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T小管膜去极化导致肌浆网(SR)钙释放的信号转导机制，我们研制了“纵向切割”肌纤维制剂，其表面和T小管膜的性质保持不变，但细胞内介质的组成仍然很容易改变。从两栖骨骼肌中分离出单一的肌纤维，将其在松弛溶液中切下的短节以这样的方式安装，即其表面的一小部分暴露在一个室(池E)中，而相反的表面暴露在另一个室(池I)中。电池表面的所有其他部分都用硅油密封。用皂素或膜上的划痕对面向池I的膜进行处理，使其对各种溶液具有渗透性。借助放置在池I中的电极，…更多面向E池的e膜被电压钳制。当膜电位去极化时，可观察到早期内向电流和延迟外向电流。延迟外向电流的幅度通过在几分钟内改变池I中的钾浓度而改变。通过细胞内引入的Fura-2荧光强度的变化来监测SR释放的大量钙，荧光是用带外照射的荧光显微镜测量的。钙释放对T管去极化程度的依赖性与在完整或“横向切断”纤维中的报道相似。利用这种“纵向切割”的纤维制剂，研究了增加细胞内培养液中钙缓冲液浓度对钙释放的影响，发现不会减少钙的释放。因此，我们认为钙诱导的钙释放机制在生理钙释放中不起重要作用的观点得到了支持。改变细胞内环境对T小管去极化释放钙的影响的进一步实验正在进行中。较少

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