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

为了探讨目前兴奋-收缩偶联中最大的问题，即信号转导机制是T-小管膜去极化引起肌浆网（SR）钙释放，我们研制了“纵切”肌纤维标本，其中表面和T-小管膜的性质保持完整，但细胞内介质的组成仍然可以容易地改变。从两栖动物骨骼肌中分离出单个肌纤维，并将其在松弛溶液中切出的短节段以这样的方式固定，即其表面的一小部分暴露于腔室（池E），而相反的表面暴露于另一个腔室（池I）。电池表面的所有其他部分用硅脂密封。用皂苷处理面向池I的膜，或在膜上划一划，使其对各种溶质具有渗透性。 ...更多信息 对面向池E的膜进行电压钳位。当膜电位去极化时，观察到早期内向和延迟外向电流。延迟外向电流的幅度被改变池I中的钾浓度在几分钟内。从SR释放的钙的量通过引入细胞内的fura-2的荧光强度的变化来监测，使用具有落射照明的荧光显微镜测量荧光。钙释放的依赖性T-小管去极化的幅度上获得的是类似的完整的或“trans-cut”的纤维报告。使用这种“纵向切割的”纤维制品，检测了增加细胞内介质中钙缓冲液浓度对钙释放的影响，发现并没有减少钙释放的量。因此，我们的论点，钙诱导的钙释放机制不发挥重要作用的生理钙释放得到支持。细胞内环境的改变对T-小管去极化释放钙的影响的进一步实验正在进行中。少

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