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RESEARCH PRODUCT

The new radioligand [H-3]-L 748,337 differentially labels human and rat beta(3)-adrenoceptors

Martina B. Michel-reherJan-peter Van WieringenToshiki HatanakaKoji UeshimaMartin C. Michel

subject

MaleStereochemistryUrinary BladderCHO CellsIn Vitro TechniquesAminophenolsBinding CompetitiveRats Sprague-Dawley03 medical and health sciencesRadioligand Assay0302 clinical medicineCricetulusRadioligandAnimalsHumans030304 developmental biologyPharmacology0303 health sciencesSulfonamidesbiologyChemistryChinese hamster ovary cellHEK 293 cellsAntagonistMuscle SmoothTransfectionbiology.organism_classificationMolecular biologyRadioligand AssayRatsMembraneHEK293 CellsReceptors Adrenergic beta-3CricetulusAdrenergic beta-3 Receptor Antagonists030217 neurology & neurosurgery

description

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.

10.1016/j.ejphar.2013.10.039http://dx.doi.org/10.1016/j.ejphar.2013.10.039