title: The new radioligand [(3)H]-L 748,337 differentially labels human and rat β3-adrenoceptors.
abstract: As no suitable radioligand exists for the detection of β3-adrenoceptors, we have explored the radioligand binding properties of a tritiated version of the selective β3-adrenoceptor antagonist L 748,337. Kinetic and equilibrium saturation and competition binding experiments were performed with [(3)H]-L 748,337 on membrane fractions of HEK and CHO cells stably transfected with human and rat β-adrenoceptor subtypes. Based on both association/dissociation kinetic and equilibrium saturation binding studies in transfected HEK cells, [(3)H]-L 748,337 exhibited an affinity of approximately 2 nM for human β3-adrenoceptors. Competition studies with agonists and subtype-selective antagonists validated its binding to β3-adrenoceptors. In CHO cells transfected with human β3-adrenoceptors similar saturable high-affinity of [(3)H]-L 748,337 was observed. While some isoprenaline-sensitive [(3)H]-L 748,337 binding was also observed in CHO cells transfected with human β1- or β2-adrenoceptors, this was not saturable in a similar concentration range and/or not sensitive to the antagonists propranolol and SR 59,230, indicating that it did not primarily involve β-adrenoceptors. In CHO cells transfected with rat β3-adrenoceptors [(3)H]-L 748,337 exhibited a considerably lower affinity than with the human subtype (12-95 nM). Low affinity for the rat β3-adrenoceptor was also found with unlabelled L 748,337 in rat bladder strip relaxation experiments. We conclude that L 748,337 apparently has lower affinity for the rat than the human β3-adrenoceptors and that [(3)H]-L 748,337 can bind to a low-affinity site distinct from the orthosteric pocket of β-adrenoceptors. Nevertheless, [(3)H]-L 748,337 appears to be the most promising radioligand for the selective labelling of human β3-adrenoceptors reported to date.
authors.affiliation: Dept. Nuclear Medicine, Academic Medical Center, University of Amsterdam, The Netherlands.
authors.forename: Jan-Peter
authors.initials: JP
authors.lastname: van Wieringen
authors.forename: Martina B
authors.initials: MB
authors.lastname: Michel-Reher
authors.forename: Toshiki
authors.initials: T
authors.lastname: Hatanaka
authors.forename: Koji
authors.initials: K
authors.lastname: Ueshima
authors.forename: Martin C
authors.initials: MC
authors.lastname: Michel
mesh_terms: D058673:Adrenergic beta-3 Receptor Antagonists / Q000494:pharmacology*; D000627:Aminophenols / Q000494:pharmacology*; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D003412:Cricetulus; D057809:HEK293 Cells; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D009130:Muscle, Smooth / Q000187:drug effects / Q000502:physiology; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D022702:Receptors, Adrenergic, beta-3 / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology*; D001743:Urinary Bladder / Q000187:drug effects / Q000502:physiology
chemical_list: D058673:Adrenergic beta-3 Receptor Antagonists; D000627:Aminophenols; C120576:L 748,337; D022702:Receptors, Adrenergic, beta-3; D013449:Sulfonamides
keywords: Radioligand binding; Rat urinary bladder; [(3)H]-L 748,337; β(3)-Adrenoceptor