The achievements of Natori and Ebashi, which greatly contributed to the progress in studies of excitation–contraction coupling, were reviewed. Natori succeeded in removing the cell membrane of an isolated fiber of skeletal muscle to prepare a skinned fiber, which still responded to an electrical stimulation with propagated contraction. Skinned fibers showed elastic extensibility beyond the elastic limit of intact muscle fibers. Based on this elasticity Natori predicted the presence of an elastic components, later found as connectin. Skinned fibers, an excellent experimental system, contributed greatly to the progress in subsequent studies. Ebashi showed that the essential principle of the relaxing factor was not the ATP-regenerating enzymes as generally thought, but a particulate fraction with MgATPase. Then he clearly showed that a minute amount of Ca2+ is necessary for the contractile reaction of actomyosin, and that the relaxing factor strongly accumulates Ca2+ in the presence of ATP and causes relaxation by the removal of Ca2+. He further discovered that the Ca2+-induced regulation of the contractile reaction of the myosin–actin system requires the presence of tropomyosin and a new protein, troponin. Troponin binds to a specific site on tropomyosin, which in turn binds to actin in the thin filament. Troponin is the Ca2+-receptive protein, and changes in troponin molecules upon Ca2+ binding is transmitted to actin through tropomyosin to regulate the actin–myosin interaction. Through these findings, the excitation was connected by Ca2+ with the contraction.

Calcium-induced calcium release (CICR) was first discovered in skeletal muscle. CICR is defined as Ca 2+ release by the action of Ca 2+ alone without the simultaneous action of other activating processes. CICR is biphasically dependent on Ca 2+ concentration; is inhibited by Mg 2+ , procaine, and tetracaine; and is potentiated by ATP, other adenine compounds, and caffeine. With depolarization of the sarcoplasmic reticulum (SR), a potential change of the SR membrane in which the luminal side becomes more negative, CICR is activated for several seconds and is then inactivated. All three types of ryanodine receptors (RyRs) show CICR activity. At least one RyR, RyR1, also shows non-CICR Ca 2+ release, such as that triggered by the t-tubule voltage sensor, by clofibric acid, and by SR depolarization. Maximum rates of CICR, at the optimal Ca 2+ concentration in the presence of physiological levels of ATP and Mg 2+ determined in skinned fibers and fragmented SR, are much lower than the rate of physiological Ca 2+ release. The primary event of physiological Ca 2+ release, the Ca 2+ spark, is the simultaneous opening of multiple channels, the coordinating mechanism of which does not appear to be CICR because of the low probability of CICR opening under physiological conditions. The coordination may require Ca 2+ , but in that case, some other stimulus or stimuli must be provided simultaneously, which is not CICR by definition. Thus CICR does not appear to contribute significantly to physiological Ca 2+ release. On the other hand, CICR appears to play a key role in caffeine contracture and malignant hyperthermia. The potentiation of voltage-activated Ca 2+ release by caffeine, however, does not seem to occur through secondary CICR, although the site where caffeine potentiates voltage-activated Ca 2+ release might be the same site where caffeine potentiates CICR.

OBJECTIVE In skeletal muscles, dantrolene inhibits the exercise-induced membrane translocation of GLUT4. It has been postulated that the inhibitory action of dantrolene on Ca2+ release from the sarcoplasmic reticulum (SR) causes inhibition of exercise-induced glucose uptake; however, the precise mechanism has not been adequately studied. RESEARCH DESIGN AND METHODS We discovered that dantrolene can bind to skeletal-type neuroendocrine-specific protein-like 1 (sk-NSPl1) with photoreactive dantrolene derivatives. In sk-NSPl1–deficient muscles, we examined the change in glucose uptake and the membrane translocation of GLUT4. In addition, we examined the change in blood glucose and also measured the glycogen level in both isolated and in situ skeletal muscles after electrical stimulation using our mutant mouse. RESULTS In sk-NSPl1–deficient muscles, exercise-induced glucose uptake was totally abolished with no change in insulin-induced glucose uptake. The Ca2+ release mechanism and its inhibition by dantrolene were completely preserved in these muscles. The expression of GLUT4 in the mutant muscles also appeared unchanged. Confocal imaging and results using the membrane isolation method showed that exercise/contraction did not enhance GLUT4 translocation in these sk-NSPl1–deficient muscles under conditions of adequate muscle glycogen consumption. The blood glucose level in normal mice was reduced by electrical stimulation of the hind limbs, but that in mutant mice was unchanged. CONCLUSIONS sk-NSPl1 is a novel dantrolene receptor that plays an important role in membrane translocation of GLUT4 induced by contraction/exercise. The 23-kDa sk-NSPl1 may also be involved in the regulation of glucose levels in the whole body.

The processes by which Professor Setsuro Ebashi accomplished his great work are described. Independently of Marsh, Ebashi discovered the relaxing factor in homogenized muscle and showed that it has a lipid-containing particulate fraction with ATPase activity, later identified as the sarcoplasmic reticulum. He then solved the mechanism of relaxation of the relaxing factor through the following findings. A minute amount of calcium ion (Ca(2+)) is necessary for the physiological contractile reaction. The relaxing factor strongly accumulates Ca(2+) in the presence of ATP and sufficiently removes Ca(2+) from the contractile system to bring about relaxation. Ebashi found that the contractile reaction of myosin and actin is regulated by Ca(2+) only in the presence of a tropomyosin-like protein factor, which he later showed to be a complex of tropomyosin and a new protein, troponin. He proved that troponin is the Ca(2+)-receptive protein and proposed the correct scheme for the molecular mechanism of regulation of contraction and relaxation.

In early 1960s clear evidence was presented by Professor S. Ebashi for the fact that contraction-relaxation cycle of living muscle is regulated by calcium ion (Ca2+) (cf. Ebashi and Endo, 1968). He then inquired into the mechanism of the action of Ca2+ and disclosed that the regulation of contractile reaction by Ca2+ requires the presence of a protein component other than myosin and actin (Ebashi, 1963). A few years later, he showed that the protein component is a complex of a known protein, tropomyosin, and a new protein, troponin (Ebashi and Kodama, 1965).

I first met Dr. Setsuro Ebashi in 1954, when he was an instructor (or assistant professor) in the Department of Pharmacology, the University of Tokyo, and I was an undergraduate student. Our group of students was doing some experiments on dogs in the corner of the department, which Professor Ebashi had kindly allowed us to use. He was quite kindhearted, but when talking with him one immediately recognized his brilliance, and his penetrating eyes aroused a feeling of awe in us. I did not realize it then, but this was the time when he was establishing the fact that the essential principle of Marsh’s relaxing factor is not a soluble enzyme such as creatine kinase or myokinase, but a microsomal ATPase described by Kielley and Myerhof in 1948.1

Several endogenous peptides for G-protein-coupled receptors have been found to play physiological roles in muscle contraction in addition to their well-demonstrated actions in other tissues. To further identify such peptides, we screened over 400 peptides using an isometric tension assay of rat papillary muscle. Here, we report that kyotorphin, which is known as an analgesic dipeptide, has a cardiac effect. Although kyotorphin had no effect on the twitch tension itself, it inhibited beta-adrenergic agonist isoprenaline-induced increases in twitch tension in a dose-dependent manner. Leu-Arg, a selective antagonist of kyotorphin, reversed this inhibitory effect. The inhibitory effect was also reversed by naloxone, an opioid receptor antagonist. These results suggest that kyotorphin may release opioid peptides from rat cardiac muscle and have an indirect regulatory role in beta-adrenergic action through cross-talk with opioid receptors.

Calcium release from the SR in skeletal muscle is brought about by opening of RyR/calcium release channels. RyR can be activated by Ca2+ (CICR), but physiological opening of RyR is not mediated by Ca2+. RyR is considered to open physiologically as a result of interaction with voltage sensor protein in the T-tubule membrane, and its opening mode is quite different from that of CICR. The physiological mode of opening is mimicked by clofibric acid and possibly by cerivastatin in the absence of Ca2+. On the other hand, CICR mode of opening of RyR in skeletal muscle plays the major role in MH or during caffeine action. The exact operation of RyRs in physiological calcium release remains to be solved.

Ryanodine receptors (RyRs), which form Ca2+ channels in the membrane of the endoplasmic reticulum, consist of three subtypes (RyR1, RyR2, and RyR3). The RyRs release Ca2+ from the endoplasmic reticulum into the cytoplasm and thus play an important role, especially in the contraction of skeletal and cardiac muscle cells. The genes of these RyRs are also expressed in many non-muscle tissues, but the role played by RyRs in non-muscle cells is not fully understood. In the present study, we examined the morphological changes in such cells caused by a deficiency of RyRs genes using three mutant mice lacking RyR1, RyR3, or both RyR1 and RyR3. The results showed morphological abnormalities in the adrenal cortical cells in all three mutant mice. In addition, an excessive accumulation of glycogen granules in hepatic cells, and a hypertrophy of the liver were both present in those mutant mice lacking both RyR1 and RyR3. We discuss the relationship between the morphological abnormalities of the adrenal cortex and liver induced by a deficiency of RyRs, and the possible causes of these abnormalities.