SPECTRUM OF THE μ‐, δ‐ AND κ‐BINDING SITES IN HOMOGENATES OF RAT BRAIN

Abstract

In homogenates of rat brain, the binding characteristics of tritiated opiates and opioid peptides were examined and the relative capacities of μ‐, δ‐ and κ‐binding sites of the opiate receptor determined by saturation analysis. In competition experiments, binding of the selective μ‐ligand [3 H]‐[d‐Ala 2,MePhe 4,Gly‐ol 5]enkephalin at the μ‐site was displaced by [d‐Ala 2,d‐Leu 5]enkephalin with rather low affinity (KI= 12.6 n m) and more readily by the ketazocine‐like compounds (−)‐ethylketazocine (KI= 3.1 n m) and (−)‐bremazocine (KI= 0.32 n m), which also displaced the binding of [3 H]‐[d‐Ala 2,d‐Leu 5]enkephalin from the δ‐site. In contrast, the binding to the κ‐site was easily displaced by ethylketazocine (1.0 n m) and bremazocine (0.37 n m) but not by the μ‐ligand [d‐Ala 2,MePhe 4,Gly‐ol 5]enkephalin (KI= 2000–3000 n m) or the δ‐ligand [d‐Ala 2,d‐Leu 5]enkephalin (KI> 20,000 n m). The dissociation equilibrium constant (KD) and the binding capacity (pmol/g) of the μ‐binding site were determined with the selective μ‐ligand [3 H]‐[d‐Ala 2,MePhe 4,Gly‐ol 5]enkephalin. For the δ‐site, [3 H]‐[d‐Ala 2,d‐Leu 5]enkephalin was used in the presence of unlabelled [d‐Ala 2,MePhe 4,Gly‐ol 5]enkephalin in order to suppress cross‐reactivity to the μ‐binding site. For the estimation of κ‐binding, [3 H]‐(±)‐ethylketazocine or [3 H]‐(−)‐bremazocine were used in the presence of unlabelled μ‐ and δ‐ligands for the suppression of cross‐reactivities to the μ‐ and δ‐binding sites. In rat brain the capacity of the μ‐binding site was 7.3 pmol/g brain, that of the δ‐binding site 6.7 pmol/g brain and that of the κ‐binding site 2.0 pmol/g brain. Thus, the κ‐binding site had the lowest value whereas in the guinea‐pig brain the capacity of the μ‐binding site was lower than that of the δ‐ or κ‐binding site.