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 (K_D) of 550 pM and 17.2 pM for (−)³H-dihydroalprenolol (³H-DHA) and (+/-) ¹²⁵I-cyanopindolol (¹²⁵I-cyp). The binding capacity B_max 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 B_max 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 K_D and B_max 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 K_D and B_max 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 B_max unchanged. Beta-adrenergic binding was studied in clinical investigation to compare young and old, normal and hypertensive subjects. B_max and _D were similar in MNL homogenates of 27 young and 13 older subjects. B_max as measured with ³H-DHA, but not with ¹²⁵I-CYP was slightly higher in 12 hypertensive than in normal subects.