Nicotinic acetylcholine receptor expression in human airway correlates with lung function

Abstract

Nicotine and its derivatives, by binding to nicotinic acetylcholine receptors (nAChRs) on bronchial epithelial cells, can regulate cellular signaling and inflammatory processes. Delineation of nAChR subtypes and their responses to nicotine stimulation in bronchial epithelium may provide information for therapeutic targeting in smoking-related inflammation in the airway. Expression of nAChR subunit genes in 60 bronchial epithelial biopsies and immunohistochemical staining for the subcellular locations of nAChR subunit expression were evaluated. Seven human bronchial epithelial cell lines (HBECs) were exposed to nicotine in vitro for their response in nAChR subunit gene expression to nicotine exposure and removal. The relative normalized amount of expression of nAChR α4, α5, and α7 and immunohistochemical staining intensity of nAChR α4, α5, and β3 expression showed significant correlation with lung function parameters. Nicotine stimulation in HBECs resulted in transient increase in the levels of nAChR α5 and α6 but more sustained increase in nAChR α7 expression. nAChR expression in bronchial epithelium was found to correlate with lung function. Nicotine exposure in HBECs resulted in both short and longer term responses in nAChR subunit gene expression. These results gave insight into the potential of targeting nAChRs for therapy in smoking-related inflammation in the airway.

Keywords: bronchial epithelium; lung function; nicotine; nicotinic acetylcholine receptor; quantitative polymerase chain reaction.

Fig. 1.

Side-by-side comparison of endoscopic view, with A and B showing ordinary white-light bronchoscopy (WLB) and the corresponding view under autofluorescence imaging (AFI), with the green fluorescence in AFI representing normal bronchial epithelium while the magenta areas (arrow) indicate the potential sites of abnormalities not visualized on the corresponding WLB views. Bronchial biopsies for this study were taken adjacent to AFI magenta areas where the areas appeared green in fluorescence under AFI.

Fig. 2.

A: immunostaining pattern of bronchial epithelial cells. Arrowheads, apical cell membrane (bm); long arrows, cytosol (bc). There is accentuation of the staining at the apical cell membrane and terminal web (arrows) of the ciliated bronchial cells compared with the cytosol. Immunostain for nAChRs α4, Nikon Eclipse Ni-U with Plan Achromat ×40 objective, c-mount 0.7×, and 2/3″ charge-coupled device (CCD) camera with 5 megapixels. B: immunostaining pattern of bronchial gland acini. Arrowheads, serous cells (sc); long arrows mucous cells (mc). The staining reaction is seen mainly within the cytosol. Immunostain for nAChRs α3, Nikon Eclipse Ni-U with Plan Achromat ×20 objective, c-mount 0.7×, and 2/3″ CCD camera with 5 megapixels.

Fig. 3.

Scatterplots showing significant correlations of forced expiratory volume in 1 s (FEV₁) with relative normalized amount of CHRNA4 (A), CHRNA5 (B), and CHRNA7 (C) mRNA expression level.

Fig. 4.

Graphic representations of the results of nicotine exposure for 9 days in immortalized bronchial epithelial cells [with the solid lines summarizing mean ± SE of cell lines exposed to nicotine (treatment group) and the dashed lines representing the respective cell lines without exposure to nicotine (control group)] showing sharp rise in nAChR α5 subunit gene expression upon exposure to nicotine (A), with a similar sharp but a slightly delayed rise in nAChR α7 subunit gene expression (C). Both these responses dropped with withdrawal of nicotine on day 9. B: there was also a sharp but not sustained rise in nAChR α6 subunit upon nicotine exposure but it dropped after day 6 before nicotine was removed on day 9.