Effects of reducing agents and oxidants on excitation-contraction coupling in skeletal muscle fibres of rat and toad.
1. The mechanically skinned fibre technique was used to examine the role of oxidation-reduction in the control of Ca2+ release and contraction in rat and toad skeletal muscle fibres under physiological conditions of myoplasmic [Mg2+] and [ATP] and sarcoplasmic reticulum (SR) Ca2+ load. 2. None of the reducing agents, dithiothreitol (DTT, 10 mM), glutathione (GSH, 10 mM) or cysteine (1 and 5 mM), had any detectable effect on the peak force, duration or the total number of depolarization-induced responses that could be elicited in skinned fibres from either toad or rat muscle, except for a slight alteration in one case (GSH on the duration of the response in rat fibres) caused by an effect of the agent of the Ca2+ sensitivity of the contractile apparatus. 3. Application of the reactive disulphide, 2,2'-dithiodipyridine (DTDP, 100 microM), a potent oxidizing agent, never induced any measurable force response or noticeable depletion of SR Ca2+ in any fibre under the conditions used. When all Ca2+ uptake was prevented, DTDP treatment of rat fibres was found to cause a 2- to 3-fold increase in the low rate of Ca2+ "leak' from the SR. DTDP treatment also increased the responsiveness of toad muscle fibres to 1 or 2 mM caffeine. These effects could be largely reversed by treatment with DTT. These results indicate that oxidation of the Ca2+ release channel does not cause substantial channel opening under physiological conditions. 4. Depolarization-induced force responses in both rat and toad fibres were rapidly abolished in the presence of DTDP (10 or 100 microM), in a manner favoured by inactivation of the voltage sensors. The relatively impermeant oxidant, 5,5'-dithionitrobenzoic acid (DTNB, 100 microM), had an effect very similar to DTDP if applied intracellularly, but unlike DTDP, had little or no effect if applied extracellularly (at 5 mM) before skinning. Depolarization-induced responses could be restored by treatment with DTT (10 mM). Intracellular application of the sulfhydryl-alkylating agent, N-ethylmaleimide (NEM, 100 microM), had effects very similar to DTDP and DTNB. 5. These results are not consistent with the proposal that excitation-contraction coupling in skeletal muscle primarily involves the oxidative linkage of the voltage sensors to the Ca2+ release channels, but do show that oxidation of an intracellularly accessible site can interfere with the coupling, in a process made more sensitive by voltage sensor inactivation.
Posterino
G S
GS
School of Zoology, La Trobe University, Bundoora, Victoria, Australia.
Lamb
G D
GD
D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002024:Bufo marinus; D002118:Calcium / Q000378:metabolism*; D003545:Cysteine / Q000494:pharmacology; D004220:Disulfides / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology; D066298:In Vitro Techniques; D008564:Membrane Potentials / Q000187:drug effects; D009119:Muscle Contraction / Q000187:drug effects*; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000502:physiology*; D018482:Muscle, Skeletal / Q000187:drug effects / Q000502:physiology*; D016877:Oxidants / Q000494:pharmacology*; D051381:Rats; D019163:Reducing Agents / Q000494:pharmacology*; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D004220:Disulfides; D016877:Oxidants; D019163:Reducing Agents; D013439:Sulfhydryl Reagents; C001105:2,2'-dipyridyl disulfide; D015082:2,2'-Dipyridyl; D004228:Dithionitrobenzoic Acid; D005978:Glutathione; D003545:Cysteine; D002118:Calcium; D004229:Dithiothreitol