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

  • 1 May 1990
    • journal article
    • research article
    • Vol. 37  (5) , 694-703
Abstract
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. Thus, computer analyses have helped to eastablish the multiplicity of .kappa. receptors in the guinea pig spinal cord. Results obtained in the presence of guanyl-5''-yl imidodiphosphate further lead to the conclusion that the interaction of opioids at the .kappa.2 receptors is more complex then was originally described. Finally, the relative proportions of the opioid receptors in whole spinal cord are as follows: 28% .mu., 9% .delta., 12% .kappa.1, and 51% .kappa.2 (.kappa.2h plus .kappa.2L).