Posterino G S, Cellini M A, Lamb G D
Department of Zoology, La Trobe University, Melbourne, Victoria, 3086, Australia.
J Physiol. 2003 Mar 15;547(Pt 3):807-23. doi: 10.1113/jphysiol.2002.035204. Epub 2003 Jan 31.
In this study the effects of oxidation and reduction on various steps in the excitation-contraction (E-C) coupling sequence was examined in mammalian skeletal muscle. In mechanically skinned fast-twitch fibres, electric field stimulation was used to generate action potentials in the sealed transverse-tubular (T-) system, thereby eliciting twitch responses, which are a sensitive measure of Ca2+ release. Treatment of fibres with the oxidant H2O2 (200 microM and 10 mM) for 2-5 min markedly potentiated caffeine-induced Ca2+ release and the force response to partial depolarisation of the T-system (by solution substitution). Importantly, such H2O2 treatment had no effect at all on any aspect of the twitch response (peak amplitude, rate of rise, decay rate constant and half-width), except in cases where it interfered with the T-system potential or voltage-sensor activation, resulting in a reduction or abolition of the twitch response. Exposure to strong thiol reductants, dithiothreitol (DTT, 10 mM) and reduced glutathione (GSH, 5 mM), did not affect the twitch response over 5 min, nor did varying the glutathione ratio (reduced to oxidised glutathione) from the level present endogenously in the cytosol of a rested fibre (30:1) to the comparatively oxidised level of 3:1. In fibres that had been oxidised by H2O2 (10 mM) (or by 2,2'-dithiodipyridine, 100 microM), exposure to GSH (5 mM) caused potentiation of twitch force (by approximately 20 % for H2O2); this effect was due to the increase in the Ca2+ sensitivity of the contractile apparatus that occurs under such circumstances and was fully reversed by subsequent exposure to 10 mM DTT. We conclude that: (a) the redox potential across the sarcomplamsic reticulum has no noticeable direct effect on normal E-C coupling in mammalian skeletal muscle, (b) oxidising the Ca2+-release channels and greatly increasing their sensitivity to Ca2+-induced Ca2+ release does not alter the amount of Ca2+ released by an action potential and (c) oxidation potentiates twitches by a GSH-mediated increase in the Ca2+ sensitivity of the contractile apparatus.
在本研究中，研究了氧化和还原对哺乳动物骨骼肌兴奋 - 收缩（E - C）偶联序列中各个步骤的影响。在机械去膜的快肌纤维中，使用电场刺激在封闭的横管（T -）系统中产生动作电位，从而引发抽搐反应，这是Ca2 +释放的敏感指标。用氧化剂H2O2（200 microM和10 mM）处理纤维2 - 5分钟，可显著增强咖啡因诱导的Ca2 +释放以及对T系统部分去极化（通过溶液置换）的力反应。重要的是，这种H2O2处理对抽搐反应的任何方面（峰值幅度、上升速率、衰减速率常数和半高宽）均无影响，除非它干扰了T系统电位或电压传感器激活，导致抽搐反应减弱或消失。暴露于强硫醇还原剂二硫苏糖醇（DTT，10 mM）和还原型谷胱甘肽（GSH，5 mM）5分钟对抽搐反应无影响，将谷胱甘肽比率（还原型谷胱甘肽与氧化型谷胱甘肽）从静息纤维胞质中内源性存在的水平（30:1）改变为相对氧化的水平3:1也无影响。在已被H2O2（10 mM）（或2,2'-二硫代二吡啶，100 microM）氧化的纤维中，暴露于GSH（5 mM）会使抽搐力增强（H2O2处理后约增强20%）；这种效应是由于在这种情况下收缩装置对Ca2 +的敏感性增加所致，随后暴露于10 mM DTT可使其完全逆转。我们得出以下结论：（a）肌浆网的氧化还原电位对哺乳动物骨骼肌的正常E - C偶联没有明显的直接影响；（b）氧化Ca2 +释放通道并大大增加其对Ca2 +诱导的Ca2 +释放的敏感性，不会改变动作电位释放的Ca2 +量；（c）氧化通过GSH介导的收缩装置对Ca2 +敏感性增加来增强抽搐。
