Control of calcium release and the effect of ryanodine in skinned muscle fibres of the toad.

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Abstract
1. Skinned muscle fibres from the toad were used to investigate the roles of T-system membrane potential and Ca2+ in controlling the calcium release channels of the sarcoplasmic reticulum (SR). Replacement of K+ in the bathing solution with Na+ produced a large contraction which could last for 30 s or more under certain circumstances. This prolonged contraction could be quickly and completely terminated by repolarizing the fibre in the K+ solution and then immediately re-initiated by returning to the Na+ solution. These data indicate that the membrane potential tightly controlled the substantial and prolonged release of calcium. 3. T-system depolarization in the presence of 10 mM-free EGTA (pCa > 9) markedly depleted the SR of Ca2+. This implies that depolarization of the T-system can still trigger substantial release of Ca2+ from the SR even when the myoplasmic [Ca2+] is very low and very heavily buffered by EGTA. 4. When the SR was heavily loaded with Ca2+, substitution of a weakly buffered high [Ca2+] solution (pCa 5.4, 50 .mu.M-EGTA) could produce a small to moderate, transient contraction taking between 3 and 12 s to reach a peak and lasting 30 s or more. 5. This contraction may be produced at least partly by ''calcium-induced calcium release'' as ruthenium red (2 .mu.M) completely blocked the responses. Moreover, repeated substitutions produced successively smaller responses in parallel with the ''run-down'' of the depolarization-induced contractions. 6. Depolarization could always produce an additional large and fast response at any stage during a ''Ca2+-induced'' response. 7. In the presence of 25 .mu.M-ryanodine, the rapid contraction produced by T-system depolarization was prolonged and could not be stopped by repolarization. During and after this contraction no depolarizing stimulus could induce a further contraction, even though in some fibres addition of 30 mM-caffeine produced a maximum response which indicated that there was still a substantial amount of calcium in the SR. 8. At pCa 6.4, 25 .mu.M-ryanodine could itself induce a substantial slow contracture in a normally polarized fibre within 30-60 s, after which little or no response could be induced by T-system depolarization. At higher concentrations (25 .mu.M) ryanodine produced anear-maximum contraction in only a few seconds. Ryanodine did not produce such contractures in the presence of low myoplasmic [Ca2+] (pCa > 8, 0.5 mM-EGTA) and the drug could be washed out without subsequent effects, indicating that it had not bound. 9. Thus, it appears that the major Ca2+ release channels involved in depolarization-induced contractions can be opened by Ca2+ and that ryanodine initially keeps the channels blocked open, thereby potentiating calcium-induced calcium release and producing a contracture, but then blocks them permanently shut. 10. Ryanodine could still induce a contracture, even after the rapid depolarization-induced responses had been abolished by inactivating the voltage sensor by (a) a prolonged depolarization, (b) D600 or (c) soaking the fibre in low [Ca2+] before skinning. Similarly, these procedures also did not noticeably affect the responses to 1 mM-caffeine. 11. Together with other data, these results suggest that in a functioning muscle fibre, depolarization induces Ca2+ release from the SR through a single type of release channel which is influenced primarily by the voltage sensor in the T-system membrane but which is also affected by myoplasmic Ca2+ and by caffeine and other agents.