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. 2013 Mar 5;110(10):4099-104.
doi: 10.1073/pnas.1216939110. Epub 2013 Feb 19.

Contribution of α7 nicotinic receptor to airway epithelium dysfunction under nicotine exposure

Kamel Maouche  1 , Kahina Medjber, Jean-Marie Zahm, Franck Delavoie, Christine Terryn, Christelle Coraux, Stéphanie Pons, Isabelle Cloëz-Tayarani, Uwe Maskos, Philippe Birembaut, Jean-Marie Tournier
Affiliations

Contribution of α7 nicotinic receptor to airway epithelium dysfunction under nicotine exposure

Kamel Maouche et al. Proc Natl Acad Sci U S A. .

Abstract

Loss or dysfunction of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) leads to impairment of airway mucus transport and to chronic lung diseases resulting in progressive respiratory failure. Nicotinic acetylcholine receptors (nAChRs) bind nicotine and nicotine-derived nitrosamines and thus mediate many of the tobacco-related deleterious effects in the lung. Here we identify α7 nAChR as a key regulator of CFTR in the airways. The airway epithelium in α7 knockout mice is characterized by a higher transepithelial potential difference, an increase of amiloride-sensitive apical Na(+) absorption, a defective cAMP-dependent Cl(-) conductance, higher concentrations of Na(+), Cl(-), K(+), and Ca(2+) in secretions, and a decreased mucus transport, all relevant to a deficient CFTR activity. Moreover, prolonged nicotine exposure mimics the absence of α7 nAChR in mice or its inactivation in vitro in human airway epithelial cell cultures. The functional coupling of α7 nAChR to CFTR occurs through Ca(2+) entry and activation of adenylyl cyclases, protein kinase A, and PKC. α7 nAChR, CFTR, and adenylyl cyclase-1 are physically and functionally associated in a macromolecular complex within lipid rafts at the apical membrane of surface and glandular airway epithelium. This study establishes the potential role of α7 nAChR in the regulation of CFTR function and in the pathogenesis of smoking-related chronic lung diseases.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Bioelectric properties and mucociliary transport of murine airway epithelium are altered in α7−/− mice and in nicotine-exposed α7+/+ mice. α7+/+ or α7−/− mice were exposed to saline (control) or nicotine (three 1-mg/kg i.p. injections of nicotine 24 h, 16 h, and 1 h before the measurements) and the following parameters were recorded: nasal transepithelial PD (A), changes in nasal transepithelial PD upon amiloride and amiloride + forskolin exposure (B), mucociliary transport (C), and ciliary beat frequency (D). (E) Ionic composition of tracheal airway mucus in α7+/+ and α7−/− mice. Results are presented as median, with maximal and minimal values, and compared with the Mann–Whitney test (*P < 0.05, **P < 0.01).
Fig. 2.
Fig. 2.
α7 nAChR inhibition with αBTX or prolonged nicotine exposure alters CFTR function in air–liquid interface HAEC cultures. (A) Representative Isc tracing from air–liquid interface HAEC cultures in baseline condition, in the presence of 0.1 mM amiloride and 0.1 mM amiloride and 25 µM forskolin. (B and C) Changes in Isc upon amiloride and amiloride + forskolin exposure, after a 3 h-incubation with αBTX (0–10 µM) (B) or an overnight incubation with nicotine (0–10 µM) (C), added either apically or basally. (D) SPQ fluorescence variations in air–liquid interface HAEC cultures, induced by 25 µM forskolin in the presence of 0.1 mM amiloride: effect of a preincubation with 10 µM CFTRinh-172 for 1 h and with αBTX (0–10 µM) for 3 h. CFTRinh-172 was added during the last 30 min of the 3-h incubation with αBTX. Results are presented as median, with maximal and minimal values, for six (B and C) or five (D) HAEC cultures derived from different patients and compared with the Mann–Whitney test to the corresponding control in the absence of drug (*P < 0.05, **P < 0.01).
Fig. 3.
Fig. 3.
α7 nAChR activation induces increases of [Ca2+]i, [cAMP]i, and chloride secretion in airway epithelial cells. MM39 cells were exposed to 10 µM PHA 568487 (white diamonds) and [Ca2+]i (A) and SPQ fluorescence in the presence of 0.1 mM amiloride (C) were monitored for 10 min. [cAMP]i was also discontinuously measured (B). Controls consisted of 0.1% DMSO (black triangles). Results are expressed as mean ± SD for 15 different cells (calcium and SPQ) or five independent experiments (cAMP). (D–F) Effect of different inhibitors on PHA 568487-induced α7 nAChR activation-dependent increases of [Ca2+]i, [cAMP]i, and chloride secretion. MM39 cells were exposed for 60 min to 10 µM αBTX or for 15 min to one of the following drugs: 1 mM EGTA, 50 µM SQ22536, 1 µM thapsigargin, 10 µM CGS9343B, 2 µM GF109203X, 1 µM KT5720, or 50 µM PD98059. A total of 10 µM PHA 568487 was then added and [Ca2+]i and SPQ fluorescence were monitored for 10 min. [Ca2+]i (D), [cAMP]i (E), and SPQ fluorescence variations (F) were measured 3 min after PHA 568487 addition. Results correspond to four different experiments and were compared with the control exposed only to 10 µM PHA 568487.
Fig. 4.
Fig. 4.
Chronic nicotine exposure mimics αBTX in inhibiting PHA 568487-induced α7 nAChR activation-dependent increases of [Ca2+]i and [cAMP]i in human airway epithelial cells. Air–liquid interface HAEC cultures were apically incubated for 3 h with αBTX (0–10 µM) or overnight with nicotine (0–10 µM). Three minutes after the addition of PHA 568487 (10 µM), [Ca2+]i (A) and [cAMP]i (B) were measured (Fig. 3 A and B). Results correspond to five HAEC cultures derived from different patients and were compared with the corresponding control exposed to only 10 µM PHA 568487.
Fig. 5.
Fig. 5.
Effect of cholesterol depletion on α7 nAChR, CFTR, and AC-1 distribution and on PHA 568487-induced α7 nAChR activation-dependent increases of [Ca2+]i, [cAMP]i, and chloride secretion in airway epithelial cells. (A) Distribution of α7 nAChR (green) and of CFTR and caveolin-1 (red) at the apical membrane of ciliated cells in human bronchial tissue samples. Arrowheads point to colocalization of α7 nAChR with CFTR or caveolin-1. (B) α7 nAChR, CFTR, and AC-1 were localized in control human bronchial tissue samples (Left column) or after 1-h incubation at 37 °C in the presence of 10 mM ΜβCD either alone (Center column) or with 20 µg/mL cholesterol (Right column). (C–E) MM39 cells were similarly incubated for 1 h in the presence of ΜβCD, cholesterol (Chol), or ΜβCD with cholesterol. Then, 10 µM PHA 568487 (PHA) was added and [cAMP]i was measured 3 min after PHA 568487 addition (C). [Ca2+]i (D) and chloride secretion (SPQ fluorescence variation in the presence of 0.1 mM amiloride) (E) were monitored for 6 min after PHA 568487 addition, and the magnitude of calcium and SPQ fluorescence increases at 3 min (Fig. 3 A and C) was determined. Results correspond to four (C), five (D), and six (E) different experiments. [Scale bars, 8 µm (A) and 20 µm (B).]
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