The Dissociation Rate of Unlabelled Dopamine Antagonists and Agonists from the Dopamine-D² Receptor, Application of an Original Filter Method

Abstract

A filtration technique was developed to measure the dissociation rate of unlabelled drugs from membrane receptors. Receptor preparations saturated with unlabelled drug were adsorbed to glass fibre filters positioned on a filtration apparatus. Dissociation of the drug from the receptor sites was achieved by repeatedly applying small buffer samples on the filter, this was followed by short incubation on the filter with a [³H]ligand. Rat striatal membrane preparations adsorbed on filters retained the same dopamine-D² receptor binding properties as the tissue in aqueous suspension. Stereospecific [³H]haloperidol binding was maximal with 20 mg tissue per filter and 5 min incubation, K_D = 2.8 nM and B_max = 24 fmoles/mg tissue was found. The dissociation from the dopamine-D₂ receptor for 18 dopamine antagonists and 3 agonists followed first order reaction kinetics. Half-lifes of dissociation ranged from 3.5 min for azaperone to 233 min for metitepine. There was no strict relationship between dissociation half-life and apparent equilibrium binding affinity or lipophilicity of the drugs. Half-life of receptor dissociation appeared neither to be a primary determining factor in the duration of pharmacological action of the drugs. The importance of the drug receptor dissociation rate for binding experiments in vitro as well as for chronic drug treatment is discussed.