Evidence for Functional Atypical Nicotinic Receptors That Activate K+-Dependent Cl- Secretion in Mouse Tracheal Epithelium

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The present study focused on the influence of nicotinic acetylcholine receptors (nAChR) on ion transport processes in mouse tracheal epithelium. RT-PCR experiments revealed expression of the α3, α4, α5, α7, α9, α10, β2, and β4 nAChR subunits in mouse tracheal epithelium. In Ussing chamber recordings of mouse tracheae, apically applied nicotine (100 μM) induced a dose-dependent increase of the transepithelial short-circuit current (EC50: 14.6 μM). The nicotine-induced effect (INIC) was attenuated by mecamylamine (25 μM, apical) and methyllycaconitine (1 μM, apical). The nAChR agonist 1.1-dimethyl-4-phenylpiperatinium iodide (DMPP) (100 μM) revealed apical and basolateral location of the receptors. INIC was not affected by the sodium channel inhibitor amiloride (10 μM, apical) or the cystic fibrosis transmembrane conductance regulator inhibitor CFTRinh-172 (20 μM, apical) but was reduced by the chloride channel inhibitor 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 μM, apical), the Na+/K+/2Cl- cotransporter inhibitor bumetanide (200 μM, basolateral), the potassium channel inhibitor Ba2+ (5 mM, basolateral), and 4.4′-diisothiocyanatostilbene-2.2′-disulfonate (100 μM, apical), indicating a contribution of Ca2+-activated chloride channels and potassium channels. Removal of extracellular Na+ (apical) or Ca2+ (apical) did not influence INIC but reduced the DMPP effect. Experiments with the Ca2+-ionophore A23187, a mix of 3-isobutyl-1-methylxanthine and forskolin, or the inositol-1,4,5-triphospate (IP3) receptor inhibitor 2-aminoethyl-diphenyl-borinate (75 μM, apical) decreased INIC, indicating a nicotine-mediated increase of intracellular Ca2+ and cAMP levels involving the IP3 signaling pathway. These findings indicate the activity of Ca2+-permeable nAChRs and alternative metabotropic pathways by nAChR activation that mediate Cl- and K+ transport in tracheal epithelium.

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