ORIGINAL ARTICLE Comparison of three radioligands for the labelling of human β-adrenoceptor subtypes Nadja Niclauß & Martina B. Michel-Reher & Astrid E. Alewijnse & Martin C. Michel Received: 30 May 2006 / Accepted: 7 August 2006 / Published online: 7 October 2006 # Springer-Verlag 2006 Abstract We have compared the ability of three radio- ligands, [ 125 I]-cyanopindolol, [ 3 H]-CGP 12,177 and [ 3 H]- dihydroalprenolol, to label the three human β-adrenoceptor subtypes. Saturation and competition binding experiments were performed using membrane preparations from Chinese hamster ovary cells stably transfected with the three subtypes. While [ 3 H]-CGP 12,177 had very similar affinity for β 1 - and β 2 -adrenoceptors (about 40 pM), [ 125 I]-cyano- pindolol and [ 3 H]-dihydroalprenolol had 4- to 6-fold higher affinity for β 2 - as compared to β 1 -adrenoceptors (10 vs 45 and 187 vs 1,021 pM, respectively). The affinity of [ 125 I]- cyanopindolol at β 3 -adrenoceptors was considerably lower (440 pM) than at the other two subtypes. The β 3 - adrenoceptor affinity of [ 3 H]-CGP 12,177 and [ 3 H]-dihydro- alprenolol was so low that it could not be estimated within the tested range of radioligand concentrations (up to 4,000 pM and 30,000 pM for [ 3 H]-CGP 12,177 and [ 3 H]- dihydroalprenolol, respectively). We conclude that all three radioligands are ill-suited to label β 3 -adrenoceptors, partic- ularly in preparations co-expressing multiple subtypes. In the absence of alternatives, [ 125 I]-cyanopindolol appears the least unsuitable to label β 3 -adrenoceptors. There is a need for high-affinity radioligands which are either selective for β 3 -adrenoceptors or reasonably non-selective among all three β-adrenoceptor subtypes. Keywords β 1 -adrenoceptor . β 2 -adrenoceptor . β 3 -adrenoceptor . [ 125 I]-cyanopindolol . [ 3 H]-CGP 12,177 . [ 3 H]-dihydroalprenolol Introduction Three subtypes of β-adrenoceptors exist which are desig- nated β 1 , β 2 and β 3 (Bylund et al. 1994). Each has a distinct tissue distribution and mediates distinct functions. While physiological functions of β 1 - and β 2 -adrenoceptors have been well established, those of β 3 -adrenoceptors are only starting to become elucidated, e.g. in the cardiovascu- lar and in the urogenital system (Michel and Vrydag 2006; Rozec and Gauthier 2006). A better understanding of the physiological and pathophysiological roles of β 3 -adreno- ceptors requires tools for their detection and quantification. This includes an assessment of the relative expression of β 3 -adrenoceptors in tissues expressing multiple subtypes, and of alterations of such expression related to diseases, drug treatment and/or polymorphisms of the corresponding genes (Leineweber et al. 2004). In the absence of well validated and subtype-specific antibodies, the most frequently used approach to quantify β-adrenoceptor expression at the protein level is radio- ligand binding. Unfortunately, the radioligands routinely used in β-adrenoceptor research are not well suited for the detection of β 3 -adrenoceptors. Thus, the most frequently used radioligand in this field, [ 125 I]-cyanopindolol ([ 125 I]- CYP) (Engel et al. 1981), has considerably lower affinity for β 3 -adrenoceptors than for β 1 - and β 2 -adrenoceptors, respec- tively (Hoffmann et al. 2004). An alternative radioligand for the labelling of β-adrenoceptors is [ 3 H]-CGP 12,177 (Staehelin et al. 1983), but a recent study suggests that this ligand also has much lower affinity for β 3 -adrenoceptors Naunyn-Schmiedeberg’ s Arch Pharmacol (2006) 374:99–105 DOI 10.1007/s00210-006-0104-z N. Niclauß : M. B. Michel-Reher : A. E. Alewijnse : M. C. Michel (*) Department Pharmacology and Pharmacotherapy, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands e-mail: m.c.michel@amc.uva.nl