Georg Sager

Variable binding of propranolol in human serum.

Human serum proteins were fractionated by ultracentrifugation and gel filtration. Binding of propranolol was determined by equilibrium dialysis. Propranolol was distributed to lipoproteins independent of drug concentration. Two groups of propranolol binding sites were found to be present in the protein preparation containing albumin, α1-acid glycoprotein, transferrin and prealbumin. The first binding site with a dissociation constant of 7.5 × 10−7 was present in number equivalent to concentration of α1-acid glycoprotein. The propranolol binding to serum samples from 21 healthy males expressed as binding ratio B/F and per cent binding ranged from 7.5 to 19.2 and 88.2 to 95.0 respectively. The binding ratio was correlated to concentration of α1-acid glycoprotein (r = 0.85, P < 0.001), but not to concentrations of albumin and lipoproteins. The results indicate that α1-acid glycoprotein is the main propranolol binding protein in human serum.

RECEPTOR BINDING SITES FOR BETA-ADRENERGIC LIGANDS ON HUMAN ERYTHROCYTES

Affinity, specificity and kinetics for [3H]-DHA binding to human red cell ghost were determined by ultra-filtration. At 2 degree an apparent dissociation constant of 0.96 nM was found with maximum specific binding of 29 fmoles per mg protein. The low dissociation constant was confirmed by kinetic studies with a value of 0.86 nM. Propranolol and isoproterenol inhibited [3H]-DHA binding stereo specifically. Agonist potency (IPR greater than EPI greater than NE) indicated that human erythrocytes had an adrenergic receptor of beta-2 subtype. Isoproterenol in the presence of theophylline resulted in a concentration-dependent increase of intracellular cAMP levels in intact cells. Basal and maximal levels were 2.3 and 7.5 pmoles/108 cells respectively after 2.5 min stimulation. EC50 for isoproterenol was 0.27 microM. Propranolol shifted the isoproterenol concentration response curve to the right. The present results show that human erythrocytes possess recognition sites for beta-adrenergic ligands with binding characteristics similar to that of adrenergic receptors of beta-2 subtype. At least a small number of these binding sites are functionally coupled to adenylate cyclase.

Elevated level of β-adrenergic receptors in hepatocytes from regenerating rat liver: Time study of [125I]iodocyanopindolol binding following partial hepatectomy and its relationship to catecholamine-sensitive adenylate cyclase

Hepatocytes from regenerating rat liver show an enhanced epinephrine-sensitive adenylate cyclase activity and cAMP response, which may be involved in triggering of the cell proliferation. We have determined adrenergic receptors and adenylate cyclase activity in hepatocytes isolated at various time points after partial hepatectomy. The number of beta-adrenergic receptors, measured by binding of [125I]iodocyanopindolol ([125I]CYP) to a particulate fraction prepared from isolated hepatocytes, increased rapidly after partial hepatectomy as compared with sham-operated or untreated controls. The maximal increase, which was observed at 48 h, was between 5- and 6-fold (from approximately 1 800 to approximately 10 500 sites per cell). Thereafter, the number of beta-adrenergic receptors decreased gradually. Competition experiments indicated beta 2-type receptors. Parallelism was found between the change in the number of beta 2-adrenergic receptors and the isoproterenol-responsive adenylate cyclase activity. The number of alpha 1-adrenergic receptors, determined by binding of [3H]prazosin, was transiently lowered by about 35% at 18-24 h, with no significant change in Kd. Although the results of this study do not exclude the possibility of post-receptor events, they suggest that the increased number of beta 2-adrenergic receptors is a major factor responsible for the enhanced catecholamine-responsive adenylate cyclase activity in regenerating liver.

Plasma dependent adrenergic ligand binding to human erythrocytes.

Abstract High affinity binding sites resembling beta-adrenergic receptor binding on human erythrocytes have been identified by using (±)-propranolol and (−)-alprenolol. Experiments were also conducted to study the influence of plasma on this binding. Human blood was obtained from 20 healthy subjects. Adrenergic ligand binding was studied in whole blood and in suspensions of washed erythrocytes in Krebs Ringer buffer or in plasma at 22°. (±)-Propranolol was bound to erythrocytes in plasma with K diss = 13± 2 nM and N = 5400 ±1000 sites per cell and (−)-alprenolol with K diss = 5.5 ± 1.5 nM and N = 6100 ± 1200 sites per cell. Binding of these beta-adrenergic antagonists were competitively inhibited by (±)-isoprenaline and (±)-salbutamol. By washing of cells the number of binding sites was reduced from about 6000 to 600 while dissociation constants were unaltered. Number of binding sites was re-established when washed cells were resuspended in plasma. These results support the assumption of beta -adrenergic receptor binding sites on human erythrocytes and of a water-soluble component essential for the high affinity binding sites, present on cells and in plasma.