Characterization of membrane-bound and soluble D2 receptors in canine caudate using [125I]IBZM

BS Schonwetter, RR Luedtke, MP Kung, J Billings, HF Kung and PB Molinoff

Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia.

(S)-(-)-3-iodo-2-hydroxy-6-methoxy-N-[(1-ethyl-2-pyrrolidinyl) methyl]benzamide (IBZM) was shown to be a high-affinity antagonist selective for the D2 subtype of dopamine receptor. Binding sites for the radioligand [125I]IBZM were characterized with membranes and digitonin-solubilized extracts of canine caudate enriched by chromatography on heparin-agarose. Nonspecific binding, defined using 2 microM (+)-butaclamol, was less than 10% of the total ligand bound at the Kd of the receptor for [125I]IBZM. Direct binding, competition and kinetic experiments indicated that [125I]IBZM bound to a homogeneous population of binding sites. The rank order of potency for inhibition of the binding of [125I]IBZM by antagonists and agonists was found to be consistent with the pharmacological profile expected of a D2 receptor. The affinities of [125I]IBZM for membrane-associated and detergent-solubilized binding sites were essentially identical (Kd congruent to 0.4 nM). This result contrasts with findings obtained in studies with [3H]spiroperidol, where a marked decrease in the affinity of the receptor for the ligand has been observed during detergent solubilization and purification of the receptor. The high selectivity, nanomolar affinity and high specific activity of [125I]IBZM and the results obtained in studies with detergent extracts suggest that [125I]IBZM will be a particularly useful ligand for studies of D2 receptors in the presence of detergents.

Volume 250, Issue 1, pp. 110-116, 07/01/1989
Copyright © 1989 by American Society for Pharmacology and Experimental Therapeutics