Pye Deborah, Palomero Jesus, Kabayo Tabitha, Jackson Malcolm J
Division of Metabolic and Cellular Medicine, School of Clinical Sciences, University of Liverpool, Liverpool L69 3GA, UK.
J Physiol. 2007 May 15;581(Pt 1):309-18. doi: 10.1113/jphysiol.2006.125930. Epub 2007 Mar 1.
Nitric oxide (NO) is thought to play multiple roles in skeletal muscle including regulation of some adaptations to contractile activity, but appropriate methods for the analysis of intracellular NO activity are lacking. In this study we have examined the intracellular generation of NO in isolated single mature mouse skeletal muscle fibres at rest and following a period of contractile activity. Muscle fibres were isolated from the flexor digitorum brevis muscle of mice and intracellular NO production was visualized in real-time using the fluorescent NO probe 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA). Some leakage of DAF-FM was apparent from fibres loaded with the probe, but they retained sufficient probe to respond to changes in intracellular NO following addition of the NO donor 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) up to 30 min after loading. Electrically stimulated contractions in isolated fibres increased the rate of change in DAF-FM fluorescence by approximately 48% compared to non-stimulated fibres (P < 0.05) and the rate of change in DAF-FM fluorescence in the stimulated fibres returned to control values by 5 min after contractions. Treatment of isolated fibres with the NO synthase inhibitors NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) or NG-monomethyl-L-arginine (L-NMMA) reduced the increase in DAF-FM fluorescence observed in response to contractions of untreated fibres. Treatment of fibres with the cell-permeable superoxide scavenger 4,5-dihydroxy-1,3-benzenedisulphonic acid (Tiron) also reduced the increase in fluorescence observed during contractions suggesting that superoxide, or more probably peroxynitrite, contributes to the fluorescence observed. Thus this technique can be used to examine NO generation in quiescent and contracting skeletal muscle fibres in real time, although peroxynitrite and other reactive nitrogen species may potentially contribute to the fluorescence values observed.
一氧化氮（NO）被认为在骨骼肌中发挥多种作用，包括调节对收缩活动的一些适应性反应，但目前缺乏分析细胞内NO活性的合适方法。在本研究中，我们检测了分离的单个成熟小鼠骨骼肌纤维在静息状态下以及经过一段时间收缩活动后的细胞内NO生成情况。从小鼠的趾短屈肌中分离出肌肉纤维，并使用荧光NO探针4-氨基-5-甲基氨基-2'，7'-二氟荧光素二乙酸酯（DAF-FM DA）实时观察细胞内NO的产生。用该探针加载的纤维中明显存在一些DAF-FM泄漏，但它们保留了足够的探针，以便在加载后长达30分钟内对添加NO供体3-（2-羟基-1-甲基-2-亚硝基肼基）-N-甲基-1-丙胺（NOC-7）后细胞内NO的变化做出反应。与未刺激的纤维相比，分离纤维中的电刺激收缩使DAF-FM荧光的变化率增加了约48%（P < 0.05），并且刺激纤维中DAF-FM荧光的变化率在收缩后5分钟恢复到对照值。用NO合酶抑制剂盐酸Nω-硝基-L-精氨酸甲酯（L-NAME）或Nω-单甲基-L-精氨酸（L-NMMA）处理分离的纤维，可减少在未处理纤维收缩时观察到的DAF-FM荧光增加。用细胞可渗透的超氧化物清除剂4,5-二羟基-1,3-苯二磺酸（Tiron）处理纤维也可减少收缩期间观察到的荧光增加，这表明超氧化物，或者更可能是过氧亚硝酸盐，对观察到的荧光有贡献。因此，尽管过氧亚硝酸盐和其他活性氮物质可能潜在地影响观察到的荧光值，但该技术可用于实时检测静止和收缩骨骼肌纤维中的NO生成。
