NADPH Oxidase-dependent Acid Production in Airway Epithelial Cells

Abstract

The purpose of this study was to determine the role of NADPH oxidase in H ؉ secretion by airway epithelia. In whole cell patch clamp recordings primary human tracheal epithelial cells (hTE) and the human serous gland cell line Calu-3 expressed a functionally similar zincblockable plasma membrane H ؉ conductance. However, the rate of H ؉ secretion of confluent epithelial monolayers measured in Ussing chambers was 9-fold larger in hTE compared with Calu-3. In hTE H ؉ secretion was blocked by mucosal ZnCl 2 and the NADPH oxidase blockers acetovanillone and 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), whereas these same blockers had no effect in Calu-3. We determined levels of transcripts for the NADPH oxidase transmembrane isoforms (Nox1 through -5, Duox1 and -2, and p22 phox ) and found Duox1, -2, and p22 phox to be highly expressed in hTE, as well as the intracellular subunits p40 phox , p47 phox , and p67 phox . In contrast, Calu-3 lacked transcripts for Duox1, p40 phox , and p47 phox . Anti-Duox antibody staining resulted in prominent apical staining in hTE but no significant staining in Calu-3. When treated with amiloride to block the Na ؉ /H ؉ exchanger, intracellular pH in hTE acidified at significantly higher rates than in Calu-3, and treatment with AEBSF blocked acidification. These data suggest a role for an apically located Duox-based NADPH oxidase during intracellular H ؉ production and H ؉ secretion, but not in H ؉ conduction.

NADPH oxidases in lung biology and pathology: Host defense enzymes, and more

“…2). As a result, activation of NOX leads to the activation of voltage-gated H + channels and/or other ion channels (18)(19)(20), as mechanisms of charge compensation. Original studies of NOX1 gene regulation suggested the presence of a short NOX1 splice variant with intrinsic H + channel activity (8), although this was later realized to be an artifact due to the formation of a stable loop within NOX1 mRNA (6).…”

“…The fact that ROS production by polarized epithelia occurred only apically and could be stimulated by activators of protein kinase C (82), known to activate the phagocyte NADPH oxidase (5), suggested regulated and compartmentalized epithelial ROS production by an NADPH oxidase. The source of epithelial ROS was identified only a few years ago, after initial discovery of prominent expression of the NOX homologs DUOX1 and DUOX2 within the lung (35), and studies on lung tissues using in situ hybridization and immunohistochemistry, which revealed the presence of DUOX1 at the apical surface of tracheobronchial epithelial cells, and DUOX2 within salivary and submucosal glands (19,79,83,84 (88,89), which is due to the presence of DUOX1 (90). Although DUOX expression in the lung is primarily localized to airway or alveolar epithelial cells, recent studies have also suggest the presence of DUOX proteins in lymphocytes (51,91).…”

“…Acid secretion and acidification of the airway surface fluid by epithelial DUOX activation (Fig. 2) may further promote oxidant-mediated bacterial killing (19,102).…”

Expression of Nox1 in 3T3 cells increases cellular acid production but not proton conductance

“…Cells were whole cell patch-clamped as previously described [20]. Briefly, cells were bathed in (in mM): 120 N- [2-hydroxyethyl] Note that the solutions were Cl -free in order to exclude the contribution of Cl -currents during the measurements of the relatively small H + currents.…”

“…Measurements of intracellular pH (pH i ) were done as described [20]. Briefly, BCECF-AM (2', 7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxy-methyl ester)-loaded cells were investigated on an inverted microscope at 400x magnification, excited at 440 nm and 495 nm and emission was collected between 525 nm and 550 nm with a cooled CCD camera (Photometrics CoolSnap HQ, Roper Scientific) controlled by a computerized imaging system (Metafluor, Universal Imaging Corp.).…”