Choline uptake and acetylcholine synthesis in synaptosomes: investigations using two different labeled variants of...

Using sequential incubations in media of different K+ composition, we investigated the dynamics of choline (Ch) uptake and acetylcholine (ACh) synthesis in rat brain synaptosomal preparations, using two different deuterated variants of choline and a gas chromatographic-mass spectrometric (GC-MS) assay for ACh and Ch. Synaptosomes were preincubated for 10 min in a Krebs medium with or without high K+ and with 2 micrometer-[(2)H(9)]Ch. At the end of the preincubation al variants of ACh and Ch were measured in samples of the pellet and medium. In the second incubation (4 min) samples of synaptosomes were resuspended in normal or high K+ solutions containing [(2)H(4)]Ch (2 micrometer) and all variants of ACh and Ch were measured in the pellet and medium at the end of this period. This protocol allowed us to compare the effects of preincubation in normal or high K+ solution on the metabolism during a second low or high K+ incubation of a [(2)H(9)]Ch pool accumulated during the preincubation period. Moreover, we were able to compare and contrast the effects of this protocol on [(2)H(9)]Ch metabolism versus [(2)H(4)]Ch metabolism. The most striking result we obtained was that [(2)H(9)]Ch that had been retained by the synaptosomes after the preincubation was not acetylated during a subsequent incubation in normal or high K+ media. This result suggests that if an intraterminal pool of Ch is involved in ACh synthesis, the size of this pool is below the limits of detection of our assay. We have confirmed the observation that a prior depolarizing incubation results in an enhanced uptake of Ch during a second incubation in normal K+ Krebs. Moreover, Ch uptake is stimulated by prior incubation under depolarizing conditions relative to normal preincubation when the second incubation is in a high K+ solution. These results are discussed in terms of current models of the regulation of ACh synthesis in brain.