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1. The turnover of synaptosomal (vesicular-cytoplasmic) and stable-bound (vesicular) acetylcholine isolated from cortical tissue was investigated after the administration, under local anaesthesia, of [N-Me-(3)H]choline into the lateral ventricles of guinea pigs. 2. Radioactive acetylcholine and choline present in acid extracts of subcellular fractions were separated by a combination of liquid and column ion-exchange and thin-layer chromatography. 3. The specific radioactivity and pattern of labelling of acetylcholine present in a fraction of monodisperse synaptic vesicles was found to be essentially the same as that of synaptosomal acetylcholine. 4. The specific radioactivity of stable-bound acetylcholine present in partially disrupted synaptosomes (fraction H) at short times (10-20min) after the injection of [N-Me-(3)H]choline was very variable and inversely related to the yield of acetylcholine in that fraction. 5. Evidence was found for the existence of two small, but highly labelled pools of acetylcholine, one which could be isolated in fraction H and the other which was lost when synaptosomes, after isolation by gradient centrifugation, were left at 0 degrees C or pelleted. 6. It is concluded that the results are best explained by metabolic differences among the nerve-ending compartments (thought to be vesicles) which contain stable-bound acetylcholine. Computer simulation of our experiments supports this possibility and suggests that the highly labelled pool in fraction H is present in vesicles close to the external membrane.

Choline metabolism in the cerebral cortex of guinea pigs. Stable-bound acetylcholine