Do parasympatholytic effects of long-acting beta 2-sympathomimetics contribute to their relaxant effects in airway smooth muscle cells?

To address the hypothesis of whether functional antagonistic effects of long-acting beta 2-sympathomimetics (formoterol and salmeterol) might be supported by direct antagonistic effects on muscarinic acetylcholine receptors (mAChR), we performed radioligand binding experiments with 3H-quinuclidinyl-benzilate in membranes of airway smooth muscle cells (calf tracheal myocytes). beta 2-Sympathomimetics (short- and long-acting) were compared to catechol-ethanolamines and catechol-ethylamines. Tracheal myocytes were characterized by a high density of mAChR 1017 +/- 17 fmol/mg, which exceeds that of beta 2-adrenoceptors 20-fold. The affinities of drugs were determined by competition binding. Dissociation constants ¿pKD-values) of formoterol (5.04 +/- 0.05) and salmeterol (5.24 +/- 0.04) matched that of ACh (5.37 +/- 0.03) and were significantly higher than that of the mAChR-agonist carbachol (4.65 +/- 0.03). pKD-values of mAChR-agonists were strictly dependent on GTP-concentration (> 50-fold difference between high- and low-affinity states), in contrast to those of formoterol, thereby characterizing formoterol as an mAChR-antagonist. A 10-fold lower affinity of the related compound fenoterol (3.94 +/- 0.02) hinted at the formyl-amino moiety of formoterol as a structural determinant of high-affinity whereas a 100-fold lower affinity of salbutamol as compared to salmeterol suggested the aliphatic side chain was a structural determinant of high affinity. The high affinity of dobutamine (4.96 +/- 0.02) and dopexamine (6.20 +/- 0.02) provided evidence that high affinity can be found not only for catechol-ethanolamines with long side chains but also for catechol-ethylamines. The question of whether high local concentrations after inhalation of long-acting beta 2-sympathomimetics could contribute to their therapeutical effects by antagonism with ACh on mAChR remains to be answered.