氧化氮（NO）的生成情况。分别使用荧光探针2,7 - 二氯荧光素二乙酸酯/2,7 - 二氯荧光素（DCFH - DA/DCFH）和4,5 - 二氨基荧光素二乙酸酯/4,5 - 二氨基荧光素（DAF - 2 - DA/DAF - 2）来检测H₂O₂和NO。与未受刺激的对照细胞相比，对肌肉细胞进行强电刺激后，细胞内和细胞外的DCF荧光分别增加了171%和105%（p <.05）。在电刺激前添加谷胱甘肽（GSH）或钛铁试剂可抑制细胞内DCFH的氧化（p <.05），而添加谷胱甘肽过氧化物酶（GSH - PX）+ GSH则可抑制细胞外DCFH的氧化（p <.05）。强电刺激还分别使细胞内和细胞外的DAF - 2荧光信号增加了56%和20%（p <.05）。添加N(G)-硝基-L-精氨酸（L-NA）可完全消除细胞内和细胞外的DAF - 2荧光信号。我们的结果表明，收缩过程中骨骼肌细胞会生成H₂O₂和NO，提示H₂O₂和NO的快速释放可能构成一种重要的防御机制，以抵御细胞内羟自由基（*OH）和过氧亚硝酸盐（*ONOO）的形成。此外，我们的数据表明，DCFH和DAF - 2是适用于检测骨骼肌细胞培养物中细胞内和细胞外活性氧（ROS）及NO的探针。
