SELECTIVITY OF HEMICHOLINIUM MUSTARD, AN AFFINITY LIGAND, FOR THE HIGH-AFFINITY CHOLINE TRANSPORT-SYSTEM

The selectivity of the irreversible inhibition of high-affinity cholin e uptake (HACU) by hemicholinium mustard (HCM; enylene)bis[2-hydroxy-4 -(2-bromoethyl)-morpholine] hydrochloride) with respect to other choli nergic proteins and other sodium-dependent transport systems was exami ned. Preincubation of rat forebrain membranes with HCM, followed by wa shing and measurement of [H-3]-hemicholinium-3 binding to the high-aff inity choline transporter, was shown to decrease binding capacity (B-m ax) by 70% without affecting the apparent affinity of the ligand. Howe ver, a similar preincubation, wash and binding experiment using [H-3]- NMS as a Ligand for muscarinic receptors showed no HCM effect on bindi ng parameters. To measure the effects of HCM on choline acetyltransfer ase (ChAT), synaptosomes were incubated in HCM, then washed. The synap tosomes were lysed and ChAT activity was measured. Treatment with 50 m u M HCM, a concentration that inhibits 100% of synaptosomal HACU, resu lts in a 24% decrease in ChAT activity. HCM demonstrates little residu al inhibition of other sodium-dependent neurotransmitter transporter t ransporters: preincubation with 50 mu M HCM results in a decrease of 1 2% in transport of [H-3]-dopamine and a decrease of 6% in the transpor t of [H-3]-GABA. The binding of HCM, Like that of hemicholinium-3 is s odium-dependent. HCM preincubation in the presence of sodium results i n 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 irrev ersible inhibition produced by HCM. Irreversible inhibition by HCM can also be prevented by co-incubation with hemicholinium-3. Go-incubatio n with hemicholinium-3 results in residual HACU inhibition that decrea ses from 51% (HCM alone) to 28% (HCM + hemicholinium-3). When atropine instead of hemicholinium-3 is co-incubated with HCM, HCM still inhibi ts 40% of transport, demonstrating the pharmacological specificity of the protective effect of hemicholinium-3. Experiments in the guinea-pi g myenteric plexus preparation demonstrate a gradual recovery from the residual effects of HCM. Evoked ACh release decreases to 24% immediat ely following treatment with 1 mu M HCM. After 2 hr of recovery, tissu es have recovered to about 50% of control levels, after which recovery continues at a slower rate. (C) 1998 Elsevier Science Ltd. All rights reserved.