Differential Labeling of Depot and Active Acetylcholine Pools in Nondepolarized and Potassium-Depolarized Rat Brain Synaptosomes

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Abstract
To test the hypothesis that a pool of newly synthesized acetylcholine (ACh) turns over independently of preformed ACh, compartmentation and K+-evoked release of ACh were examined in perfused synaptosomal beds intermittently stimulated by 50 mM K+. In resting synaptosomes, endogenous and labeled ACh was distributed between synaptic vesicles and the cytoplasm in a dynamic equilibrium ratio of 4:6. In the absence of new ACh synthesis, 5 sequential K+-depolarizations caused a decremental release of performed labeled ACh totaling 30% of the initial transmitter store. Further depolarization evoked little additional release, despite the fact that 60% of the labeled ACh remained in these preparations. Release of the preformed [14C]ACh was unaltered while new ACh was being synthesized from exogenous [3H]choline. Since the evoked release of [3H]ACh was maintained while that of [14C]ACh was decreasing, the [3H]ACh/[14C]ACh ratio in perfusate increased with each successive deplorization. This ratio was 6-10 times higher than the corresonding ratio in vesicles or cytoplasm. The newly synthesized ACh did not equilibrate with either the depot vesicular or cytoplasmic ACh pools prior to release.