Research On The Mechanism Of Ca～(2+) Released From The Sarcoplasmic Reticulum In Skeletal Muscle

The course of skeletal muscular contraction includes latency, systole and period of relaxation. During the course, the skeletal muscle must have a series of structural and functional changes. In skeletal muscular fibers, the T-tube, sarcoplasmic reticulum(SR) and Ca~2+ ion play a key role during the excitation- contraction (e-c) coupling course, but it was still not clear what was the mechanism of Ca~2+ releasing and what happened structurally and functionally during SR releasing Ca~2+ ion. All these concerned are very heatedly discussed in cell biology, physiology and biophysics. The time course of skeletal muscular e-c coupling is very short (measured at millisecond), so it can't reserve the ultrastructure as functional changes quickly by the conventional method of chemical fixation (measured at minute). Because of the restriction of these researching methods, it can't obtain the skeletal muscular structural changes in millisecond level conventional measure of studying on cellular ultrastructure. However, we can study skeletal ultrastructure in quiensce condition and its changes in different time of EC course by ultra-hypothermy quick freezing fixation at millisecond level technique, freezing displacement and TEM.Calcium, acting as a common second messenger, plays an important regulating role in many life processes. For example: muscle contraction, gland secretion, synaptic transmit, fucundation, nuclear pore complex regulation, transcriptional regulation .Cell lives in high concentration of calcium, however, the concentration in cytoplasm is particular low. Na~+-Ca~2+ exchange system and ion channel change the concentration of calcium by calcium pump, which provides biological sign dependent of calcium. Notonly membrane promotes Ca~2+ signal transduction ,but some special structrure-Ca~2+storage or Ca~2+ releasing pool provide local quick Ca~2+ signal .The major Ca~2+ storages in skeletal muscle are sarcoplasmic reticulum (SR) and mitochodria. Ca~2+ exchange systerm includes Ca~2+ pump and Ca~2+ releasing channel by which maintain the Ca~2+ balance and induce local quick Ca~2+ signal. Ca~2+ in plasm can be retained in low level, due to Ca~2+ pump's transport function. However, the releasing Ca~2+ in plasm operates biology effect by different regulating machanism.We could study the ultrastructural time-morphological changes of SR and T-tube of the skeletal muscle in latency and systole during the e-c coupling course when the skeletal muscle is fixed on different time spots (Oms to 24ms) after electrical stimulated, then we would acquire the morphological structure, calcium concentration and distribution situs changes at the moment of calcium releasing from SR when induced. Also we could study the RyR ultrastrcture by homogenization and centrifugation .Combination with confocal microscopy, the entireness of Ca~2+spark could be observed on submicroscopic level, and the mechanism of calcium releasing would be analyzed. Colligating the two methods, the mechanism of calcium releasing from SR would be proved in morphology when the e-c coupling takeing place.To study the controlling of calcium channels in sarcoplasmic reticulum could make clear the ultrastructural and functional changes in e-c coupling result from calcium induced calcium release. We have developed a procedure to isolate, from skeletal muscle, enriched terminal cisternae of SR, which retain morphologiclly intact junctional "feet" stuctrures. The fraction is largely devoid of transverse tubule , plasma membrane ,mitochondria,triads and long cisternae. The terminal cisternae vesicles have distinctive morphological characteristics that differ from the isolated longitudinal cisternae obtained from the same gradient. The terminal cisternae consist of two distinct types of membranes ,ie,the junctional face membrane and the Ca~2+ pump protein-containing membrane ,whereas the longitudinal cisternae contain only the Ca~2+ pump protein-containing membrane .The junctional face membrane of the terminal cisternae contains feet structures that can be clearly visualized by electron microscopy. The intravesicular content of the terminal cisternae, mostly Ca~2+-binding protein (calsequestrin),is organized in the form of strands, and attached to the inner face of the membrane in the vicinity of the junctional feet. We also have obtained the ultrastructural images on different time spots in latency of the e-c coupling course after a single electrical stimulation in skeletal muscle by two-way infrared detectors with a computer to control the signal of electric stimulation and ultra-hypothermy quick freezing fixation at millisecond level technique, freezing displacement and TEM. In the area of SOjum from frozen side, the skeletal muscular ultrastructure is protected very well, myofibril arranging regularly, I-band; A-band, Z-line and M-line visible; the structures of triad,myofibril, glycogen particle, mitochondria, nucleolus very clear; T-tube presenting circle or ellipse with section on Z-line level; SR affluent. With the time went on, the quantity of the vesicula in cytoplasm increased gradually and some vesiculas coalesce each other, and then dismissed. At 10ms after electric stimulation sarcoplasmic reticulum recovered to straight. The substance in SR distributed uniformly and there were no obvious changes in SR and T-tube during the course of 0ms—2ms after electric stimulation, but some deep electron density substance could be observed in SR and moved to T-tube direction little by little from 2ms—10ms. At 2 ms, a round transparent domain (called core cylinder) emerged in the middle of SR and the deep electron density substance distributed uniformly in SR except core cylinder area; Then at 4.6 ntis, some homologous deep density substances were parallelism to inside and outside the membrane of SR, and close to this domain, some substance located in outer face of T-tube membrane in the same density. But after 10ms, the morphology of SR and T-tube regained, and the substance dismissed. So we can say the CICR mechanism exists in skeletal muscle indeed and a series of ultrastructural changes occur in e-c coupling course induced by calcium, meanwhile, it may be a powerful proof of CICR on the other aspect.Fluorescence probe — Fluo-3AM? can luminate in confocal microscope after combining Ca2+, so we can measure and test dissociative Ca2+ in cytoplasm by this way. Some confocal laser line-scan images were obtained after calcium signed with Fluo-3AM in muscle fibers. A group of luminescent substance emerged close to sarcoplasmic reticulum intracellulare at the 2nd second after electron stimulation, and the volume enlarged and the brightness augmented with the time passing. At the 7th second, the volume reached maximum and then began to diminution and the brightness tenuated accordingly, and at the 14th second, the luminescent substance dismissed. That is the Ca2+spark changing with time in skeletal muscle.All results manifest: a series of time phase ultrastructural changes existing in e-c coupling course, especially in triad, and the feet structures must have concern with e-c coupling of skeletal muscle. The phenomenon of deep electron density substance emergence and displacement, the appearance of core cylinder and the generation of Ca + spark are necessarily related with the calcium release from SR. So we need to confirm it further by calcium determination and physiological experiments, which may be a...