Irreversible blockade of high-affinity choline uptake in rat brain by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ).

N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), an agent that causes irreversible covalent modification of protein carboxyl residues, has been used previously to produce irreversible occlusion of neurotransmitter receptors as well as other cellular proteins. The present investigation was undertaken to ascertain the mechanism by which EEDQ inhibits stimulus-dependent acetylcholine (ACh) release from rat brain hippocampal synaptosomes. Brief pretreatment with EEDQ (up to 100 microM) eliminated completely calcium-evoked [3H]acetylcholine ([3H]ACh) release and reduced de novo synthesis of transmitter by greater than 90%. Studies revealed that pretreatment with EEDQ in vitro caused a time- and concentration-dependent inhibition of high-affinity [3H]choline uptake (HACU) by synaptosomes. EEDQ-induced inhibition of HACU was not reversed by repeated tissue washing; however, co-incubation with hemicholinium-3, a highly specific and reversible inhibitor of HACU, protected against EEDQ-induced inhibition of HACU, as well as the loss of stimulus-dependent [3H]-ACh release. In vivo administration of EEDQ (20 mg/kg, s.c.) to rats caused marked reductions (46-65%) in synaptosomal HACU as well as the number of membrane binding sites for the muscarinic cholinergic antagonist L-[benzilic-4,4'-3H]quinuclidinyl benzilate ([3H]QNB) in the hippocampus and striatum. Treatment with atropine (100 mg/kg) prevented the reduction in [3H]QNB binding but did not influence EEDQ-induced inhibition of HACU. Taken together, these results indicate that EEDQ causes a direct and irreversible inhibition of high-affinity choline transporters on CNS cholinergic nerve terminals and, therefore, may be a useful investigational tool for characterization of the turnover and regulation of this transporter protein in vivo.