Stimulation of Cholinergic Synaptosomes Isolated fkom Torpedo Electric Organ

Abstract

In this chapter the isolation and characterization of pure cholinergic synaptosomes from the electric organ of Torpedo is described. A technique has been developed to study the release of ACh from these synaptosomes. K + depolarization triggers a Ca 2+ dependent release. The good time resolution (4-5 sec) permits further investigations on the mechanisms of ACh release. The two main characteristics of the isolation procedure are the gradual comminution of the tissue by successive filtrations, and the use of centrifugation media having an ionic composition and osmolarity close to plasma. Torpedo synaptosomes are able to synthesize ATP from extracellular adenosine. A facilitated diffusion mechanism for adenosine uptake has been described. Most of the adenosine taken up is converted to ATP. A high affinity uptake of choline in Torpedo synaptosomes is also characterized. Twenty percent of the incorporated choline is converted to ACh. Acetate that is a good precursor of ACh in Torpedo electric organ and in neuromuscular junctions is efficiently incorporated into the ACh of synaptosomes. For extracellular acetate concentrations lower than 50 μM, as much as 95% of incorporated radioactivity was found as ACh. Since all the synaptosomal radioactivity is found as ACh, it should be possible to follow the release of ACh by measuring the efflux of radioactivity.