Properties of β-adrenoceptor sites in metabolizing and nonmetabolizing rat reticulocytes and in resealed reticulocyte ghosts

Intact cells and resealed ghosts of a homogeneous reticulocyte population isolated from the blood of phenylhydrazine-treated rats bound β-adrenergic ligands reversibly, stereospecifically and with high affinity. Maximal specific binding capacity under control conditions (37° C), corresponded to 9.9 ± 0.8 fmol/μl cells (∼ 600 sites/cell) and was similar in intact cells and ghosts. Pretreatment with metabolic inhibitors decreased the density of binding sites in intact cells to 6.48 ± 1.1 fmol/μl cells, but had no effect in ghosts. Incubation at 1°C reduced specific binding in paired experiments by 68 and 44% in intact cells and ghosts, respectively. Rewarming to 37°C increased specific binding in cells and ghosts by 270 and 190%, respectively. A temperature shift from 1 to 37°C reduced the KD value for the antagonist (-)timolol from 8.6 ± 1.5 to 1.1 ± 0.3 nmol/l in intact cells, while no significant reduction in KD was observed with ghosts. Under all conditions the receptor population was homogeneous with respect to antagonist affinity but inhomogenous with respect to agonist affinity. Low affinity agonist binding sites predominated in native cells and in GTP- loaded resealed ghosts (apparent KD values 447 and 680 nmol/l, respectively). High affinity binding sites predominated in both preparations at 1°C (KD 29 and 14 nmol/l, respectively). β-Adrenoceptor sites in starved cells and ghosts at 37°C showed intermediate apparent KD values. GTP had no effect on antagonist affinity or on the density of β-adrenoceptors. The results suggest that intact metabolizing cells can regulate β-adrenoceptor density by mechanisms which are not shared by ghost cells. The fractional contribution of high and low affinity states of the receptor to the overall binding of agonists seemed to be determined largely by the intracellular GTP concentration.