title: Effects of oxidation and cytosolic redox conditions on excitation-contraction coupling in rat skeletal muscle.
abstract: 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.
authors.affiliation: Department of Zoology, La Trobe University, Melbourne, Victoria, 3086, Australia.
authors.forename: G S
authors.initials: GS
authors.lastname: Posterino
authors.forename: M A
authors.initials: MA
authors.lastname: Cellini
authors.forename: G D
authors.initials: GD
authors.lastname: Lamb
mesh_terms: D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004558:Electric Stimulation; D005978:Glutathione / Q000378:metabolism; D019803:Glutathione Disulfide / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology*; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D051381:Rats; D020318:Rats, Long-Evans
chemical_list: D016877:Oxidants; D010726:Phosphodiesterase Inhibitors; D002110:Caffeine; D006861:Hydrogen Peroxide; D005978:Glutathione; D002118:Calcium; D004229:Dithiothreitol; D019803:Glutathione Disulfide