Inhibition Of High Affinity Choline Uptake Research Articles

Choline availability and acetylcholine synthesis in the hippocampus of acetylcholinesterase-deficient mice

Mice deficient for acetylcholinesterase (AChE) have strongly increased extracellular levels of acetylcholine (ACh) in the dorsal hippocampus [Hartmann, J., Kiewert, C., Duysen, E.G., Lockridge, O., Greig, N.H., Klein, J., 2007. Excessive hippocampal acetylcholine levels in acetylcholinesterase-deficient mice are moderated by butyrylcholinesterase activity. J. Neurochem. 100, 1421–1429]. Using microdialysis, we found that increased ACh levels are accompanied by decreased levels of extracellular choline which were 1.60 μM in AChE-deficient mice and 4.36 μM in wild-type mice. Addition of choline (10 μM) to the perfusion fluid, while ineffective in wild-type animals, more than doubled extracellular ACh levels in AChE-deficient mice. High-affinity choline uptake (HACU), as measured ex vivo in corticohippocampal synaptosomes, was more than doubled in AChE-deficient mice. Inhibition of HACU by hemicholinium-3 (HC-3) in vivo reduced extracellular levels of ACh by 60% in wild-type mice but by more than 90% in AChE-deficient mice. Decreased ACh levels caused by infusion of HC-3 or tetrodotoxin (TTX) were accompanied by increased levels of free choline. Infusion of scopolamine (1 μM) caused a fivefold increase of ACh levels in wild-type animals but only a 50% increase in AChE-deficient mice. In conclusion, absence of AChE causes dynamic changes in the ratio of choline to ACh. High levels of extracellular ACh are accompanied by reduced levels of extracellular choline, and ACh release becomes strongly dependent on choline availability. Similar changes may take place in patients chronically exposed to AChE inhibitors.

Classification of the mode of inhibition of high-affinity choline uptake using capillary electrophoresis with electrochemical detection at an enzyme-modified microelectrode

A nonradiochemical in vitro assay using capillary electrophoresis with electrochemical detection at an enzyme-modified microelectrode has been developed to evaluate the inhibition of high-affinity choline transport in synaptosomes. Quantitative analysis of high-affinity choline transporter rates as a function of inhibitor and substrate concentrations allowed determination of the mode of inhibition for the quaternary ammonium-catechol-based inhibitors 3-[(trimethylammonio)methyl]catechol, N,N-dimethylepinephrine, and 6-hydroxy- N,N-dimethylepinephrine. The results are compared to the well-characterized inhibitor of choline transport, hemicholinium-3.

Role of high-affinity choline uptake on extracellular choline and acetylcholine evoked by NMDA.

Previous studies have shown that NMDA evokes a calcium-dependent and region-specific increase in extracellular choline that is associated with a reduction of membrane phosphatidylcholine and precedes neuronal cell death. We investigated, using in vivo microdialysis, the contribution of high-affinity choline uptake on the increase in extracellular choline evoked by NMDA. Dialysis was performed in the presence of Neostigmine (0.5 microM), an acetylcholinesterase inhibitor, in prefrontal cortex or hippocampus of freely moving rats. Drugs were administered through the dialysis probe. In cholinergic denervation experiments, rats were subjected to sham or AMPA-induced lesion of cholinergic nuclei at least 2 weeks before microdialysis. Excitotoxic lesion of the medial septum / ventral diagonal band nuclei reduced hippocampal choline acetyltransferase activity by 74%, [(3)H]hemicholinium-3 binding by 32%, and completely abolished potassium-evoked acetylcholine release. Despite this reduction of presynaptic cholinergic function, perfusion of NMDA (300 microM) by retrodialysis produced an increase in hippocampal extracellular choline (249 +/- 22% of basal levels) that was similar to that observed in sham controls (301 +/- 35%). Inhibition of choline uptake with hemicholinium-3 in nonlesioned rats produced a sustained increase in dialysate choline (163 +/- 8%) and reduced acetylcholine to 33 +/- 2% of basal levels, consistent with a depletion of the acetylcholine pool due to precursor deficit. Simultaneous perfusion of hemicholinium-3 and NMDA produced a synergistic increase in dialysate choline (664 +/- 95% of basal levels), indicating that part of the choline released by NMDA is taken up. In contrast, NMDA antagonized the decrease of acetylcholine produced by hemicholinium-3. These results show that NMDA-evoked choline release is not mediated by inhibition of high-affinity choline uptake and indicate that choline released by NMDA can be used to sustain acetylcholine synthesis when there is a precursor deficit secondary to uptake inhibition.

Selectivity of hemicholinium mustard, an affinity ligand, for the high-affinity choline transport system

The selectivity of the irreversible inhibition of high-affinity choline uptake (HACU) by hemicholinium mustard (HCM; 2,2′-(4,4′-biphenylene)bis[2-hydroxy-4-(2-bromoethyl)-morpholine] hydrochloride) with respect to other cholinergic proteins and other sodium-dependent transport systems was examined. Preincubation of rat forebrain membranes with HCM, followed by washing and measurement of [ 3H]-hemicholinium-3 binding to the high-affinity choline transporter, was shown to decrease binding capacity (B max) by 70% without affecting the apparent affinity of the ligand. However, a similar preincubation, wash and binding experiment using [ 3H]-NMS as a ligand for muscarinic receptors showed no HCM effect on binding parameters. To measure the effects of HCM on choline acetyltransferase (ChAT), synaptosomes were incubated in HCM, then washed. The synaptosomes were lysed and ChAT activity was measured. Treatment with 50 μM. HCM, a concentration that inhibits 100% of synaptosomal HACU, results in a 24% decrease in ChAT activity. HCM demonstrates little residual inhibition of other sodium-dependent neurotransmitter transporter transporters: preincubation with 50 μM HCM results in a decrease of 12% in transport of [ 3H]-dopamine and a decrease of 6% in the transport of [ 3H]-GABA. The binding of HCM, like that of hemicholinium-3 is sodium-dependent. HCM preincubation in the presence of sodium results in inhibition of HACU to 32% of control; in the absence of sodium HACU is 65% of control. This represents a loss of 51% of the observed irreversible inhibition produced by HCM. Irreversible inhibition by HCM can also be prevented by co-incubation with hemicholinium-3. Co-incubation with hemicholinium-3 results in residual HACU inhibition that decreases from 51% (HCM alone) to 28% (HCM + hemicholinium-3). When atropine instead of hemicholinium-3 is co-incubated with HCM, HCM still inhibits 40% of transport, demonstrating the pharmacological specificity of the protective effect of hemicholinium-3. Experiments in the guinea-pig myenteric plexus preparation demonstrate a gradual recovery from the residual effects of HCM. Evoked ACh release decreases to 24% immediately following treatment with 1 μM HCM. After 2 hr of recovery, tissues have recovered to about 50% of control levels, after which recovery continues at a slower rate.

Inhibition of high-affinity choline uptake and acetylcholine synthesis by quinuclidinyl and hemicholinium derivatives.

Choline uptake into cholinergic neurons for acetylcholine (ACh) synthesis is by a specific, high-affinity, sodium- and temperature-dependent transport mechanism (HAChU). To assess the role of choline availability in regulation of ACh synthesis, the structure-activity relationships of several hemicholinium (HC) and quinuclidinyl analogs were evaluated in a dose response manner. As confirms previous studies, the HCs, e.g., HC-3, acetylsecohemicholinium, and HC-15 are potent inhibitors of HAChU, HC-3 being the most potent (I50 = 6.1 X 10(-8) M). In the present study, the most potent quinuclidinyl derivative was the N-methyl-3-quinuclidinone (I50 = 5.6 X 10(-7) M). This compound had approximately 100-fold greater inhibitory activity than the corresponding racemic alcohol, suggesting that the 3-hydroxyl functional group is not absolutely essential for activity. Increasing the size of the N-functional group from a methyl to an allyl in the alcohol led to a 10-fold increase in activity. However, removal of the quaternizing N-methyl group yielding the tertiary amine, 3-quinuclidinol hydrochloride, greatly reduced its capacity to inhibit HAChU. Of the 2-benzylidene-3-quinuclidinone derivatives studied, only the m-chloro derivative significantly reduced HAChU.

Chemical Modification of Taipoxin and the Consequences for Phospholipase Activity, Pathophysiology, and Inhibition of High‐Affinity Choline Uptake

Treatment of taipoxin with p-bromophenacyl bromide resulted in modification of single histidine residues in the alpha and beta subunits. The modification decreased the neurotoxicity (lethality) 350-fold, but the inhibitory action on high-affinity choline transport was reduced only threefold. The phospholipase activity and Ca2+-association constants for taipoxin and its subunits were determined. A model for the neurotoxicity of taipoxin indicates the alpha subunit as the ultimate cause of the disruption of synaptic transmission.