Comparative pharmacology of human �-adrenergic receptor subtypes?characterization of stably transfected receptors in CHO cells

Abstract

Although many beta1-receptor antagonists and beta2-receptor agonists have been used in pharmacotherapy for many years their pharmacological properties at all three known subtypes of beta-adrenergic receptors are not always well characterized. The aim of this study was, therefore, to provide comparative binding characteristics of agonists (epinephrine, norepinephrine, isoproterenol, fenoterol, salbutamol, salmeterol, terbutalin, formoterol, broxaterol) and antagonists (propranolol, alprenolol, atenolol, metoprolol, bisoprolol, carvedilol, pindolol, BRL 37344, CGP 20712, SR 59230A, CGP 12177, ICI 118551) at all three subtypes of human beta-adrenergic receptors in an identical cellular background. We generated Chinese hamster ovary (CHO) cells stably expressing the three beta-adrenergic receptor subtypes at comparable levels. We characterized these receptor subtypes and analyzed the affinity of routinely used drugs as well as experimental compounds in competition binding studies, using the non-selective antagonist 125I-cyanopindolol as a radioligand. Furthermore, we analyzed the beta-receptor-mediated adenylyl cyclase activity in isolated membranes from these cell lines. The results from our experiments show that all compounds exhibit distinct patterns of selectivity and activity at the three beta-receptor subtypes. In particular, a number of beta2- or beta3-receptor agonists that are inverse agonists at the other subtypes were identified. In addition, beta1-receptor antagonists with agonistic activity at beta2- and beta3-receptors were found. These specific mixtures of agonism, antagonism, and inverse agonism at different subtypes may have important implications for the therapeutic use of the respective compounds.

Tools to study β3-adrenoceptors

“…For example, the prototypical antagonist propranolol was reported to have K i values at β 1 -, β 2 ,-and β 3 -adrenoceptors of 1.8, 0.8, and 186 nM, respectively (Table 2). Similarly, much lower β 3 -than β 1 -and β 2 -adrenoceptor affinity was also reported for the clinically used drugs sotalol, alprenolol, carvedilol, metoprolol, atenolol, and bisoprolol in most studies (Baker 2005;Hoffmann et al 2004), although one study reported high β 3 -adrenoceptor affinity of carvedilol (Candelore et al 1999). Among frequently used experimental antagonists, the selectivity of ICI 118,551 for β 2 -adrenoceptors relative to β 3 -adrenoceptors is comparable to that relative to β 1 -adrenoceptors (Table 2) (Baker 2005;Hoffmann et al 2004).…”

“…Similarly, much lower β 3 -than β 1 -and β 2 -adrenoceptor affinity was also reported for the clinically used drugs sotalol, alprenolol, carvedilol, metoprolol, atenolol, and bisoprolol in most studies (Baker 2005;Hoffmann et al 2004), although one study reported high β 3 -adrenoceptor affinity of carvedilol (Candelore et al 1999). Among frequently used experimental antagonists, the selectivity of ICI 118,551 for β 2 -adrenoceptors relative to β 3 -adrenoceptors is comparable to that relative to β 1 -adrenoceptors (Table 2) (Baker 2005;Hoffmann et al 2004). Similarly, the selectivity of CGP 20,712A for β 1 -adrenoceptors relative to β 3 -adrenoceptors is comparable to that relative to β 2 -adrenoceptors (Table 2) (Baker 2005;Hoffmann et al 2004).…”

“…"Classical" antagonists A key reason that β 3 -adrenoceptors were identified so much later than β 1 -and β 2 -adrenoceptors is that many classical β-adrenoceptor antagonists have much lower affinity for them (Baker 2005;Hoffmann et al 2004). For example, the prototypical antagonist propranolol was reported to have K i values at β 1 -, β 2 ,-and β 3 -adrenoceptors of 1.8, 0.8, and 186 nM, respectively (Table 2).…”

“…Among frequently used experimental antagonists, the selectivity of ICI 118,551 for β 2 -adrenoceptors relative to β 3 -adrenoceptors is comparable to that relative to β 1 -adrenoceptors (Table 2) (Baker 2005;Hoffmann et al 2004). Similarly, the selectivity of CGP 20,712A for β 1 -adrenoceptors relative to β 3 -adrenoceptors is comparable to that relative to β 2 -adrenoceptors (Table 2) (Baker 2005;Hoffmann et al 2004). Thus, most well-established β-adrenoceptor antagonists are unsuitable to also block β 3 -adrenoceptors.…”

“…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).…”