Comparison of three radioligands for the labelling of human β-adrenoceptor subtypes

Abstract

We have compared the ability of three radioligands, [(125)I]-cyanopindolol, [(3)H]-CGP 12,177 and [(3)H]-dihydroalprenolol, to label the three human beta-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 beta(1)- and beta(2)-adrenoceptors (about 40 pM), [(125)I]-cyanopindolol and [(3)H]-dihydroalprenolol had 4- to 6-fold higher affinity for beta(2)- as compared to beta(1)-adrenoceptors (10 vs 45 and 187 vs 1,021 pM, respectively). The affinity of [(125)I]-cyanopindolol at beta(3)-adrenoceptors was considerably lower (440 pM) than at the other two subtypes. The beta(3)-adrenoceptor affinity of [(3)H]-CGP 12,177 and [(3)H]-dihydroalprenolol 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 beta(3)-adrenoceptors, particularly in preparations co-expressing multiple subtypes. In the absence of alternatives, [(125)I]-cyanopindolol appears the least unsuitable to label beta(3)-adrenoceptors. There is a need for high-affinity radioligands which are either selective for beta(3)-adrenoceptors or reasonably non-selective among all three beta-adrenoceptor subtypes.

Tools to study β3-adrenoceptors

“…(Baker 2005;Hoffmann et al 2004;Niclauß et al 2006;Tate et al 1991). While [ 3 H]-dihydroalprenolol has been proposed to label β 3 -adrenoceptors in porcine lower urinary tract tissues (Yamanishi et al 2002a,b), a systematic comparison of its binding to all three cloned human subtypes found only very poor labeling of β 3 -adrenoceptors (Niclauß et al 2006). This is entirely consistent with the low β 3 -adrenoceptor affinity of unlabeled alprenolol (Hoffmann et al 2004 (Roberts et al 1993).…”

“…Subsequently, it has been used in numerous studies in which inhibition of a functional response by this compound has been taken as evidence for mediation of this response by a β 3 -adrenoceptor (Sarma et al 2003;Yamanishi et al 2002bYamanishi et al , 2003. However, more recent radioligand binding studies with cloned human β-adrenoceptor subtypes have reported that SR 59,230 is not selective for β 3 -adrenoceptors and, if anything, has slightly lower affinity for this subtype than for β 1 -and β 2 -adrenoceptors (Table 2) (Candelore et al 1999;Hoffmann et al 2004;Niclauß et al 2006). Moreover, SR 59,230 can exhibit agonist rather than antagonist properties and, in some systems, even behave as a full agonist (Horinouchi and Koike 2001;Hutchinson et al 2005).…”

“…However, it can exhibit agonistic effects in some cases. While some of these may relate to a second ligand recognition site on β 1 -adrenoceptors (Joseph et al 2004a,b), CGP 12,177 can also act on β 3 -adrenoceptors (Table 2), albeit at least 20-fold higher concentrations than those required to inhibit the classical catecholamine recognition site on β 1 -and β 2 -adrenoceptors (Baker 2005;Hoffmann et al 2004;Niclauß et al 2006). Indeed, a tritiated form of CGP 12,177 has been used as a radioligand to label β 3 -adrenoceptors (Feve et al 1991), but its affinity is much lower than for the other subtypes (Baker 2005).…”

“…When used in autoradiographic techniques, they can identify the location of receptors, and when used in binding assays with intact cells or homogenates of cells or tissues, they can be used to determine the expression density of a given receptor and its regulation by physiological and pathophysiological conditions as well as by drug treatment. (Baker 2005;Hoffmann et al 2004;Niclauß et al 2006;Tate et al 1991). While [ 3 H]-dihydroalprenolol has been proposed to label β 3 -adrenoceptors in porcine lower urinary tract tissues (Yamanishi et al 2002a,b), a systematic comparison of its binding to all three cloned human subtypes found only very poor labeling of β 3 -adrenoceptors (Niclauß et al 2006).…”

Pharmacological profile of β3-adrenoceptor agonists in clinical development for the treatment of overactive bladder syndrome

“…In isolated human bladder, KUC-7322 was slightly less potent than in the monkey bladder and much less potent than in the rat bladder in two independent studies performed against either passive tension of KCl-induced tone but remained a full agonist relative to isoprenaline . The response against KClinduced tone was not affected by the β 1 -adrenoceptor antagonist CGP 20,712A or the β 2 -adrenoceptor antagonist ICI 118,551,but surprisingly also not by the general β-adrenoceptor antagonist SR 59,230A (Niclauß et al 2006). In contrast to the other agonists, bladder relaxation by solabegron has not been reported in rats as the primary animal species but has been in dogs (Hicks et al 2007).…”