Characterization of Opioid Binding Sites in Rat Spinal Cord

Abstract

Binding sites were characterized in rat whole spinal cord crude membrane preparations using selective labelling techniques with multiple methods of mathematical analysis of experimental curves. Mathematical analysis of single [³H]-[D-Ala², MePhe⁴, Gly-ol⁵] enkephalin (DAGO) saturation curves suggested binding of the [³H]-ligand at one site, while displacement curves of low concentrations of [³H]-DAGO with selective μ-ligands indicated the presence of high- and low-affinity sites. All the [³H]-DAGO curves processed simultaneously by LIGAND analysis showed the presence of high (27%) and low (73%) affinity components, with a total B_max of 3.19 pmol/g tissue. Eighty percent of [³H]-[D-Ala², D-Leu⁵] enkephalin (DADLE) binding was displaced by DAGO with high affinity, indicating that a high percentage of [³H]-DADLE binding was at μ-sites. Saturation curves of [³H]-DADLE after inhibition of μ-sites by unlabelled DAGO (∂-sites) were monophasic with non-linear fitting analysis and the B_max was 0.90 pmol/g tissue. Most mathematical analysis of single saturation curves of [³H]-(-)-bremazocine indicated binding at more than one site. DAGO, DADLE, U-69,593 and PD 117302 displaced 0.15 nM of [³H]-(-)-bremazocine biphasically: the percentages of displacement calculated with the non-linear fitting program were respectively 50 (μ-sites), 64 (μ+∂)-sites), 18 and 25 (kappa-sites). Haloperidol displaced [³H]-(-)-bremazocine only at μM concentrations, suggesting no binding at sigma-sites. In the presence of 225 nM of DAGO, DADLE displaced only 21% of [³H]-(-)-bremazocine 0.15 nM binding (∂-sites). Most mathematical analysis of saturation curves of [³H]-(-)-bremazocine, after inhibition of binding at μ- and ∂-sites by DAGO and DADLE, still indicated binding at more