Interaction between [3H]ethylketocyclazocine and [3H]etorphine and opioid receptors in membranes from rat brain. A kinetic analysis.

Scatchard analysis of the binding to opioid receptors of [3H]ethylketocyclazocine ([3H]EKC) and [3H]etorphine at equilibrium yielded biphasic plots and computer fitting of the data resulted in a minimal model of two independent saturable binding sites. The KD values for the high- and low-affinity sites were 0.58 and 38 nM for [3H]EKC, and 0.13 and 22 nM for [3H]etorphine. The corresponding density of binding sites was 157 and 418 fmol/mg protein for [3H]EKC, and 220 and 289 fmol/mg protein for [3H]etorphine. The KD values calculated from the association and dissociation rate constants corresponded to those observed at equilibrium. In the course of equilibrium binding, various opioids competed with [3H]EKC and [3H]etorphine preferentially at the high-affinity opioid receptor sites. No difference between the competition patterns of putative mu and kappa ligands was observed. The kinetics of association and dissociation of [3H]EKC and [3H]etorphine revealed that the apparently homogeneous high-affinity binding site observed at equilibrium consisted of two components characterized by their fast and slow equilibrium times, respectively. While none of the mu and kappa opiates investigated altered the rate of dissociation of [3H]EKC or [3H]etorphine, in the presence of sodium ions the rapidly dissociating binding component of [3H]etorphine became refractory to inhibition by mu but not kappa agonists. The results underline the advantages of evaluating both equilibrium binding and the kinetics of ligand-receptor interactions.