Pharmacology of high-affinity binding of [3H](+/-)2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (ADTN) to bovine caudate nucleus tissue

Abstract

High-affinity binding of [3H](+/-)2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene [( 3H]-ADTN) was improved by use of a subcellular fraction (P4) of tissue obtained from calf brain. The highest concentration of binding sites was found in caudate nucleus which was evaluated extensively. Binding of 0.5 nM [3H]-ADTN was optimal at 25 degrees and pH 7.5 to 8.0 when a cation-free medium containing antioxidants was used and was 82-87% displaceable ("specific"). The T1/2 for association was 20 min, and for dissociation 38 min, under these conditions. Analysis of association-dissociation kinetics and of ligand saturation isotherms revealed an apparent affinity (Kd) of 1-2 nM and a binding maximum (Bmax) of 422 fmoles/mg protein. The process proved to be reversible by, and monophasically competitive with, several potent dopamine agonists, with Hill constants close to unity. Stereoselectivity was found with eight isomer-pairs. Binding of [3H]-ADTN was selective for 3,4-dihydroxyphenethylamines and 10,11-dihydroxyaporphines with potent dopamine-agonist actions, but not for adrenergic catecholamines or other catechols, or blockers of dopamine uptake. Dopamine antagonists competed more weakly and in poor correspondence with their in vivo activities. There was a close correspondence between IC50 values obtained for fifty of the agents tested with both [3H]-ADTN and [3H]-(-)apomorphine (r and slope greater than 0.9), supporting impressions of the structure-activity characteristics of dopamine agonist binding sites based on prior studies with [3H]-apomorphine.