TABLE 1

Comparison of functional properties of the anticholinergics at the hM3-R in different assays

pA2 values of the different antagonists were determined in CHO-hM3 cells with the InsP accumulation and the AP-1-driven luciferase reporter gene assays. pA2 values were experimentally obtained by extrapolation of agonistic (carbachol) concentration response shifts, according to Schild analysis, as shown in Fig. 3. Values shown are the average of at least three independent experiments ± S.E.M., with each point determined in triplicate. Schild slopes were not significantly different from unity. The inverse agonistic potencies (pIC50 ± S.E.M.) of the different anticholinergics were determined as the concentration that blocks 50% of the hM3-R constitutive activity, as measured in the AP-1-driven luciferase assay in the absence of any added agonist.

InsP3 pA2Reporter-gene pA2Reporter-gene pIC50
Atropine9.21 ± 0.059.01 ± 0.089.14 ± 0.10
NMS9.58 ± 0.099.83 ± 0.049.35 ± 0.05†
Pirenzepine6.67 ± 0.09*7.09 ± 0.066.80 ± 0.10
Ipratropium9.21 ± 0.079.25 ± 0.039.10 ± 0.07
Tiotropium10.68 ± 0.1010.52 ± 0.099.61 ± 0.06†
Aclidinium10.04 ± 0.07*9.23 ± 0.109.72 ± 0.24
Glycopyrrolate9.63 ± 0.079.83 ± 0.079.61 ± 0.10
4-DAMPN.D.9.23 ± 0.109.28 ± 0.06

  • N.D., not determined.

  • ↵* Significant differences between pA2 values obtained with the reporter gene assay and the InsP3 assay (P < 0.05; Student's t test).

  • ↵† Significant differences between pA2 and pIC50 values obtained with the reporter gene assay (P < 0.05; Student's t test).