Modes of Determining β‐Adrenoceptor Number in Human Mononuclear Leucocytes

Abstract
Inhibition of total 125I-ICYP binding to intact human mononuclear leucocytes at 32 by propranolol and (.+-.) CGP-12177 was biphasic. The high affinity component of 125I-CYP binding, representing approximately 30% of total, was stereospecific, while the low affinity binding site was inhibited without stereospecificity. (-) Isoproterenol inhibited the high affinity component of 125I-ICYP binding only, with low affinity. By performing binding studies in intact cells at 4.degree. or in broken cell preparations at 37, the fraction of total 125I-ICYP binding representing specific binding was increased, and agonist affinity was high. Inhibition of 3H-CGP-12177 binding to intact cells at 32.degree. demonstrated a high fraction of specific binding and high agonist affinity. Computer-assisted analysis of total radioligand binding determined over a broad concentration range revealed two populations of saturable 125I-CYP binding sites in intact cells as well as in broken cell preparations, while 3H-CGP-12177 binding demonstrated only one saturable binding site. The number of high affinity 125I-CYP binding sites was comparable to the number of saturable 3H-CGP-12177 binding sites. Receptor numbers determined by analysis of total radioligand binding were comparable to receptor numbers determined by subtraction of non-specific binding, determined in the presence of a high concentration of competing ligand. Analysis of total radioligand binding was found to be a better procedure because it eliminates the use of an arbitrary concentration of unlabelled ligand and improves the accuracy of the assay.