Multiple opioid binding sites and their ligands
The presence of μ-, δ- and κ-binding sites in homogenates of guinea-pig brain was demonstrated by the use of selective labelling techniques. In saturation experiments, the tritiated ligands [^3H]-[D-Ala^2, MePhe^4, Gly-ol^5]enkephalin, [^3H]-dihydromorphine, [^3H]-morphine and [^3H]-dihydronormorphine labelled only the μ-binding site. The δ-binding site could be labelled selectively with [^3H]-[D-Pen^2, D-Pen^5]enkephalin. However, the less selective δ-ligand, [3H]-[D-Ala2, D-Leu5] enkephalin, could only be used when its μ-binding was blocked with the unlabelled μ-ligand [D-Ala2, MePhe4, Gly-ol5]enkephalin. Selective labelling of the κ-binding site was more of a problem since the non-selective ligands [^3H]-etorphine, [^3H]-(±)-ethylketazocine and [3H]-(-)-bremazocine bind to the μ-, δ- and κ-sites. Therefore, the κ-binding site could only be labelled selectively when the binding of the tritiated ligands to the μ- and δ-sites was prevented by addition of the unlabelled μ-ligand [D-Ala^2, MePhe^4, Gly-ol^5]enkephalin and the unlabelled δ-ligand [D-Ala2, D-Leu5]enkephalin. By analysis of the saturation curves obtained using these selective labelling techniques, the proportion of binding sites in homogenates of guinea-pig brain at 25°C was 24% μ-sites; 32% δ-sites and 44% κ-sites. The selective labelling techniques were also used to label the μ, δ- and κ-sites in displacement assays. The compounds with the highest degree of preference for each binding site were: for the μ-site, [D-Ala^2, MePhe^4, Gly-ol^5]enkephalin and Tyr-Pro-MePhe-D-Pro-NH_2; for the δ-site, [D-Pen2, L-Pen5]enkephalin, [D-Pen2, D-Pen5]enkephalin and ICI 174864 and for the κ-site, U-50,488H and U-69,593. As far as antagonists were concerned, naloxone displayed the highest preference for the μ-binding site and Mr 2266 had a preference for the κ-binding site but neither compound was highly selective unlike the δ-antagonist ICI 174864. The effect of pre-incubation with β-funaltrexamine on opioid binding was investigated in homogenates of guinea-pig brain and myenteric plexus-longitudinal muscle.