Kinetic analysis of the interactions of agonists and antagonists with beta adrenergic receptors

The affinity of the beta adrenergic receptor for antagonists is frequently higher than that for agonists. It has been assumed that the binding of agonists and antagonists is diffusion limited and that the high affinity of the receptor for typical antagonists is due to slow rates of dissociation. To test this hypothesis, the kinetics of binding of unlabeled agonists and antagonists were determined using the method described by Motulsky and Mahan (Mol. Pharmacol. 25: 1-9, 1984). The time course of the binding of a radioligand in the presence of a competing unlabeled ligand was analyzed in terms of rate constants of association (kon) and dissociation (koff) for binding of the radioligand and the competitor. This approach was validated by showing that the rate constants for binding of [3H]dihydroalprenolol and [3H]CGP-12177 [[3H]-4-(3-tertiarybutylamino-2-hydroxypropoxy)-benzimidazole-2-on ] determined directly were similar to values determined when the binding of [125I]iodopindolol was measured in the presence of [3H]dihydroalprenolol or [3H]CGP-12177. Computer simulations suggested that this method was experimentally limited to competing ligands with rate constants of dissociation below approximately 0.50 min-1. The apparent rate constants for binding of four unlabeled agonists and eight antagonists were determined experimentally at 10 degrees C. Although the values of koff for agonists and antagonists were similar, the values for kon for binding of agonists were consistently lower than the values for binding of antagonists. The relatively slow rate constant for association of agonists may be explained by a two-step mechanism or may involve agonist-induced isomerization of the receptor.