Acid secretion and proton conductive pathways across primary human airway surface epithelial cultures were investigated with the pH stat method in Ussing chambers and by single cell patch clamping. Cultures showed a basal proton secretion of 0.17 Ϯ 0.04 mol ⅐ h Ϫ1 ⅐ cm Ϫ2 , and mucosal pH equilibrated at 6.85 Ϯ 0.26. Addition of histamine or ATP to the mucosal medium increased proton secretion by 0.27 Ϯ 0.09 and 0.24 Ϯ 0.09 mol ⅐ h Ϫ1 ⅐ cm Ϫ2 , respectively. Addition of mast cells to the

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... medium of airway cultures similarly activated proton secretion. Stimulated proton secretion was similar in cultures bathed mucosally with either NaCl Ringer or ion-free mannitol solutions. Proton secretion was potently blocked by mucosal ZnCl2 and was unaffected by mucosal bafilomycin A1, Sch-28080, or ouabain. Mucosal amiloride blocked proton secretion in tissues that showed large amiloride-sensitive potentials. Proton secretion was sensitive to the application of transepithelial current and showed outward rectification. In whole cell patch-clamp recordings a strongly outward-rectifying, zincsensitive, depolarization-activated proton conductance was identified with an average chord conductance of 9.2 Ϯ 3.8 pS/pF (at 0 mV and a pH 5.3-to-pH 7.3 gradient). We suggest that inflammatory processes activate proton secretion by the airway epithelium and acidify the airway surface liquid. primary airway cultures; JME airway cells; pH stat; patch clamp; Ussing chamber THE MUCOSA OF THE AIRWAY SURFACE epithelium is lined with a thin layer of fluid called the airway surface liquid (ASL). The composition of the ASL affects its physiological functions, the most important of which are removal of inhaled particles and antimicrobial activity (36). Active transport of Na ϩ and Cl Ϫ by the airway epithelium have been well characterized and recognized as critical determinants of ASL composition and depth. In contrast, the regulation of the H ϩ concentration in the ASL has received little attention. Acidic luminal pH has been shown to inhibit ciliary beating (4) and to cause bronchoconstriction (1), cough (39), loosening of the epithelial cells from one another (13), and detachment from the basement membrane (17). Acidic ASL has been implicated in airway diseases such as asthma (19, 29) and cystic fibrosis (CF) (33). Although the airway epithelium has been shown to secrete HCO 3 Ϫ , normal ASL in ferret trachea was previously reported as slightly acidic (pH 6.85) compared with plasma (26). In human primary airway cultures a pH of 6.9 was recently found with the use of either pH-selective microelectrodes (5) or a pH-sensitive fluorophore (22) . The concentration of HCO 3 Ϫ is one determinant of the pH of ASL, and active secretion of HCO 3 Ϫ across primary airway cultures or cell lines has been demonstrated (28, 33), presumably reflecting the HCO 3 Ϫ permeability of the apical CF transmembrane conductance regulator (CFTR) Cl Ϫ channel (31). Jayaraman et al. (22) estimated a HCO 3 Ϫ concentration of 8 mM in the ASL from their measurements of pH and a partial pressure of CO 2 of 5 kPa. With that HCO 3 Ϫ concentration and with an average CO 2 level in the airways in vivo of 2.2 kPa, the ASL is predicted to be slightly alkaline (pH ϳ7.3), which is in contrast to the reported relative acidity of ASL. Thus we hypothesized that the airway epithelium expresses a regulated mechanism to secrete H ϩ into the ASL. Because the volume of the ASL is small (ϳ1 l/cm 2 ) it can be expected to be rapidly acidified by cellular H ϩ transport. Recently, Hunt et al. (19) found that the exhaled breath of asthmatic patients during an attack is markedly acidic (pH 5.2). We hypothesized that during an inflammatory challenge the airway epithelium secretes increased amounts of acid into the ASL. In this report we measured acid secretion by human airway epithelial cultures in vitro and investigated its mechanism and regulation. We conclude that mucosal histamine, ATP, or mast cells activate epithelial acid secretion that is mediated mainly by an electrogenic apical proton conductance. METHODS Cell cultures. Human tracheal primary cultures were isolated and cultured as previously described (40). In brief, strips of epithelium were removed from the underlying tissues and treated with protease overnight. The resulting isolated, dispersed cells were plated on permeable filter sup-