Quantitative analysis of multiple kappa-opioid receptors by selective and nonselective ligand binding in guinea pig spinal cord: resolution of high and low affinity states of the kappa 2 receptors by a computerized model-fitting technique

M Tiberi and J Magnan

Departement de pharmacologie, Universite de Montreal, Quebec, Canada.

The binding characteristics of selective and nonselective opioids have been studied in whole guinea pig spinal cord, using a computer fitting method to analyze the data obtained from saturation and competition studies. The delineation of specific binding sites labeled by the mu- selective opioid [3H]D-Ala2,MePhe4,Gly-ol5-enkephalin (Kd = 2.58 nM, R = 4.52 pmol/g of tissue) and by the delta-selective opioid [3H]D-Pen2, D-Pen5-enkephalin (Kd = 2.02 nM, R = 1.47 pmol/g of tissue) suggests the presence of mu and delta-receptors in the spinal cord tissue. The presence of kappa receptors was probed by the kappa-selective opioid [3H]U69593 (Kd = 3.31 nM, R = 2.00 pmol/g of tissue). The pharmacological characterization of the sites labeled by [3H]U69593 confirms the assumption that this ligand discriminates kappa receptors in guinea pig spinal cord. The benzomorphan [3H]ethylketazocine labels a population of receptors with one homogeneous affinity state (Kd = 0.65 nM, R = 7.39 pmol/g of tissue). The total binding capacity of this ligand was not different from the sum of the binding capacities of mu, delta-, and kappa-selective ligands. Under mu- and delta-suppressed conditions, [3H]ethylketazocine still binds to receptors with one homogeneous affinity state (Kd = 0.45 nM, R = 1.69 pmol/g of tissue). Competition studies performed against the binding of [3H]ethylketazocine under these experimental conditions reveal that the pharmacological profile of the radiolabeled receptors is similar to the profile of the kappa receptors labeled with [3H]U69593. Saturation studies using the nonselective opioid [3H]bremazocine demonstrate that this ligand binds to spinal cord membranes with heterogeneous affinities (Kd1 = 0.28 nM, R1 = 7.91 pmol/g of tissue; Kd2 = 3.24 nM, R2 = 11.2 pmol/g of tissue). The total binding capacity obtained with [3H]bremazocine (Rtotal = 19.1 pmol/g of tissue) was different from either the sum of the binding capacities of mu-, delta, and kappa- selective ligands or the binding capacity of [3H]ethylketazocine obtained under unsuppressed conditions. These results suggest that [3H]bremazocine labels additional opioid sites, namely the kappa 2 receptors, in contrast to kappa 1 sites labeled with [3H]U69593. In experimental conditions where the binding of [3H]bremazocine at mu, delta, and kappa 1 receptors was quenched by selective blockers, [3H]bremazocine recognizes the kappa 2 receptors with one homogeneous affinity state (Kd = 3.45 nM, R = 8.23 pmol/g of tissue). However, competition studies suggest that some opioids bind to these kappa 2 receptors with heterogeneous affinity states (high and low affinity states), whereas others bind with one apparently homogeneous affinity state.(ABSTRACT TRUNCATED AT 400 WORDS)

Volume 37, Issue 5, pp. 694-703, 05/01/1990
Copyright © 1990 by American Society for Pharmacology and Experimental Therapeutics

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