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Cholinergic neurons rely on the sodium-dependent choline transporter CHT to provide choline for synthesis of acetylcholine. CHT cycles between cell surface and subcellular organelles, but little is known about regulation of this trafficking. We hypothesized that activation of protein kinase C with phorbol ester modulates choline uptake by altering the rate of CHT internalization from or delivery to the plasma membrane. Using SH-SY5Y cells that stably express rat CHT, we found that exposure of cells to phorbol ester for 2 or 5 min significantly increased choline uptake, whereas longer treatment had no effect. Kinetic analysis revealed that 5 min phorbol ester treatment significantly enhanced V(max) of choline uptake, but had no effect on K(m) for solute binding. Cell-surface biotinylation assays showed that plasma membrane levels of CHT protein were enhanced following 5 min phorbol ester treatment; this was blocked by protein kinase C inhibitor bisindolylmaleimide-I. Moreover, CHT internalization was decreased and delivery of CHT to plasma membrane was increased by phorbol ester. Our results suggest that treatment of neural cells with the protein kinase C activator phorbol ester rapidly and transiently increases cell surface CHT levels and this corresponds with enhanced choline uptake activity which may play an important role in replenishing acetylcholine stores following its release by depolarization., (Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

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)

The objective of this study was to measure the uptake and metabolism of [3H]choline mustard aziridinium ion in rat brain synaptosomes. In previous investigations, we showed that this compound binds irreversibly to the choline carrier thereby inhibiting choline transport into nerve terminals; it also acts as both a substrate and inhibitor of the acetylcholine biosynthetic enzyme choline acetyltransferase. We now report that [3H]choline mustard aziridinium ion was transported into purified rat brain synaptosomes by a hemicholinium-sensitive mechanism, but at only a fraction of the rate of uptake of [3H]choline. Following 5 min incubation with the nerve terminal preparation, uptake of [3H]choline mustard aziridinium ion was 20% of that of [3H]choline transport, but this fell to 10% of [3H]choline accumulation at 30 min incubation. Apparent Michaelis constants derived from double reciprocal plots of velocity of transport versus substrate concentration revealed that the apparent affinity constants (Km) of the high-affinity choline carrier for [3H]choline mustard aziridinium ion and [3H]choline were not different (1.44 +/- 0.15 and 2.14 +/- 0.80 microM for choline and choline mustard aziridinium ion, respectively). Increasing the incubation time from 5 to 30 min, during which time a proportion of the high-affinity choline carriers were irreversibly inactivated by choline mustard aziridinium ion, did not alter the binding affinity for this compound. The maximum velocity of transport (Vmax) for the two compounds were significantly different with the maximum uptake of [3H]choline mustard aziridinium ion being 19.5% of that for choline at 5 min incubation, and falling to only 10.6% of the maximum rate of choline transport by 30 min incubation. [3H]Choline mustard aziridinium ion transported into synaptosomes on the high-affinity choline carrier was metabolized, with 27% being recovered as [3H]acetylcholine mustard aziridinium ion, 27% as [3H]phosphorylcholine mustard aziridinium ion, 7% as unmetabolized [3H]choline mustard aziridinium ion and 16% recovered as an unidentified metabolite. In parallel samples, [3H]choline taken up into synaptosomes was recovered as [3H]acetylcholine (71%) and unmetabolized [3H]choline (18%) with no net production of [3H]phosphorylcholine. Acetylation of [3H]choline mustard aziridinium ion amounted to only 7.6% of [3H]acetylcholine synthesized under the same conditions. These results show clearly that choline mustard aziridinium ion was accumulated into the cholinergic nerve terminals by the high-affinity choline carrier, but the amount was small relative to the uptake of choline and probably restricted by progressive inactivation of the transporters through covalent bond formation.