The subsynaptosomal distribution and release of [3H]acetylcholine synthesized by rat cerebral cortical synaptosomes

Synaptosomes were prepared from rat cerebral cortex and incubated in ³H]choline for periods ranging from 1 to 90 min. The ³H]ACh synthesized during this period was found only in the cytoplasm and in a membrane-associated fraction. A negligible amount of the newly formed ³H]ACh was recovered in the vesicular fraction despite concerted efforts to protect a hypothetical population of labile vesicles. The specific activity of the membrane-associated component, accounting for 21% of the total ³H]ACh, was by far the highest. This membrane-associated fraction was not released by hypotonic shock or homogenization and apparently was not in association with the monodisperse synaptic vesicles. The ³H]ACh was released in a calcium dependent manner. This investigation has determined that the ACh synthesized by synaptosomes is localized in only two fractions, cytoplasmic and membrane-associated; that this newly synthesized ACh can be released from synaptosomes by a process consistent with physiological release; and that at least part of the ACh released was originally present in the cytoplasm.