Abstract
Background and Purpose Mucociliary clearance is an innate immune process of the airways, essential for removal of respiratory pathogens. It depends on ciliary beat and ion and fluid homeostasis of the epithelium. We have shown that nicotinic ACh receptors (nAChRs) activate ion transport in mouse tracheal epithelium. Yet the receptor subtypes and signalling pathways involved remained unknown.
Experimental Approach Transepithelial short circuit currents (I-SC) of freshly isolated mouse tracheae were recorded using the Ussing chamber technique. Changes in [Ca2+](i) were studied on freshly dissociated mouse tracheal epithelial cells.
Key Results Apical application of the nAChR agonist nicotine transiently increased I-SC. The nicotine effect was abolished by the nAChR antagonist mecamylamine. alpha-Bungarotoxin (alpha 7 antagonist) had no effect. The agonists epibatidine (alpha 3 beta 2, alpha 4 beta 2, alpha 4 beta 4 and alpha 3 beta 4) and A-85380 (alpha 4 beta 2 and alpha 3 beta 4) increased I-SC. The antagonists dihydro-beta-erythroidine (alpha 4 beta 2, alpha 3 beta 2, alpha 4 beta 4 and alpha 3 beta 4), alpha-conotoxin MII (alpha 3 beta 2) and alpha-conotoxin PnIA (alpha 3 beta 2) reduced the nicotine effect. Nicotine- and epibatidine-induced currents were unaltered in beta 2(-/-)mice, but in beta 4(-/-) mice no increase was observed. In the presence of thapsigargin (endoplasmatic reticulum Ca2+-ATPase inhibitor) or the ryanodine receptor antagonists JTV-519 and dantrolene there was a reduction in the nicotine-effect, indicating involvement of Ca2+ release from intracellular stores. Additionally, the PKA inhibitor H-89 and the TMEM16A (Ca2+-activated chloride channel) inhibitor T16Ainh-A01 significantly reduced the nicotine-effect.
Conclusion and Implications alpha 3 beta 4 nAChRs are responsible for the nicotine-induced current changes via Ca2+ release from intracellular stores, PKA and ryanodine receptor activation. These nAChRs might be possible targets to stimulate chloride transport via TMEM16A.