Table 2

Potencies of selected β2-AR ligands at β2-ARGs α as assessed by AC activity in presence of GTP, ITP, or XTP and by GTPase and ITPase activity and agonist affinities

β2-AR Ligandβ2-AR Ligand Potency/Affinity
(−)-ISO(+)-ISOSALDOBEPHDCI
EC50 (nM)
 AC + GTP18  ± 8115  ± 763  ± 20300  ± 1108400  ± 110038  ± 12
 GTPase13  ± 3150  ± 1593  ± 1090  ± 19560  ± 18029  ± 4
 AC + ITP233  ± 341600  ± 7501700  ± 200850  ± 20010000  ± 200048  ± 10
 ITPase57  ± 101100  ± 4601200  ± 420310  ± 805400  ± 210043  ± 10
 AC + XTP416  ± 445700  ± 2100900  ± 3003500  ± 800N.A.N.A.
K h(nM)2.0  ± 1.330  ± 1144  ± 2061  ± 12
K l (nM)130  ± 622200  ± 6101700  ± 4203200  ± 9405100  ± 1300160  ± 49
Ratio EC50
 AC + GTP/EC50GTPase  1.390.77 0.68 3.3315.01.31
 AC + ITP/EC50ITPase  4.091.45 1.41 2.74  1.851.12
 AC + ITP/EC50 AC + GTP12.913.9  27.0  2.83  1.191.26
 AC + XTP/EC50 AC + GTP23.149.6  14.3 11.7 N.A.N.A.
 ITPase/EC50GTPase  4.387.3312.9  3.44  9.641.48

AC activity was determined in membranes expressing β2-ARGs α (2.3–2.7 pmol/mg) in the presence of GTP (1 μM), ITP (10 μM), or XTP (100 μM). GTPase activity was determined in membranes expressing β2-ARGs α (7.0–7.5 pmol/mg) in the presence of 100 nM [γ-32P]GTP. ITPase activity was determined in membranes expressing β2-ARGs α (7.0–7.5 pmol/mg) in the presence of 3 μM [γ-32P]ITP. Reaction mixtures contained ligands at 0.1 nM to 1 mM as appropriate to obtain saturated concentration-response curves. The EC50 values of ligands were calculated by nonlinear regression analysis. To facilitate the analysis of the different potencies of ligands for various parameters, we also calculated potency ratios by dividing EC50 values for two parameters (dimensionless). For comparison of agonist potencies with agonist affinities, the K i values for high-affinity (K h) and low-affinity (K l) agonist binding are shown as well (expressed in nM). When distinct high- and low-affinity sites could not be discriminated,K i values are listed as K lvalues. Data for ligand affinities were taken from Seifert et al. (1998a).

    • N.A., not applicable because effects of EPH and DCI on AC in the presence of XTP were too small to reliably calculate EC50values.