Further characterization of [3H]U69593 binding sites in the rat heart

K binding sites in the crude membrane preparation of the rat heart homogenate were further characterized by a displacement binding assay of [3H]-U69593 with specific κ ligands and a direct binding assay with [3H]-etorphine. Scatchard analysis of specific [3H]-U69593 binding showed that the Kd and Bmax were 6.4±1.0 nm and 97±8 fmol/mg protein, respectively. The binding of [3H]-U69593 was effectively displaced by the selective κ1 ligands, U-69593 and U-50488H, but only weakly displaced by Met5-enkephalin-Arg6-Phe7, a selective κ2 ligand, which showed only 11±3% inhibition of [3H]-U69593 binding at the concentration of 1 μm. In addition, there was no binding site for [3H]-etorphine, known to bind to μ, δ and κ2 binding sites, but not κ1 binding sites. The findings suggest that the κ binding sites in the rat heart most likely belong to the κ1 subtype. The binding sites have high and low affinity components as nonlinear regression analysis of the competition curves is best fit by two components with IC50 values of 11±2 and 62±7 nm for U-69593, and 9.9±1.5 and 414±108 nm for U-50488H. Furthermore, the binding of [3H]-U69593 were inhibited by both monovalent cations (Na+, Li+) and divalent cations (Mg2+, Mn2+ and Ca2+).