DIFFERENCES BETWEEN AGONIST AND ANTAGONIST BINDING TO ALPHA-1-ADRENERGIC RECEPTORS OF INTACT AND BROKEN-CELL PREPARATIONS

Abstract

.alpha.1-Adrenergic receptors of a nonfusing muscle cell line (BC3H1) were identified on intact and broken-cell preparations using the high-affinity antagonist [3H]prazosin. In intact cells, both equilibrium and kinetic studies gave Kd values in the range of 0.07-0.12 nM. The maximal number of binding sites determined by Scatchard analysis was .apprx. 85,000 .+-. 9000 sites/cell. Antagonists [phentolamine, yohimbine, (-)alprenolol] bound to the [3H]prazosin binding sites with Michaelis-Menten characteristics, and their specificity was typical of .alpha.1-adrenergic receptors. No significant modification of antagonist binding occurred either after cell disruption or by lowering incubation temperature to 4.degree. C. In intact cells at 37.degree. C, agonist [(-)epinephrine, (+)epinephrine, (-)norepinephrine and (-)phenylephrine] competition curves were shallow with Hill coefficients of < 1. The heterogeneity of [3H]prazosin binding sits toward (-)-norepinephrine also appeard in [3H]prazosin saturation experiments carried out in the absence and in the presence of this agonist. After cell disruption, the EC50 [median effective concentration] values of agonist competition curves deceased and Hill coefficients were close to 1. When the temperature was lowered from 37.degree. to 4.degree. C, the affinity for (-)-norepinephrine in intact cells increased dramatically by 10,000 times and the Hill coefficient of the competition curve was equal to unity. This affinity shift induced by temperature was not so important in broken-cell preparations (50 times).