Characteristics of β-adrenergic-agonist binding to rat adipocyte membranes: Evidence that [formula omitted]hydroxybenzylisoproterenol interacts selectively with the adipocyte β-adrenergic receptors

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The binding characteristics of the β-adrenergic agonist (±)-[ 3 H]hydroxybenzylisoproterenol to rat adipocyte membranes were studied. Binding was rapid, reaching equilibrium within 10 min at 37°C (second order rate constant k 1=1.37·10 7·M −1·min −1). Dissociation of specific binding by 0.5 mM (−)-isoproterenol suggested dissociation from two different sites with respective dissociation rate constants k 2 of 0.106·min −1 and 0.011·min −1.[ 3H]Hydroxybenzylisoproterenol binding was saturable ( B max=690±107 fmol/mg protein), yielding curvilinear Scatchard plots. Computer modeling of these data were consistent with the existence of two classes of [ 3H]hydroxybenzylisoproterenol binding sites, one having high affinity ( K D=3.5±0.7 nM) but low binding capacity (10% of the total sites) and one haveing low affinity ( K D=101±20 nM) but high binding capacity (90% of the sites). Adrenergic ligands competed with [ 3H]hydroxybenzylisoproterenol binding with the following order of potency=(−)-propranolol>(−)-isoproterenol>(−)-norepinephrine≈ (−)-epinephrine>>(+)-isoproterenol=(+)-propranolo, which is consistent with binding to β 1-adrenergic receptors. Competition curves of [ 3H]hydroxybenzylisoproterenol binding by the β-agonist (−)-isoproterenol were shallow and modeled to two affinity states of binding, whereas, competition curves by β-antagonist (−)-propranolol were steeper with Hill number near to one. Gpp[NH]p severely reduced [ 3H]hydroxybenzyl-isoproterenol binding, an effect which apparently resulted from the reduction of the number of both the high and low affinity sites. In membranes which had been previously exposed to (−)-isoproterenol, then number of [ 3H]hydroxybenzylisoproterenol binding sites was reduced by 50%, an effect which apparently resulted from the loss of part of both the high and low affinity state binding sites. Finally, the ability of (−)-isoproterenol to stimulate adenylate cyclase correlate closely with the ability of (−)-isoproterenol to displace [ 3H]hydroxybenzylisoproterenol binding. Comparison of these findings with the binding characteristics of the β-antagonist [ 3H]dihydroalprenolol to rat adipocyte membranes, led to conclude that [ 3H]hydroxybenzylisoproterenol can be successfully used to label the β-adrenergic receptors of rat fat cells and suggests that it might be a better ligand than [ 3H]dihydroalprenolol in these cells.