The role of a low β₁-adrenoceptor selectivity of [³H]CGP-12177 for resolving subtype-selectivity of competitive ligands

Summary

On the basis of saturation binding studies on rat cardiac microsomes, which contained a mixed population of β-adrenoceptor subtypes, [³H]CGP-12177 is presumed to be a non-selective β-adrenergic radioligand. However, saturation binding studies carried out in the presence of subtype-saturating concentrations of the β₂-selective antagonist ICI 118,551 and the β₁-selective antagonist ICI 89,406, respectively, revealed a K _D for β₁-adrenoceptors of 0.33 ± 0.02 nmol/l and a K _D for β₂-adrenoceptors of 0.90 ± 0.14 nmol/l. Competition experiments with the highly selective antagonists revealed greatly different competition binding curves in the presence of either [³H]CGP-12177 or (−)[¹²⁵I]iodocyanopindolol (ICYP), a β-adrenergic radioligand considered to be as non-selective as [³H]CGP-12177. The following results are further suggestive for a selectivity of [³H]CGP-12177 for β₁-adrenoceptors: (1) Using non-linear regression analysis, a significantly lower selectivity (expressed as the ratio of the IC₅₀ for β₂-adrenoceptors to the IC₅₀ for β₁-adrenoceptors) as well as a larger proportion of β₁-adrenoceptors were calculated by competition of the β₁-selective antagonist ICI 89,406 with [³H]CGP-12177 binding than by competition of ICI 89,406 with ICYP binding; (2) reducing the [³H]CGP-12177 concentration from 2 to 0.4 nmol/l, competition experiments with ICI 89,406 led to an increase in the estimated selectivity of the competitor and in the estimated proportion of β₁-adrenoceptors; (3) reverse findings were obtained with ICI 118,551, a β₂-selective antagonist. Theoretical competition data assuming a varying selectivity of the radioligand were generated using an equation which describes inhibition of the binding of a selective radioligand by a selective competing ligand. Analysis of these data by the model which assumes that the radioligand is entirely non-selective revealed a logarithmic relationship between the distortion of the true selectivity of the competing ligand and the degree of selectivity of the radioligand. For instance, the selectivity of ICI 89,406, estimated against [³H]CGP-12177 binding, was 23% of its selectivity estimated against ICYP binding. Hence, non-linear fits of competition curves with subtype-selective unlabeled ligands will result in serious distortion in the estimates of affinity of the competing ligands and in the size of subtype populations if [³H]CGP-12177 is considered entirely non-selective.