The possibility that a chemical transmission mechanism, involving inositol (1,4,5) trisphosphate (InsP3) as a second messenger, is responsible for excitation-contraction coupling in skeletal muscle will be studied in physiological/biochemical experiments. The release of phosphoinositide-derived inositol phosphates induced by the electrical activation of skeletal muscle will be studied by the biochemical analysis of metabolic constituents obtained from rapidly frozen muscles. The ability of InsP3 and other inositol phosphates (cyclic InsP3) to rapidly induce release from the sarcoplasmic reticulum (SR) will be assessed using optical techniques in skinned skeletal muscle fibers. The modulation of this ability by several agents will also be investigated. Novel techniques to produce rapid release of Ca2+ and inositol phosphates (using photoreleasable derivatives) will be developed in order to investigate the kinetic response of the SR to putative agonists. These studies will give important kinetic information on the chemical modulation of the physiological process of Ca2+ release and they will complement our new findings about the nature of the transmission delay in E-C coupling. The role of Na and K conductances in the T-tubule depolarization and in possible ion depletion/accumulation phenomena will be studied with the aid of potentiometric dyes. The possibility that voltage dependent phospholipase C activity is endogenously present in the T tubule membranes will be directly tested in vitro. Studies in barnacle muscle fibers will allow us to evaluate the role of inositol phosphates on the E-C coupling of invertebrate muscle and will provide insights into the role of extracellular Ca in the regulation of this process.
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