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[3H]Choline mustard aziridinium ion binds irreversibly to the sodium-coupled high-affinity choline transport protein in a sodium-dependent and hemicholinium-sensitive manner, and thus is a useful affinity ligand. In rat striatal synaptosomal membranes, it radiolabels two polypeptides with apparent molecular masses of 58 and 35 kDa. Based upon the use of two different experimental approaches, it appears that neither of these polypeptides is glycosylated.

Choline uptake by cholinergic nerve terminals is increased by depolarization; the literature suggests that this results from either the appearance of occult transporters or the increased activity of existing ones. The present experiments attempt to clarify the mechanism by which choline transport is regulated by testing if the preexposure of synaptosomes to choline mustard aziridinium ion prevents the stimulation-induced appearance of hemicholinium-3 binding sites and/or choline transport activity. Choline mustard inhibited irreversibly most of the "ground-state" (basal) high-affinity choline transport but only 50% of "ground-state" hemicholinium-3 binding sites. Exposure of both striatal and hippocampal synaptosomes to the mustard, before stimulation, inhibited K(+)-stimulated increases in choline transport and of [3H]-hemicholinium-3 binding. We conclude that the mechanism by which choline transport is regulated involves the increased activity of a pool of transport sites that are occluded to hemicholinium-3 but are available to choline mustard aziridinium ion, and presumably to choline, before stimulation. However, the concentration of mustard needed to inhibit the stimulation-induced increase of [3H]-hemicholinium-3 binding and choline transport was lower for striatal synaptosomes than for hippocampal synaptosomes. In the absence of extracellular Ca2+ or presence of high Mg2+ levels, the choline mustard did not prevent the appearance of extra striatal hemicholinium-3 binding sites. Also, high Mg2+ levels removed the ability of the mustard to inhibit K(+)-stimulated increases of either [3H]-hemicholinium-3 binding or choline transport by hippocampal synaptosomes. In contrast, the preexposure of hippocampal synaptosomes to the mustard in the presence of a calcium ionophore (A23187) reduced the concentration of inhibitor needed to prevent the activation of [3H]hemicholinium-3 binding and choline uptake. Thus, we conclude that the ability of the choline mustard to alkylate the pool of choline transporters that are activated by stimulation appears dependent on the entry of extra-cellular Ca2+.

Choline acetyltransferase catalyzes the formation of acetylcholine from choline and acetyl-CoA in cholinergic neurons. The present study examined conditions for modulation of kinase-mediated phosphorylation of this enzyme. By using a monospecific polyclonal rabbit anti-human choline acetyltransferase antibody to immunoprecipitate cytosolic and membrane-associated subcellular pools of enzyme from rat hippocampal synaptosomes, we determined that only the cytosolic fraction of the enzyme (67,000 +/- 730 daltons) was phosphorylated under basal, unstimulated conditions. The quantity of this endogenous phosphoprotein was dependent, in part, upon the level of intracellular calcium, with 32Pi incorporation into the enzyme in nerve terminals incubated in nominally calcium-free medium only 43 +/- 7% of control. The corresponding enzymatic activity of cytosolic choline acetyltransferase did not appear to be altered by lowered cytosolic calcium, whereas membrane-associated choline acetyltransferase activity was decreased to 58 +/- 11% of control. Depolarization of synaptosomes with 50 microM veratridine neither altered the extent of phosphorylation or specific activity of cytosolic choline acetyltransferase, nor induced detectable phosphorylation of membrane-associated choline acetyltransferase, although the specific activity of the membrane-associated enzyme was increased to 132 +/- 5% of control. In summary, phosphorylation of choline acetyltransferase does not appear to regulate cholinergic neurotransmission by a direct action on catalytic activity of the enzyme.

Membrane carriers display structural and functional asymmetry with a substrate binding site which can be oriented alternately, but not simultaneously, to the extracellular and intracellular environment. Hemicholinium-3 is an inhibitor of the high-affinity choline carrier in cholinergic nerve terminals which binds to the transporter at the outer membrane surface but is not taken up into the cell. In the present study, we investigated the decline in choline transport which occurs during the first few minutes cholinergic nerve terminals are incubated in physiological salt solutions. Following incubation of rat hippocampal synaptosomes with hemicholinium-3, samples were washed free of the inhibitor and high-affinity choline uptake was measured. Choline uptake into hemicholinium-treated nerve terminals was significantly greater than control (132 +/- 4%). This effect appeared not to be due to an increase in uptake of choline above initial values in the hemicholinium-treated synaptosomes, but to a decrease in choline carrier activity in control samples by more than 25% during the first few minutes of incubation. Addition of hemicholinium-3 to samples after the preincubation induced decrease in choline uptake, followed by a wash period to remove the inhibitor resulted in elevation of choline uptake levels to initial levels. The effect of hemicholinium-3 was concentration-dependent, requiring near saturating concentrations of the inhibitor to elicit the effect. Measurement of acetylcholine content of synaptosomes at different points during the incubation procedure revealed that there was a trend for transmitter levels to vary inversely compared to choline uptake activity, but the differences were not statistically significant during treatments when significant changes in transport activity were measured.

NGF promotes the survival and enhances the neurotransmitter phenotype of basal forebrain and striatal cholinergic neurons in brain of rat. The objective of the present study was to determine whether stimulations of the cholinergic neuronal markers ChAT and high-affinity choline uptake are reflected in enhanced synthesis and release of ACh. Enhancement of ACh release in brain of adult and aged rats could result in increased cholinergic neurotransmission, and altered animal behavior. NGF (1.2 micrograms/d) was administered intracerebroventricularly for 2 weeks by osmotic minipump to male Fischer-344 rats aged 4 and 24 months. Cholinergic neuronal functional parameters were measured in frontal cortex, hippocampus, and striatum. In young adult rats, increased ChAT and choline uptake activities were accompanied by enhanced ACh synthesis, basal and depolarization-induced release of both endogenous and newly synthesized transmitter, with the largest effect generally being observed in striatum. In aged animals, the responses to NGF were less uniform. Whereas the pattern for changes in ChAT activity was similar to that seen in younger animals, choline uptake activity was increased only in frontal cortex and striatum. Coincidentally, ACh synthesis was also increased only in these two brain regions. ACh content of synaptosomes was not affected by age or NGF treatment, and the ACh levels in microwaved samples of striatum and basal forebrain were not affected by NGF treatment. However, profound deficits in both basal and evoked release of newly synthesized ACh were observed in the aged rats. NGF treatment had no significant effect on the basal release of newly synthesized ACh in aged rats.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

Choline mustard aziridinium is a potent, irreversible and selective blocker of sodium-dependent, high-affinity transport of choline into rat forebrain synaptosomes; it was found to be 30 times less potent against low-affinity transport of choline. The IC50 value for high-affinity transport was 0.94 microM, compared to 29 microM for low-affinity uptake. The inhibitory action of choline mustard aziridinium ion on high-affinity transport of choline was graded with respect to time; a 12-fold increase in potency was obtained by increasing the inhibitor preincubation times from 1 to 30 min. Low concentrations of choline mustard aziridinium ion could produce significant blockade of choline carriers providing the exposure time was prolonged. The characteristics of the blockade of synaptosomal high-affinity choline transport by choline mustard aziridinium ion also changed depending upon preincubation time. The kinetics of inhibition of high-affinity choline transport by choline mustard aziridinium ion showed apparent competitive inhibition initially, followed by noncompetitive characteristics at longer preincubations with inhibitor. The rate of irreversible inhibition of carriers by this nitrogen mustard analogue would appear to be rapid; the rate constant was determined to be 5 X 10(-2) s-1 for micromolar concentrations of inhibitor. This action may preclude the transport of the mustard analogue into the nerve terminal, although initially some reversible binding with the carrier may result in the translocation of some choline mustard aziridinium ion into the presynaptic ending. The progressive alkylation of high-affinity carriers by the analogue could indicate the presence of excess carrier sites in the presynaptic membrane, or subpopulations of carriers in an inactive state in equilibrium with active carriers. A model is described for the inhibitory action of choline mustard aziridinium ion on synaptosomal high-affinity choline carriers.

Sodium-dependent, hemicholinium-sensitive choline transport was measured in purified synaptosomes prepared from fresh necropsy brain of patients with senile dementia of the Alzheimer type and from control subjects. Choline transport velocity was standardized in terms of the occluded lactate dehydrogenase activity of the various synaptosomal preparations, rather than in terms of the protein content, since this enzyme is more representative of the synaptosome content of the purified homogenates. A regional difference in high-affinity choline transport was observed in purified synaptosomes prepared from brains of mentally normal controls; the velocities of sodium-dependent and hemicholinium-sensitive choline uptake into synaptosomes from hippocampus were about twice as great as that into synaptosomes from frontal cortex, indicating a greater relative density of cholinergic innervation in the hippocampus. Hippocampal and neocortical cholinergic nerve cell endings, prepared as synaptosomes, from brains of patients with Alzheimer's disease, also accumulated choline by a high-affinity mechanism; however, the velocity of uptake into both brain areas was decreased in comparison with controls. Choline transport into synaptosomes from Alzheimer frontal cortex was reduced approximately 50%, while uptake into Alzheimer hippocampal synaptosomes represented only 20% of the control activity. The reduction in synaptosomal high-affinity choline transport in Alzheimer's disease could be indicative of degeneration of cholinergic nerve terminal boutons resulting from cholinergic nerve cell death, or could result from an overall decrease in the number of carrier sites per nerve terminal or in the carrier transport velocity.(ABSTRACT TRUNCATED AT 250 WORDS)

The objectives of the present study were to validate the presence of cytoplasmic and membrane-associated pools of choline acetyltransferase (ChAT) in rat brain synaptosomes, and to evaluate inhibition of these different forms of the enzyme by the nitrogen mustard analogue of choline, choline mustard aziridinium ion (ChM Az). The relative distribution of ChAT and lactate dehydrogenase (LDH) was followed in subfractions of synaptosomes to establish whether ChAT activity associated with salt-washed presynaptic membranes represents membrane-bound protein rather than cytosolic enzyme trapped within undisrupted synaptosomes or revesiculated membrane fragments. The percentage of total synaptosomal ChAT activity (14%) recovered in the final membrane pellet always exceeded that of LDH (6%), lending support to the hypothesis that much of the ChAT associated with the membranes was a membrane bound form of the enzyme. Incubation of purified synaptosomes with ChM Az led to irreversible inhibition of ChAT activity; this loss of enzyme activity could not be accounted for by lysis of nerve terminals during incubation in the presence of the mustard analogue. Subfractionation of the ChM Az-treated nerve terminals revealed that the membrane-bound form of ChAT was inhibited to the greatest extent, followed by the ionically membrane-associated enzyme, with the activity of the water-solubilized enzyme not differing significantly from control. Preparation of the synaptosomal ChAT subfractions from untreated nerve terminals prior to incubation with varying concentrations of ChM Az or naphthylvinylpyridine revealed that under these conditions water-solubilized, ionically membrane-associated, and detergent-solubilized membrane-bound pools of ChAT were not differentially inhibited by either compound.(ABSTRACT TRUNCATED AT 250 WORDS)