The selectivity of beta-adrenoceptor agonists at human beta1-, beta2- and beta3-adrenoceptors

Abstract

Background and purpose: There are two important properties of receptor-ligand interactions: affinity (the ability of the ligand to bind to the receptor) and efficacy (the ability of the receptor-ligand complex to induce a response). Ligands are classified as agonists or antagonists depending on whether or not they have efficacy. In theory, it is possible to develop selective agonists based on selective affinity, selective intrinsic efficacy or both. This study examined the affinity and intrinsic efficacy of 31 beta-adrenoceptor agonists at the three human beta-adrenoceptors to determine whether the current agonists are subtype selective because of affinity or intrinsic efficacy.

Experimental approach: Stable clonal CHO-K1 cell lines, transfected with either the human beta(1), beta(2) or beta(3)-adrenoceptor, were used, and whole-cell [(3)H]-CGP 12177 radioligand binding and [(3)H]-cAMP accumulation were measured.

Key results: Several agonists were found to be highly subtype selective because of selective affinity (e.g. salmeterol and formoterol, for the beta(2)-adrenoceptor over the beta(1) or beta(3)), while others (e.g. isoprenaline) had little affinity-selectivity. However, the intrinsic efficacy of salmeterol, formoterol and isoprenaline was similar across all three receptor subtypes. Other ligands (e.g. denopamine for beta(1); clenbuterol, AZ 40140d, salbutamol for beta(2)) were found to have subtype-selective intrinsic efficacy. Several ligands appeared to activate two agonist conformations of the beta(1)- and beta(3)-adrenoceptors.

Conclusions and implications: There are agonists with subtype selectivity based upon both selective affinity and selective intrinsic efficacy. Therefore, there is scope to develop better selective agonists based upon both selective affinity and selective intrinsic efficacy.

Figure 2

Inhibition of [³H]-CGP 12177 binding to whole cells by AZ 40140d in (A) CHO β₁ cells, (B) CHO β₂ cells and (C) CHO β₃ cells. The columns represent total [³H]-CGP 12177 binding and non-specific binding determined in the presence of 10 µM propranolol. The concentrations of [³H]-CGP 12177 present in each case are (A) 1.41 nM, (B) 1.29 nM and (C) 19.31 nM. Data points are mean ± SEM of triplicate determinations. These single experiments are representative of (A) 8, (B) 10 and (C) 7 separate experiments.

Figure 5

[³H]-cAMP accumulation in response to pindolol after 5 h incubation in (A) CHO β₁ cells, (B) CHO β₂ cells and (C) CHO β₃ cells. The columns represent basal [³H]-cAMP accumulation and that in response to 10 µM isoprenaline. Data points are mean ± SEM of triplicate determinations. These single experiments are representative of (A) six, (B) nine and (C) six separate experiments.

Figure 1

Inhibition of [³H]-CGP 12177 binding to whole cells by salmeterol in (A) CHO β₁ cells, (B) CHO β₂ cells and (C) CHO β₃ cells. The columns represent total [³H]-CGP 12177 binding and non-specific binding determined in the presence of 10 µM propranolol. The concentrations of [³H]-CGP 12177 present in each case are (A) 0.90 nM, (B) 1.39 nM and (C) 17.9 nM. Data points are mean ± SEM of triplicate determinations. These single experiments are representative of (A) 11, (B) 10 and (C) 8 separate experiments.

Figure 3

[³H]-cAMP accumulation in response to fenoterol after 10 and 30 min, and 5 h incubation in (A) CHO β₁ cells, (B) CHO β₂ cells and (C) CHO β₃ cells. The columns represent basal [³H]-cAMP accumulation and that in response to 10 µM isoprenaline at each time-point. Data points are mean ± SEM of triplicate determinations. These single experiments are representative of (A) four, (B) four and (C) three separate experiments.

Figure 4

[³H]-cAMP accumulation in response to salmeterol after 10 and 30 min, and 5 h incubation in (A) CHO β₁ cells, (B) CHO β₂ cells and (C) CHO β₃ cells. The columns represent basal [³H]-cAMP accumulation and that in response to 10 µM isoprenaline at each time-point. Data points are mean ± SEM of triplicate determinations. These single experiments are representative of (A) four, (B) four and (C) three separate experiments.

Figure 6

[³H]-cAMP accumulation in response to carazolol after 5 h incubation in (A) CHO β₁ cells, (B) CHO β₂ cells and (C) CHO β₃ cells. The columns represent basal [³H]-cAMP accumulation and that in response to 10 µM isoprenaline. Data points are mean ± SEM of triplicate determinations. These single experiments are representative of (A) eight, (B) four and (C) eight separate experiments.

Figure 7

(A) Plot of log K_D for compounds from Table 1 for human β₁- versus human β₂-adrenoceptors. The line is through the origin and represents equal affinity, so compounds occurring to the right and below are β₂-selective. Thus, salmeterol can be seen to be highly β₂-selective based on affinity, whereas isoprenaline, denopamine and AZ 40140d have little selectivity based on affinity. (B) Plot of efficacy values (log K_D/log EC₅₀) for the same compounds (taken from Tables 3 and 4). The line is that of best fit – the slope is not 1 nor does it go though the origin as this represents a function of efficacy (i.e. differences in cell line which include receptor number, receptor–effector coupling, etc.). Denopamine can be seen to be β₁-selective based on efficacy, whereas AZ 40140d is β₂-selective, and isoprenaline and salmeterol were non-selective based on efficacy.