A physiological and pharmacological study of excitation-contraction coupling by using single skeletal muscle fibres whose intracellular medium can be exchanged.

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In order to approach to the biggest problem in excitation-contraction coupling at present, the mechanism of signal transduction throuth which depolarization of the T-tubule membrane causes a release of calcium from the sarcoplasmic reticulum (SR), we have developed "longitudinally cut" muscle fibre preparation, in which the properties of the surface and the T-tubule membrane are kept intact but still the composition of the intracellular medium can easily be altered. A single muscle fibre was isolated from amphibian skeletal muscle and its short segment cut out in a relaxing solution was mounted in such a way that a small area of its surface was exposed to a chamber (pool E) while the opposite surface was exposed to another chamber (pool I). All the other parts of the cell surface were sealed with silicone grease. A treatment of the membrane facing pool I with saponin or a scratch on the membrane rendered it permeable to various solutes.With the help of electrodes placed in the pools th … More e membrane facing pool E was voltage-clamped. When membrane potential was depolarized, an early inward and a delayed outward current were observed. The amplitude of the delayed outward current was altered by changing K concentration in the pool I within a few minutes. Amound of calcium released from the SR was monitored by the change in fluorescence intensity of fura-2 introduced intracellularly, the fluorescence being measured using a fluorescence microscope with epi-illumination. The dependence of calcium release on the magnitude of t-tubule depolarization obtained was similar to that reported in intact or "trans-versely cut" fibres. using this "longitudinally cut" fibre preparation, the effect of increasing calcium buffer concentration in the intracellular medium on calcium release was examined and found not to reduce the amount of calcium released. Thus, our contention that calcium-induced calcium release mechanism does not play an important role in physiological calcium release was supported. Further experiments on the influence of changing the intracellular environments on calcium release by T-tubule depolarization are now being carried out. Less