A reliable assay for beta-adrenoceptors in intact isolated human fat cells with a hydrophilic radioligand, [3H]CGP-12177.
The beta-adrenergic receptors of isolated human fat cells were identified using a new hydrophilic beta-adrenergic radioligand (+/-)[3H]CGP-12177. The results were compared with those from [3H]dihydroalprenolol binding to fat cells and membranes. [3H]CGP-12177 binding to isolated fat cells showed lower nonspecific binding (less than 15% of total binding) than the lipophilic [3H]dihydroalprenolol (40-60%) at 3 times the KD. At 37 degrees C, [3H]CGP-12177 binding was rapid, reversible, of high affinity (1.2 +/- 0.3 nM) and saturable. The total number of binding sites per cell in subcutaneous adipocytes was 25,000 +/- 6,000 and was equivalent to that found using membrane fractions. Displacement of [3H]CGP-12177 bound to adipocytes by propranolol was stereoselective, consistent with competition at a single site, and had the same characteristics as in membranes. The displacement curves of the beta 1-selective antagonists (atenolol and betaxolol) were biphasic, the high affinity displacement accounting for 70% of the total binding sites. Beta-adrenergic agonists also competed with [3H]CGP-12177 binding in the order of potency: (-) isoproterenol greater than (-) norepinephrine greater than (-) epinephrine, similar to that found in membranes and in in vitro studies on the lipolytic activity of isolated fat cells. This study demonstrates that the sites specifically labeled by [3H]CGP-12177 are the physiological beta-adrenoceptors and also shows that the ligand is better than [3H]dihydroalprenolol for the accurate identification of these receptors in intact human adipocytes. The methodology, which requires biopsies of less than 1 gram of adipose tissue, can be of potential interest for clinical studies investigating the status of fat cell beta-adrenoceptors in various pathophysiological situations.
Lacasa
D
D
Mauriège
P
P
Lafontan
M
M
Berlan
M
M
Giudicelli
Y
Y
D000273:Adipose Tissue / Q000032:analysis / Q000166:cytology*; D000319:Adrenergic beta-Antagonists / Q000494:pharmacology*; D000328:Adult; D000368:Aged; D001667:Binding, Competitive; D002395:Catecholamines / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002469:Cell Separation; D006801:Humans; D007700:Kinetics; D008066:Lipolysis / Q000187:drug effects; D008875:Middle Aged; D011412:Propanolamines / Q000378:metabolism; D011869:Radioligand Assay / Q000379:methods; D011943:Receptors, Adrenergic, beta / Q000032:analysis* / Q000187:drug effects; D013696:Temperature
D000319:Adrenergic beta-Antagonists; D002395:Catecholamines; D011412:Propanolamines; D011943:Receptors, Adrenergic, beta; C033394:CGP 12177