Mechanism of the Release of Acetylcholine from Presynaptic Nerve Terminals Isolated from Electric Organ of Japanese Electric Ray

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1. Action and binding of various Ca channel blockers were examined on the calcium channels triggering the release of acetylcholine (ACh) in the synaptosomes prepared form the electric organ of Japanese electric ray, Narke japonica. The data obtained suggests that the Ca channels subserving the ACh release are mainly of N- and P-types and the L-type also has a small contribution. 2. A monoclonal antibody, MCC-1, that recognizes the alpha _2 delta subunit of rabbit skeletal muscle, inhibited partially the ACh release. 3. Immunoaffinity chromatography using MCC-1 was employed to solubilized plasma membrane of the synaptosomes in order to purify proteins with affinity to MCC-1. The SDS-PAGE analysis and Western blotting of the purified fraction identified a 170 kDa polypeptide as an MCC-1 binding protein. 4. The purified fraction also contained syntaxin, which is known to be an essential protein for the membrane fusion. 5. Incorporation of the synaptosomes into a planar lipid membrane revealed the presence of a K channel but failed to show the presence of Ca channels. 6. A novel radioactive probes were developed to measure phospholipid translocation across the biomembranes. With this probe, it was found that synaptosomal plasma membrane contains a phoshatidylserine-specific translocase. On the other hand, no specific translocase was found in synaptic vesicles. 7. A simultaneous measurement of the degranulation and intracellular Ca change in rat peritoneal mast cells implicated that intracellular Ca rise is a prerequisite for degranulation. 8. Membranes of the secretory granules of mast cells contain a cation-channel which may be responsible for the formation of so-called fusion pore.