High-affinity binding of agonists to β-adrenergic receptors on intact cells

The interactions of agonists and antagonists with β-adrenergic receptors on intact 1321N1 human astrocytoma and C62B rat glioma cells were studied by using the radioligand (-)-[¹²⁵I]iodopindolol. Competition binding assays were performed at 37°C under equilibrium conditions and in short-time nonequilibrium assays that approximated initial velocity conditions for binding of the radioligand. The theoretical basis and experimental validation of the initial velocity approach for determining binding affinities of rapidly equilibrating ligands are presented. For the agonists isoproterenol and epinephrine, high binding affinities that approximated their apparent affinities for binding in membranes and for increase of cyclic AMP concentrations in intact cells could be demonstrated only in short-time assays; in contrast, much lower affinities were observed with equilibrium (60-min) assays as reported previously for various cell lines. High-affinity binding of isoproterenol to 1321N1 cells also was observed in equilibrium (6-hr) binding assays carried out on ice. These results indicate that in the native state the intact cell β-adrenergic receptor has a high binding affinity for agonists and suggest that incubation at 37°C in the presence of an agonist converts the receptors to a form with low affinity for agonists.