Espinosa Alejandra, Leiva Aida, Peña Marisol, Müller Mariolly, Debandi Anibal, Hidalgo Cecilia, Carrasco M Angélica, Jaimovich Enrique
Escuela de Tecnología Médica, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
J Cell Physiol. 2006 Nov;209(2):379-88. doi: 10.1002/jcp.20745.
Controlled generation of reactive oxygen species (ROS) may contribute to physiological intracellular signaling events. We determined ROS generation in primary cultures of rat skeletal muscle after field stimulation (400 1-ms pulses at a frequency of 45 Hz) or after depolarization with 65 mM K+ for 1 min. Both protocols induced a long lasting increase in dichlorofluorescein fluorescence used as ROS indicator. Addition of diphenyleneiodonium (DPI), an inhibitor of NAD(P)H oxidase, PEG-catalase, a ROS scavenger, or nifedipine, an inhibitor of the skeletal muscle voltage sensor, significantly reduced this increase. Myotubes contained both the p47phox and gp91phox phagocytic NAD(P)H oxidase subunits, as revealed by immunodetection. To study the effects of ROS, myotubes were exposed to hydrogen peroxide (H2O2) at concentrations (100-200 microM) that did not alter cell viability; H2O2 induced a transient intracellular Ca2+ rise, measured as fluo-3 fluorescence. Minutes after Ca2+ signal initiation, an increase in ERK1/2 and CREB phosphorylation and of mRNA for the early genes c-fos and c-jun was detected. Inhibition of ryanodine receptor (RyR) decreased all effects induced by H2O2 and NAD(P)H oxidase inhibitors DPI and apocynin decreased ryanodine-sensitive calcium signals. Activity-dependent ROS generation is likely to be involved in regulation of calcium-controlled intracellular signaling pathways in muscle cells.
活性氧（ROS）的可控生成可能有助于生理细胞内信号传导事件。我们测定了大鼠骨骼肌原代培养物在电场刺激（45 Hz频率下的400个1毫秒脉冲）或用65 mM K⁺去极化1分钟后的ROS生成情况。这两种方案均诱导用作ROS指示剂的二氯荧光素荧光持续增加。添加NAD(P)H氧化酶抑制剂二苯基碘鎓（DPI）、ROS清除剂聚乙二醇过氧化氢酶或骨骼肌电压传感器抑制剂硝苯地平，可显著降低这种增加。免疫检测显示，肌管同时含有p47phox和gp91phox吞噬性NAD(P)H氧化酶亚基。为了研究ROS的作用，将肌管暴露于浓度为（100 - 200 microM）且不改变细胞活力的过氧化氢（H₂O₂）中；H₂O₂诱导了以fluo - 3荧光测量的细胞内Ca²⁺瞬时升高。在Ca²⁺信号启动数分钟后，检测到ERK1/2和CREB磷酸化增加以及早期基因c - fos和c - jun的mRNA增加。ryanodine受体（RyR）的抑制降低了H₂O₂诱导的所有效应，NAD(P)H氧化酶抑制剂DPI和夹竹桃麻素降低了对ryanodine敏感的钙信号。活性依赖的ROS生成可能参与肌肉细胞中钙控制的细胞内信号通路的调节。
