Choline Uptake and Acetylcholine Synthesis in Synaptosomes: Investigations Using Two Different Labeled Variants of Choline

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Abstract
Using sequential incubations in media of different K+ composition, the dynamics of choline (Ch) uptake and acetylcholine (ACh) synthesis were investigated in rat brain synaptosomal preparations, using 2 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 .mu.M-[2H9]Ch. At the end of the preincubation all variants of ACh and Ch were measured in samples of the pellet and medium. In the 2nd incubation (4 min) samples of synaptosomes were resuspended in normal or high K+ solutions containing [2H4]Ch (2 .mu.M) and all variants of ACh and Ch were measured in the pellet and medium at the end of this period. This protocol allowed the comparison of the effects of preincubation in normal or high K+ solution on the metabolism during a 2nd low or high K+ incubation of a [2H9]Ch pool accumulated during the preincubation period. The effects of this protocol on [2H9]Ch metabolism vs. [2H4Ch] metabolism were compared. The most striking result obtained was that [2H9]Ch that was retained by the synaptosomes after the preincubation was not acetylated during a subsequent incubation in normal or high K+ media. If an intraterminal pool of Ch is involved in ACh synthesis, the size of this pool is below the limits of detection of this assay. A prior depolarizing incubation results in an enhanced uptake of Ch during a 2nd incubation in normal K+ Krebs. Ch uptake is stimulated by prior incubation under depolarizing conditions relative to normal preincubation when the 2nd incubation is in a high K+ solution. These results are discussed in terms of current models of the regulation of ACh synthesis in brain.