Basal synthesis of acetylcholine in hippocampal synaptosomes is not dependent upon membrane-bound choline...

Choline acetyltransferase, the enzyme which catalyses the formation of acetylcholine within cholinergic nerve terminals, exists in both cytosolic and membrane-associated subcellular pools. In the present study, alteration in nerve terminal Cl- homeostasis was used as an experimental tool to elucidate the role of membrane-bound choline acetyltransferase in regulation of the biosynthesis of acetylcholine in rat hippocampal synaptosomes under basal or resting conditions. Reduction of extracellular Cl- concentration from 131 to 48 mM through iso-osmotic replacement with isethionate ions produced a selective decrease, to approximately 50% of control, of nerve terminal membrane-associated choline acetyltransferase activity. Under these experimental conditions, there were no changes in the activity of cytosolic enzyme or high-affinity choline uptake, or in acetylcholine synthesis. Replacement of medium Cl- with Br- supported maintenance of synaptosomal membrane-bound choline acetyltransferase activity better than did I- or isethionate ions; high-affinity choline uptake activity and acetylcholine synthesis were affected similarly. Incubation of synaptosomes with low concentrations of the Cl- channel blockers 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (50 microM) and niflumic acid (100 microM) selectively decreased activity of the membrane-bound enzyme, with no effect on cytosolic choline acetyltransferase or high-affinity choline uptake activities. Acetylcholine synthesis was unchanged, even though membrane-bound choline acetyltransferase activity was decreased in some samples (250 microM 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid) to about 10% of control. Experimental manipulations designed to alter neuronal Cl- homeostasis resulted in selective changes in membrane-bound choline acetyltransferase activity, thereby allowing the first direct examination of its physiological role in regulation of acetylcholine synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)