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The calcium release process of the sarcoplasmic reticulum mammalian skeletal muscle will be examined in studies involving isolated sarcoplasmic reticulum (SR) and isolated triads (junctions of t-tubules and sarcoplasmic reticulum). The mechanism(s) of Ca++ release that contribute toward physiological contractile activation during excitation-contraction coupling will be identified by comparing the sensitivity of various ways of releasing calcium from SR to blockade by different pharmacological agents. Calcium-induced, "depolarization"-induced, alkalinization-induced, pump reversal mediated spontaneous, and various forms of drug-induced releases of calcium will be elicited from SR isolated from both rabbit skeletal and cardiac muscle. Conditions to demonstrate these releases will be optimized by variation of the following factors: the type of assay detection method, the type of SR material utilized, the extent of Ca++ preloading, possible utilization of Ca++ precipitating anions, the extravesicular free Ca++ concentration, and other ingredients of the assay medium. The sensitivity of each kind of release to different pharmacological agents (local anesthetics, other muscle relaxants, surface membrane Ca++ channel blockers, Ca++ pump inhibitors, other divalent cations, and reported blockers of one or another form of Ca++ release from isolated SR or skinned fibera) will be tested. Comparison to the sensitivity of the physiological release in muscle fibers should help assess the physiological relevance of any of these forms of calcium release and may demonstrate alternative means of opening the presumed SR Ca++ channels involved. Isolated triads will be utilized to reproduce in vitro calcium release from the SR portion of the isolated structure in response to depolarization of the associated transverse tubule membrane. Experiments with this system will assess the pharmacological sensitivity of the physiological SR calcium release process, which will then be compared to the sensitivities of various forms of calcium release elicited directly from isolated SR. These experiments will surely delineate the number of different Ca++ efflux pathways in the SR and demonstrate ways to block each. These experiments may further demonstrate whether any of the model Ca++ release mechanisms described for isolated SR participate in physiological excitation-contraction coupling.

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