RADIOLIGAND ASSAY OF BETA ADRENOCEPTORS IN LYMPHOCYTES ISOLATED FROM RAT SPLEEN

[H-3]Dihydroalprenolol ([H-3]DHA) was used to label beta-adrenoceptors in intact lymphocytes isolated from rat spleen. The binding conditions were investigated by the methods of L9 (3(4)) orthogonal analysis, the results showed the optimum binding conditions were pH 7.7, 1 x 10(8) lymphocytes . ml-1 and 10 min at 25-degrees-C for incubation. On these conditions the [H-3]DHA binding to beta-adrenoceptors was rapid (T1/2 2 min) and readily reversed by propranolol 1 mmol . L-1 (T1/2 4 min). [H-3]DHA saturation experiments indicated a single class of site with a K(D) of 7.2 +/- 2.2 nmol . L-1 and B(max) of 81 +/- 28 fmol/10(7) lymphocytes (n = 3). Computer analysis of competition experiments with isoproterenol and epinephrine revealed two classes of sites. One site had high affinity for the [H-3]DHA and comprised 60 +/- 5% of the total number of sites, whereas the other site had a lower affinity. The affinity of the high affinity site was about 3 orders of magnitude higher than that of the lower affinity site.