The new radioligand [3H]-L 748,337 differentially labels human and rat β3-adrenoceptors

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 [3H]-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, [3H]-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 [3H]-L 748,337 was observed. While some isoprenaline-sensitive [3H]-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 [3H]-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 [3H]-L 748,337 can bind to a low-affinity site distinct from the orthosteric pocket of β-adrenoceptors. Nevertheless, [3H]-L 748,337 appears to be the most promising radioligand for the selective labelling of human β3-adrenoceptors reported to date.