Β-adrenergic Receptor Binding Research Articles

Agonist-induced increase in apparent β-adrenergic receptor size

The properties of digitonin-solubilized beta-adrenergic receptors from frog erythrocyte membranes were studied by gel exclusion chromatography on AcA 34 Ultragel. beta-Adrenergic receptor binding activity in these membranes can be identified by both an agonist ligand, [(3)H]hydroxybenzylisoproterenol, and the antagonist ligands, [(3)H]dihydroalprenolol and (125)I-labeled hydroxybenzylpindolol. Occupancy of the beta-adrenergic receptors with the [(3)H]hydroxybenzylisoproterenol agonist prior to their solubilization from the membrane leads to an increase in apparent receptor size. Alterations in the molecular size of the receptor cannot be mimicked by occupancy of the binding site with the antagonist ligands. Exposure of frog erythrocyte membranes to [(3)H]hydroxybenzylisoproterenol agonist in the presence of 10 muM Gpp(NH)(p), a guanyl nucleotide analog that exerts multiple regulatory effects on the catecholamine-sensitive adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1] system, results in the elution of the [(3)H]hydroxybenzylisoproterenol radioligand in both the region characteristic of the agonist-receptor complex and the region characteristic of the antagonist-receptor complex. The precise molecular interactions responsible for the agonist-induced increase in apparent beta-adrenergic receptor size are still unresolved. However, the low concentrations of agonist that are capable of altering apparent receptor size and the sensitivity of this effect to guanyl nucleotides suggest that these phenomena may be intimately involved in eliciting the physiological effects of beta-adrenergic catecholamines at the molecular level.

β-Adrenergic receptor binding: synaptic localization in rat brain

β-Adrenergic receptor binding: synaptic localization in rat brain

Independent variation of β-adrenergic receptor binding and catecholamine-stimulated adenylate cyclase activity in rat erythrocytes

Isoproterenol-stimulated adenylate cyclase activity in membrane preparations of reticulocyte-rich (90%) erythrocytes from phenylhydrazine-treated rats is 9 times greater than in untreated animals (1% reticulocytes); basal and fluoride-stimulated activities are also enhanced 2 and 4-fold respectively. In contrast, the number of β-adrenergic receptor sites detected by the binding of 125I-hydroxybenzylpindolol ( 125I-HYP) is increased only 40% in these same preparations. The dissociation constant (K D) of 125I-HYP and the IC 50 of (-)-isoproterenol for receptor binding sites are unchanged, as is the EC 50 of (-)-isoproterenol for activation of adenylate cyclase. The disproportionately large increase in the activity of the isoproterenol-sensitive adenylate cyclase, compared with the small increase in the number of 125I-HYP binding sites indicates that the functions of catecholamine recognition and consequent adenylate cyclase response can vary independently and suggest that the receptor and the cyclase may be autonomous molecular entities.

β-Adrenergic receptors: Evidence for negative cooperativity

The specific binding of [ 3H] (−)alprenolol to sites in frog erythrocyte membranes provides a tool for directly assessing ligand binding to adenylatecyclase coupled β-adrenergic receptors. Hill Plots of such binding data yield slopes (n H=“Hill Coefficients”) less than 1.0, suggesting that negatively cooperative interactions among the β-adrenergic receptors may occur. The existence of such negative cooperativity was confirmed by a direct kinetic method. The dissociation of receptor bound [ 3H] (−)alprenolol was studied under two conditions: 1) with dilution of the ligand-receptor complex sufficient to prevent rebinding of the dissociated tracer and 2) with this same dilution in the presence of excess unlabeled (−)alprenolol. If the sites are independent, the dissociation rates must be the same in both cases. However, the presence of (−)alprenolol increases the rate of [ 3H] (−)alprenolol dissociation, indicating that negatively cooperative interactions among the β-adrenergic receptor binding sites do occur.

Stereospecific [ 3H](−)-alprenolol binding sites, β-adrenergic receptors and adenylate cyclase

The binding of [ 3H](−)-alprenolol (a potent β-adrenergic antagonist) to sites in frog erythrocyte membranes has been studied by a centrifugal assay. The specificity of the binding sites is strikingly similar to what might be expected of the β-adrenergic receptor binding sites which mediate stimulation of adenylate cyclase by catecholamines in these membranes. The sites bind β-adrenergic antagonists and agonists with affinities which are directly related to their antagonist or agonist potency on the frog erythrocyte membrane adenylate cyclase. Binding shows strict stereospecificity with (−)-isomers exhibiting two orders of magnitude higher affinities than (+)-isomers. Dissociation constants for potent β-adrenergic antagonists are in the range of 10 −9 – 10 −8M whereas those for β-adrenergic agonists are about two orders of magnitude higher (≥ 10 −6M).

"Trophic" influences of nerve on muscle.

"Trophic" influences of nerve on muscle.