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 K d and B max were 6.4±1.0 n m and 97±8 fmol/mg protein, respectively. The binding of [ 3H]-U69593 was effectively displaced by the selective κ 1 ligands, U-69593 and U-50488H, but only weakly displaced by Met 5-enkephalin-Arg 6-Phe 7, a selective κ 2 ligand, which showed only 11±3% inhibition of [ 3H]-U69593 binding at the concentration of 1 μ m. In addition, there was no binding site for [ 3H]-etorphine, known to bind to μ, δ and κ 2 binding sites, but not κ 1 binding sites. The findings suggest that the κ binding sites in the rat heart most likely belong to the κ 1 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 IC 50 values of 11±2 and 62±7 n m for U-69593, and 9.9±1.5 and 414±108 n m for U-50488H. Furthermore, the binding of [ 3H]-U69593 were inhibited by both monovalent cations (Na +, Li +) and divalent cations (Mg 2+, Mn 2+ and Ca 2+).