Human Lymphocytes as a Model for Beta-Adrenergic Receptors in Clinical Investigation

Abstract

Beta-adrenergic binding studies were performed in human mononuclear leucocytes (MNL). Different tissue preparations and radioligands were compared. Viable cells and homogenates obtained by Potter or Polytron homogenisation or ultrasonication showed similar dissociation constants (KD) of 550 pM and 17.2 pM for (−)3H-dihydroalprenolol (3H-DHA) and (+/-) 125I-cyanopindolol (125I-cyp). The binding capacity Bmax as measured with 3H-DHA or 125I-CYP was higher in intact cells than in homogenates by a factor of 3. In intact cells more 3H-DHA than 125I-CYP binding sites were measured (7.8 vs. 6.2 fmoles/106 cells), similarly in homogenates Bmax of 3H-DHA exceeded that of 125I-CYP (29.1 vs. 14.5 fmol/mg of protein). The tissue linearity of binding in MNL homogenate was assessed with 3H-DHA, (+/-) 125I-CYP and (−)125I-CYP; whereas KD and Bmax were linear with the protein concentration up to 3 mg/ml and 0.6 mg/ml for 3H-DHA and (−)125I-CYP respectively, an increase of the KD and Bmax above 0.3 mg/ml was observed for the (+/-) isomer of 125I-CYP, indicating problems with racemic radioligands at high receptor concentrations. The guanine nucleotide induced change in affinity of isoproterenol binding was absent in the crude polytron homogenate, when measured with either 3H-DHA or 125I-CYP, it was not improved by the use of the selective agonist salbutamol. There was a moderate shift in a more purified preparation. On the other hand the guanine nucleotide guanidyl imidodiphosphate (Gpp(NH)p) reduced the 125I-CYP affinity by a factor of 2, leaving Bmax unchanged. Beta-adrenergic binding was studied in clinical investigation to compare young and old, normal and hypertensive subjects. Bmax and D were similar in MNL homogenates of 27 young and 13 older subjects. Bmax as measured with 3H-DHA, but not with 125I-CYP was slightly higher in 12 hypertensive than in normal subects.