Differential labeling of depot and active acetylcholine pools in nondepolarized and potassium-depolarized rat brain synaptosomes.

To test the hypothesis that a pool of newly synthesized acetylcholine (ACh) turns over independently of performed 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, five 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 performed [14C]ACh was unaltered while new ACh was being synthesized from exogenous [3H]choline. Since the evoke 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 depolarization. This ratio was six to ten times higher than the corresponding ratio in vesicles or cytoplasm. These results indicate that the newly synthesized ACh did not equilibrate with either the depot vesicular or cytoplasmic ACh pools prior to release.