Characteristics of beta-adrenergic-agonist binding to rat adipocyte membranes. Evidence that (+/-)-[3H]hydroxybenzylisoproterenol interacts selectively with the adipocyte beta-adrenergic receptors.
The binding characteristics of the beta-adrenergic agonist (+/)-[3H]hydroxybenzylisoproterenol to rat adipocyte membranes were studied. Binding was rapid, reaching equilibrium within 10 min at 37 degrees C (second order rate constant K1 = 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 k2 of 0.106 . min-1 and 0.011 . min-1 . [3H]Hydroxybenzylisoproterenol binding was saturable (Bmax = 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 (KD = 3.5 +/- 0.7 nM) but low binding capacity (10% of the total sites) and one having low affinity (KD = 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 greater than (-)-isoproterenol greater than (-)-norepinephrine approximately (-)-epinephrine greater than greater than (+)-isoproterenol = (+)-propranolol, which is consistent with binding to beta 1-adrenergic receptors. Competition curves of [3H]hydroxybenzylisoproterenol binding by the beta-agonist (-)-isoproterenol were shallow and modeled to two affinity states of binding, whereas, competition curves by beta-antagonist (-)-propranolol were steeper with Hill number near to one. Gpp[NH]p severely reduced [3H]hydroxybenzylisoproterenol 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, the 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 correlated closely with the ability of (-)-isoproterenol to displace [3H]hydroxybenzylisoproterenol binding. Comparison of these findings with the binding characteristics of the beta-antagonist [3H]dihydroalprenolol to rat adipocyte membranes, led to conclude that [3H]hydroxybenzylisoproterenol can be successfully used to label the beta-adrenergic receptors of rat fat cells and suggests that it might be a better ligand than [3H]dihydroalprenolol in these cells.
D000273:Adipose Tissue / Q000378:metabolism*; D000318:Adrenergic beta-Agonists / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D004082:Dihydroalprenolol / Q000378:metabolism; D007545:Isoproterenol / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D011941:Receptors, Adrenergic / Q000378:metabolism*; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*
D000318:Adrenergic beta-Agonists; D011941:Receptors, Adrenergic; D011943:Receptors, Adrenergic, beta; D004082:Dihydroalprenolol; C015272:hydroxybenzylisoproterenol; D007545:Isoproterenol