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Received October 29, 2002
Accepted after revision January 6, 2003
1 Department of Zoology, La Trobe University, Bundoora, Victoria, 3086, Australia
2 Department of Zoology, La Trobe University, Victoria, 3086, Australia
* To whom correspondence should be addressed. E-mail: zoogl{at}zoo.latrobe.edu.au.
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 µM 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 2,2â-dithiodipyridine, 100 µM), exposure to GSH (5 mM) caused potentiation of twitch force (by ~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.
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