IRREVERSIBLE BLOCKADE OF HIGH-AFFINITY CHOLINE UPTAKE IN RAT-BRAIN BYN-ETHOXYCARBONYL-2-ETHOXY-1,2-DIHYDROQUINOLINE (EEDQ)

N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), an agent that c auses irreversible covalent modification of protein carboxyl residues, has been used previously to produce irreversible occlusion of neurotr ansmitter receptors as well as other cellular proteins. The present in vestigation was undertaken to ascertain the mechanism by which EEDQ in hibits stimulus-dependent acetylcholine (ACh) release from rat brain h ippocampal synaptosomes. Brief pretreatment with EEDQ (up to 100 mu M) eliminated completely calcium-evoked [H-3]acetylcholine ([H-3]ACh) re lease 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 [H-3]choline uptake (HACU) by synaptosomes. EEDQ-induced inhibition of HACU was not reversed by repeated tissue washing; however, co-incubation with hemi cholinium-3, a highly specific and reversible inhibitor of HACU, prote cted against EEDQ-induced inhibition of HACU, as well as the loss of s timulus-dependent [H-3]-ACh release. In vivo administration of EEDQ (2 0 mg/kg, s.c.) to rats caused marked reductions (46 65%) in synaptosom al HACU as well as the number of membrane binding sites for the muscar inic cholinergic antagonist L-[benzilic-4,4'-H-3] quinuclidinyl benzil ate ([H-3]QNB) in the hippocampus and striatum. Treatment with atropin e (100 mg/kg) prevented the reduction in [H-3]QNB binding but did not influence EEDQ-induced inhibition of HACU. Taken together, these resul ts indicate that EEDQ causes a direct and irreversible inhibition of h igh-affinity choline transporters on CNS cholinergic nerve terminals a nd, therefore, may be a useful investigational tool for characterizati on of the turnover and regulation of this transporter protein in vivo.