Tiotropium

In experiments evaluating the effect of tiotropium (a gift from Boehringer Ingelheim, Ingelheim Germany) or fluticasone (Sigma-Aldrich, Zwijndrecht, The Netherlands) on HRV-induced mucin production, cells were pre-treated by incubation in HC-free complete medium containing tiotropium (10–1,000 nM) or fluticasone (10–1,000 nM) prior to infection with HRV. After infection, the basal medium was replaced by fresh free-HC medium containing tiotropium or fluticasone.
In experiments aimed to investigate the role of extracellular ATP release in virus-induced mucin production, suramin (a non-selective P2R antagonist, TOCRIS) was added to basal media at 10 μM for 30 min prior to HRV treatment. To investigate the ability of tiotropium and fluticasone to inhibit ATP- induced mucin expression, cells pre-treated with tiotropium/fluticasone were stimulated with 100 μM ATP at the basal side for 24 h.
In experiments with DAPT (γ-secretase inhibitor, TOCRIS), ALI-PBEC were incubated in complete medium supplemented with either 5 μM DAPT or solvent control (0.1% DMSO) during differentiation for 14 days. On day 14, cells were infected with HRV-A16 at MOI 1, and following infection the basal medium was replaced by fresh HC-free medium containing 5 μM DAPT or solvent control and cells were incubated for another 24 h.

MCh, indomethacin, procaterol, salbutamol, isoproterenol, formoterol, atropine, and glycopyrronium were obtained from Wako pure Chemical Industries (Osaka, Japan). IbTX was obtained from Peptide Institute (Osaka, Japan). Tiotropium, verapamil, PTX, CTX, forskolin, db-cAMP and indomethacin were obtained from Sigma (St. Louis, MO, USA).