Quantitative analysis of the selectivity of radioligands for subtypes of beta adrenergic receptors

In tissues with two classes of binding sites for a drug, it is common to estimate the proportion of each class of binding site by inhibiting the binding of a radioligand with a selective unlabeled ligand. Accurate estimates of the density of each class of binding site, however, will be obtained only if the radioligand is nonselective or used at a concentration that saturates both classes of binding sites. A method of simultaneous regression analysis of multiple inhibition curves, using the program MLAB on the PROPHET system, was used to quantify the selectivity of radioligands for beta-1 or beta-2 adrenergic receptors. The selectivity of [¹²⁵I]iodopindolol, [¹²⁵I]iodocyanopindolol, [¹²⁵I]iodohydroxybenzylpindolol and [³H]dihydroalprenolol for beta-1 and beta-2 adrenergic receptors was assessed by inhibiting the binding of each radioligand with the beta-1-selective unlabeled ligand ICI 89,406 at increasing concentrations of the radioligand, using membranes prepared from C₆ glioma cells, which have both beta-1 and beta-2 adrenergic receptors. Scatchard plots for all four radioligands were linear, with correlation coefficients greater than 0.95. [¹²⁵I]Iodopindolol and [¹²⁵I]iodocyanopindolol were 3.2- and 2-fold selective, respectively, and [¹²⁵I]iodohydroxybenzylpindolol and [³H]dihydroalprenolol were 5.8- and 2.3-fold selective, respectively, for beta-2 adrenergic receptors. Values obtained for the densities of beta-1 and beta-2 adrenergic receptors and the affinities of the receptors for ICI 89,406 were independent of the radioligand used. For experiments requiring an accurate estimate of the proportion of each subtype of beta adrenergic receptors, [¹²⁵I]iodocyanopindolol may be the most appropriate ligand, as it is only 2-fold selective, has a high affinity and specific activity and has little nonspecific binding at concentrations of radioligand many times its K(D) value.