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J. Biol. Chem., Vol. 279, Issue 35, 36454-36461, August 27, 2004

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NADPH Oxidase-dependent Acid Production in Airway Epithelial Cells^*

Christian Schwarzer¶, Terry E. Machen, Beate Illek, and Horst Fischer||

From the Children's Hospital Oakland Research Institute, Oakland, California 94609 and the Department of Molecular and Cell Biology, University of California, Berkeley, California 94720

Received for publication, May 5, 2004 , and in revised form, June 14, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 ZnCl₂ 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The composition of the airway surface liquid (ASL)¹ is critically important for the normal function of the airway epithelium, including bacterial killing and mucociliary clearance (1). Recently, considerable attention has focused on the pH of the ASL (pH_ASL), which has been shown to affect various epithelial functions such as ion transport (2, 3), ciliary beating (4, 5), and epithelial integrity (6, 7). pH_ASL has been found to be slightly acidic compared with plasma. Initially, pH_ASL in the ferret trachea was found to be 6.85 using microelectrode measurements (8). Using a pH-sensitive fluorophore, pH_ASL was found to be 6.8 in bovine cultures and 7.1 in vivo in mice (9). In the inflammatory airway diseases asthma, cystic fibrosis, and chronic obstructive pulmonary disease an acidic pH_ASL or acidic exhaled breath has been demonstrated (10–12). These observations suggested a regulated mechanism of acid secretion by the airways that is up-regulated during inflammation. Recently we found that human airway cells express a voltage-activated, zinc-sensitive H⁺ conductance and secrete acidequivalents into the mucosal bath (13).

H⁺-selective, voltage-activated currents were first recorded from snail neurons (14) and since then have been found in many other cell types (15). The transmembrane protein complex NADPH oxidase has been suggested to mediate the H⁺ conductance in phagocytic cells (16), although this notion has been challenged recently (17). In phagocytic cells NADPH oxidase plays a crucial role in killing invading organisms by producing superoxide radicals () by transferring an electron from intracellular NADPH across the membrane to extracellular O₂. During the process of NADPH oxidation in neutrophils, intracellular H⁺ is released from NADPH and thereby contributes to intracellular acid production (18). The phagocytic NADPH oxidase is a multisubunit enzyme consisting of two transmembrane subunits (the cytochrome gp91^phox and p22^phox), three cytosolic subunits (p40^phox, p47^phox, and p67^phox), and a small ubiquitously expressed GTP-binding protein p21^rac (19). In phagocytes, all subunits were shown to be necessary for a regulated release of during the respiratory burst (20).

gp91^phox is a member of the Nox gene family, where it is referred to as Nox2. In addition to the small Nox homologs Nox1 through -5, two large homologs have been described recently, called dual oxidases Duox1 and -2 (21–23). Expression of Duox1 and -2 has been found in the epithelium of the thyroid gland by immunofluorescence (22), and expression of Duox1 transcript by in situ hybridization and Duox1 antisense-sensitive H₂O₂ production by airway epithelial cultures has been demonstrated (24).

Recent studies in our laboratory identified a zinc-sensitive H⁺ conductance and H⁺ secretion across the apical membrane of well differentiated human airway epithelia (13). We were intrigued by the notion that this physiological process was provided by the NADPH oxidase complex. This was addressed by studying H⁺ conductance, H⁺ secretion, NADPH oxidase expression, and intracellular H⁺ production in two functionally different types of human airway epithelia. Ciliated cells from the airway surface epithelium function to absorb fluid and acidify the airway lumen slightly (8, 9, 13), whereas cells from the submucosal serous glands secrete a bicarbonate-rich, alkaline fluid (25, 26). In this report we compare the properties of airway surface versus submucosal gland cells by using primary cultures of human tracheal surface epithelia (hTE) and the Calu-3 submucosal gland cell line. These cells were chosen to relate high (hTE) and low rates (Calu-3) of H⁺ secretion to the expression of NADPH oxidase and its function. The experiments in this report demonstrate the localization of the dual oxidase Duox in the apical membrane of hTE surface cells but not in Calu-3 gland cells. Our data suggest that a Duox-based NADPH oxidase complex supports epithelial H⁺ secretion by generating intracellular H⁺, which provides a H⁺ gradient across the apical cell membrane. The plasma membrane H⁺ conductance did not correlate with Duox expression suggesting that intracellular H⁺ exited through a parallel pathway.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Cultures—Human primary tracheal epithelial cultures (hTE) were isolated as previously described (27). Cells were cultured in Dulbecco's modified Eagle's medium:bronchial epithelial cell basal medium (1:1) as described by Gray et al. (28). Compared with our previous report (13), this is a change in culture conditions, which resulted in epithelial cultures with significantly lower transepithelial resistances (R_t = 569 ± 36 ·cm², n = 12) and higher potentials (V_t = –26.0 ± 2.1 mV). Sachs et al. described the effects of both culture conditions (29). Calu-3 cells, a human airway cell line with characteristics of airway serous cells (30), were grown in a Dulbecco's modified Eagle's medium/F-12 mix supplemented with 10% fetal bovine serum. For patch clamping or measurement of intracellular pH (pH_i), cells were seeded on collagen-coated glass coverslips and used after 1–2 days. For transepithelial measurements and for biochemical assays, cells were grown on permeable filters (Snapwell, 0.4-µm pore size, 1-cm² area; Corning Costar, Cambridge, MA) to confluency at an air-liquid interface. Confluent Calu-3 monolayers had R_t = 267 ± 34 ·cm² and V_t = –5.6 ± 0.7 mV (n = 18). Some cultures were treated for 1 h with 100 µM ZnCl₂ (mucosally) or for 14–16 h with the NADPH oxidase blockers 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF, 500 µM (31)) or acetovanillone (500 µM (32)) in the cell culture medium.

Whole Cell Patch Clamp Recordings—Cells were whole cell patchclamped as previously described (13, 33). On an inverted microscope cells were bathed in (in millimolar): 120 Hepes, 70 gluconic acid, 100 trimethylammonium hydroxide, 2 calcium gluconate, 1 magnesium gluconate (pH 7.3). Pipettes were filled with (in millimolar): 100 MES, 60 gluconic acid, 90 trimethylammonium hydroxide, 10 EGTA, 1 glucose, 1 magnesium gluconate, 3.3 magnesium ATP, 0.07 lithium GTP (pH 5.3). Junction potentials at the electrodes were measured, zeroed, and carefully observed for stability. Noted instabilities were smaller than ±2 mV. With these solutions pipette resistance was 8 M. After establishing the whole cell configuration the membrane potential was continuously clamped to –50 mV and pulsed every 14 s for 6 s to 20 mV. The access resistance (R_a) and the cell membrane capacitance (C_m) were measured by fitting the current transients caused by a 10-mV pulse with a single exponential. Current-voltage step protocols from –80 mV to +40 mV were applied, and resulting step-currents were recorded. Time constants of current activation () during voltage pulses were fitted with a single exponential of the form I(t) = A · e^(–t/) + K, where t is time, I(t) is the time-dependent current, A is the amplitude of the current transient, and K is a constant current offset. Whole cell conductance was calculated from steady-state currents as the chord conductance measured between –50 mV and +20 mV. For the calculation of the specific membrane conductance (G_m in picosiemens (pS)/picofarad (pF)) the whole cell conductance was corrected for R_a (on average, 22 ± 5 M (hTE) and 30 ± 3 M (Calu-3)) and normalized to C_m with G_m = (G_c ^–1 – R_a)^–1·C_m^–1. Patch clamp recordings and fits were done using pClamp 8 (Axon Instruments, Fremont, CA). Currents during current-voltage step protocols were sampled at 500 Hz and filtered at 200 Hz. For the plots in the figures, currents are shown sampled at 50 Hz by averaging adjacent samples. In the figures original currents are shown without leak subtraction or corrections. Measurements were done at 26 °C.

Quantification of Proton Secretion—Proton secretion was measured using the pH-stat titration technique in an Ussing chamber as described previously (13). Briefly, cultures were bathed serosally with Hepes-buffered solution and mucosally with buffer-free solution (3 ml each). Solutions were constantly gassed with oxygen and were nominally free of HCO^–₃/CO₂. Standard NaCl Ringer solutions contained (in millimolar): mucosal, 140 NaCl, 2 KCl, 15 glucose, 2 CaCl₂, 1 MgCl₂; serosal, 140 NaCl, 2 KCl, 5 glucose, 10 Hepes, 2 CaCl₂, 1 MgCl₂ (pH 7.3) with HCl/NaOH. During the experiment the pH of the mucosal solution was continuously measured and titrated to a target value of pH 7.3 (range, 7.25–7.35) with 10 mM NaOH to determine H⁺ secretion (J_H) by the cultures. Rates are expressed in nmol·h^–1·cm^–2, and positive rates refer to acidification of the mucosal medium. Measurements were done at 37 °C.

Molecular Expression of NADPH Oxidase Subunits—Total RNA was prepared from 2 x 10⁵ hTE and Calu-3 cells, respectively, grown on permeable filter supports, and poly(A) mRNA was isolated using the RNeasy and Oligotex mRNA kits (Qiagen, Valencia, CA). Reverse transcriptase-PCR was performed using 100 ng of mRNA, 40 units of avian myeloblastosis virus reverse transcriptase, 20 units of RNase inhibitor, 4 µl of 5x buffer (Roche Applied Science), 2 µl of 5 mM dNTP, and 2.5 µM random hexamer oligonucleotides (Applied Biosystems, Foster City, CA) for 60 min at 42 °C. One microliter of first strand cDNA was used as template in polymerase chain reactions with NADPH oxidase-specific oligonucleotides (see Table I). PCR was performed in a DNA thermal cycler using AmpliTaq Gold® DNA polymerase (Applied Biosystems). Following a 10-min 94 °C incubation, reactions proceeded for 30 s at 94 °C then 60 s at 60 °C for 43 cycles followed by 7 min at 72 °C. One-tenth of the amplification products were analyzed on a 1% agarose gel. For quantitative PCR total RNA was isolated from hTE and analyzed using an ABI Prizm 7700 sequence detection system (PE Applied Biosystems, Inc.). Samples were normalized to glyceraldehyde-3-phosphate dehydrogenase.

Measurement of Intracellular pH (pH_i)—Cells on glass coverslips were incubated for 30 min with 20 µM BCECF-AM (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, Molecular Probes Inc.), washed, and allowed to cleave the dye for 60 min. Cellular fluorescence was investigated in an open perfusion chamber on an inverted microscope. Excitation wavelengths were 440 and 495 nm, and emission was collected between 525 and 550 nm with a cooled charge-coupled device camera (Photometrics CoolSnap HQ, Roper Scientific) controlled by a computerized imaging system (Metafluor, Universal Imaging Corp.). Emission intensities of individual cells were background-subtracted, and ratios of 495 to 440 nm were calculated. pH_i was calculated from ratios using a four-point pH calibration in presence of 20 µM nigericin in KCl solution. Calibration points were pH 7.5, 7, 6.5, and 6.0. Resulting calibration ratios for individual cells were fitted to second order polynomials, and fitted parameters were used to calculate pH_i from measured ratios. Individual rates of acidification of cells during the first 15 min of treatment with amiloride were determined by linear regression. Dye loading and experiments were done at 26 °C. NaCl solution composition was (in millimolar) 145 NaCl, 5 KCl, 1 CaCl₂, 1 MgCl₂, 12.5 Hepes (pH 7.4). pH calibration was performed in (in millimolar) 5 NaCl, 145 KCl, 1 CaCl₂, 1 MgCl₂, and Hepes and/or MES (total buffer 12.5 mM).

Statistics—Data are presented as original values or as mean ± S.E.; n refers to the number of cells or tissues investigated. Effects were tested with t tests or with ANOVAs followed by Bonferroni-corrected t tests. Calculations were done with StatView (Abacus Concepts, Berkeley, CA). Calculated p values are given, and p < 0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Proton Conductances in Airway Cells—Single hTE and Calu-3 cells were whole cell patch-clamped to characterize and compare their H⁺ conductance under conditions selective for H⁺ currents with a pipette to bath pH gradient of 5.3 to 7.3. Calu-3 are large round cells with an average C_m of 29.9 ± 5.9 pF (n = 7). hTE were visibly smaller with a C_m of 10.4 ± 1.4 pF (n = 6, significantly lower than Calu-3, p = 0.012, t test). hTE had visible cilia on one pole of the elongated cells. Reported conductances were normalized to C_m to account for the difference in cell size.

Measured currents were identified as H⁺ currents by the established characteristics: (i) H⁺-selective reversal potentials, (ii) zinc sensitivity, and (iii) activation by depolarization with time constants in the order of seconds (34). Both airway cell types expressed whole cell H⁺ currents, H⁺-selective reversal potentials, and activation by depolarizing pulses. Continuous H⁺ current recordings and block by ZnSO₄ are shown in Fig. 1A for hTE and Fig. 1B for Calu-3. During the recordings the membrane potential was clamped to –50 mV and pulsed to 20 mV (as shown in the voltage traces, lower panels of Fig. 1, A and B) to continuously monitor the depolarization activation of H⁺ currents. Note the typically slow current activation during the pulses in both cell types. Addition of 100 µM ZnSO₄ to the bath fully blocked the depolarization-activated currents. The current responses to the voltage step protocols from Fig. 1 (A and B) are shown in detail in Fig. 1 (C and D) for both cell types before (top panels) and after (bottom panels) addition of 100 µM ZnSO₄. Outward H⁺ currents activated slowly during depolarizing potentials and were blocked after addition of ZnSO₄. Steady-state current-voltage relations are shown in Fig. 2 (E and F). Current-voltage relations recorded under control conditions (triangles) showed outward rectification and negative reversal potentials indicative for H⁺ currents. Control steady-state currents reversed at –21.6 ± 4.7 mV (hTE) and –25.9 ± 6.1 mV (Calu-3). Addition of Zn²⁺ shifted the reversal potential to positive values in both cell types (Fig. 1, E and F, circles, by on average 37.8 ± 3.5 mV, n = 11; not different between cell types). On average, hTE and Calu-3 expressed similar specific H⁺ conductances of 19.6 ± 3.1 pS/pF and 11.6 ± 2.8 pS/pF, respectively (p = 0.085, Fig. 1G).

View larger version (20K): [in this window] [in a new window]   FIG. 1. Whole cell recording of proton currents from airway cells. A and B, recordings from hTE (A) and Calu-3 (B) cells. Top panels show currents measured at holding potentials shown in the bottom panels with a pH 5.3 to 7.3 inside-to-outside gradient. 100 µM ZnSO was added where indicated; breaks in the current recording are due to ZnSO addition; the time axis is continuous. The slow upward drift of the 4baseline currents in the recordings was possibly caused by the slow dialysis of 4residual cellular ions that may have supported an inward current. C and D, current responses to voltage steps from recordings in A from –80 to 40 mV in 20-mV steps in hTE (C) and Calu-3 (D) cells; top panel, control; bottom panel, ZnSO4. E and F, steady-state current-voltage relations from corresponding step-currents in C and D; triangles, control; circles, ZnSO4. G, average specific membrane conductance was similar in hTE and Calu-3, p = 0.084.

View larger version (12K): [in this window] [in a new window]   FIG. 2. Time constants of voltage-activated H+ currents. A and B, membrane potential was stepped from –50 to 20 mV or 40 mV (as indicated) in hTE (A) or Calu-3 (B). Dots are sampled current data; the line shows the best fit to a single exponential function; the width of samples is 20 ms. Fitted time constants are: hTE, 40 = 2.23 s, 20 = 3.14 s; Calu-3, from 40 = 3.68 s, 20 = 6.9 s. C, average time constants during depolarizing pulses –50 mV to 20 mV or to 40 mV as indicated. Time constants were not different between cell types (p = 0.1) but 20 (3.9 ± 0.5 s) was significantly longer than 40 (2.8 ± 0.4 ms, p = 0.01, two-way ANOVA, n = 6 hTE and n = 7 Calu-3).

Acid Secretion by hTE and Calu-3 Cultures—Acid secretion of intact epithelia was measured using the pH-stat titration technique in Ussing chambers. Block of H⁺ channels (by ZnCl₂) and block of NADPH oxidase (by acetovanillone or AEBSF) was used to 1) determine the role of H⁺ channels in epithelial acid secretion, 2) verify an apical location of H⁺ channels in polarized epithelial monolayers, and 3) determine the role of NADPH oxidase during acid secretion. Both cell types showed basal acid secretion into the mucosal compartment. In hTE, J_H = 939 ± 49 nmol·h^–1·cm^–2, n = 12. Calu-3 showed a 9-fold smaller J_H of 106 ± 9 nmol·h^–1·cm^–2, n = 18 (p < 0.0001, Fig. 3). When cultures were pre-treated for 1 h with ZnCl₂ (100 µM mucosally), acid secretion by hTE was significantly reduced (to 480 ± 29 nmol·h^–1·cm^–2, n = 6, p < 0.0001, unpaired t test). In contrast, no significant effect of ZnCl₂ was found on acid secretion by Calu-3 (block to 84 ± 4 nmol·h^–1·cm^–2, n = 5, p = 0.2, Fig. 3).

View larger version (18K): [in this window] [in a new window]   FIG. 3. Acid secretion by airway epithelia. Acid secretion was measured using the pH-stat titration technique at a mucosal pH of 7.3 in NaCl solutions. Steady-state secretion after 45 min of recording is plotted. Some cultures were treated with 100 µM ZnCl2 (mucosal for 1 h), 500 µM AEBSF, or 500 µM acetovanillone (mucosal and serosal for 14–16 h). The numbers in parentheses give the number of tested cultures. Effects of all blockers were highly significant (p < 0.0001, ANOVA) and not different from one another in hTE, but had no effect in Calu-3 (p = 0.29, ANOVA).

Expression of NADPH Oxidase Genes in Airway Cells—We investigated the expression of described NADPH oxidase subunits, including the transmembrane subunits Nox1, Nox2, Nox3, Nox4, Nox5, Duox1, Duox2, and p22^phox, and the intracellular subunits p40^phox, p47^phox, and p67^phox. Actin and glyceraldehyde-3-phosphate dehydrogenase were included as control genes. The sequence-specific primers used for these genes are shown in Table I. Fig. 4A shows the expression pattern of mRNA of NADPH oxidase subunits in Calu-3 (top panel) and hTE (bottom panel) using high amplification (43 cycles). Transcripts for Nox1, Nox2, and Nox5 were detected in both cell types. Nox4 was expressed at low levels in hTE but not detected in Calu-3 cells. The large Nox genes, Duox1 and Duox2, were the major isoforms in hTE; in contrast, Duox1 was not detected and Duox2 was expressed at comparably low levels in Calu-3 cells. Nox3 was not detected in either cell type (not shown). The small transmembrane subunit of NADPH oxidase, p22^phox, was expressed highly in both cell types. Quantification of the relative expression of the transmembrane subunit in hTE using real-time PCR showed that in hTE transcripts for Duox1, Duox2 and p22^phox were expressed at >1000-fold higher levels than the small transmembrane Nox isoforms (Fig. 4B). The cytosolic subunits p40^phox, p47^phox, and p67^phox (Fig. 4A, right panels) were expressed in hTE, whereas Calu-3 expressed p67^phox but lacked transcripts for the two smaller subunits p40^phox and p47^phox. Thus the expression of NADPH oxidase subunits showed significant differences between hTE and Calu-3 cells. The lack of Duox1 and the comparably low expression of Duox2 in Calu-3, both of which were highly expressed in hTE, suggested a role for Duox during epithelial acid secretion.

View larger version (41K): [in this window] [in a new window]   FIG. 4. Expression of NADPH oxidase subunits in airway cells. A, PCR gels of transmembrane subunits (left panels) and intracellular subunits (right panels) of Calu-3 (top) and hTE (bottom). Arrows show the 100-bp band in the marker lanes. Glyceraldehyde-3-phosphate dehydrogenase and actin are shown as controls. Primers for the reactions are given in Table I. Reaction was run for 43 cycles to visualize also low expressing small Nox isoforms. B, quantification of expression of transmembrane subunits in hTE using real-time PCR. Note log scaling of the y-axis. Each bar represents determination in triplicate. Signal was normalized to glyceraldehyde-3-phosphate dehydrogenase.

View larger version (100K): [in this window] [in a new window]   FIG. 5. Immunostaining of airway cultures. A, Western blot for Duox on cell lysates from Calu-3 and hTE cells. B and C, confocal images showing a group of hTE cells stained with a polyclonal anti-Duox1 antibody (B) and anti-ZO1 as a marker of the luminal side (C). D, confocal side view of a group of hTE cells stained with an anti-Duox1 antibody. Note clear staining of the apical pole and the cilia. E and F, Calu-3 cell monolayer, confocal top view focused on apical membrane layer. Anti-Duox1 antibody did not result in specific staining (E), for comparison, anti-ZO1 staining of the layer shown in F.

Intracellular Acid Production by NADPH Oxidase—Although hTE and Calu-3 both expressed a similar Zn²⁺-sensitive plasma membrane H⁺ conductance, only hTE secreted acid in a Zn²⁺-blockable manner. These results suggested that the electrochemical gradient for H⁺ exit across an apical H⁺ conductance was larger in hTE than in Calu-3. In the following experiments we tested the notion that the activity of NADPH oxidase generates an intra- to extracellular H⁺ gradient that drives H⁺ through a plasma membrane H⁺ conductance. Under control conditions cytosolic pH in hTE, Calu-3, or in hTE treated with AEBSF was not significantly different (Fig. 6C, black bars). Treatment of cells with amiloride (1 mM) acidified all cells (Fig. 6, A and C), but hTE acidified at a significantly higher rate than Calu-3 (Fig. 6B), and after 90 min of amiloride treatment pH_i was significantly more acidic in hTE than in Calu-3 (Fig. 6C, gray bars). The amiloride-induced acidification was significantly reduced by treating hTE with AEBSF (Fig. 6A), which reduced the rate of acidification (Fig. 6B) and increased pH_i after 90 min of amiloride treatment over untreated hTE (Fig. 6C, gray bars), suggesting that the activity of NADPH oxidase significantly contributed to the intracellular acid production of hTE. These measurements are consistent with an NADPH oxidase-mediated acidification of pH_i in hTE. This process likely provides the driving force for H⁺ exit through apical membrane H⁺ channels in intact epithelia. For comparison, Calu-3 showed small rates of intracellular acid production and thus may not generate an appreciable H⁺ gradient across an apical membrane H⁺ conductance, consistent with the low rates of H⁺ secretion (Fig. 3) by these cells.

View larger version (14K): [in this window] [in a new window]   FIG. 6. Intracellular acid production in airway cells. A, measurement of cytosolic pH in hTE, Calu-3, and in hTE treated with 500 µM AEBSF. Amiloride was added to cells where indicated. Lines are averages ± S.E. of n = 33 Calu-3 cells, n = 29 hTE cells, and n = 30 hTE cells pre-treated with AEBSF in the displayed experimental runs. B, amiloride-induced initial rates of acidification of pHi. Rates were calculated from the first 20 min of treatment. All rates were significantly different from one another (ANOVA followed by t tests, p < 0.0001). C, average intracellular pH (pHi) under control conditions (black bars) and 90 min after amiloride treatment (gray bars). Control pHi was not different between treatment groups (p = 0.092). Treatment with amiloride acidified all treatment groups (p < 0.0001). After amiloride, hTE were significantly more acidic than Calu-3 or AEBSF-treated hTE (p < 0.0001). hTE, n = 128 cells in five experimental runs; Calu-3, n = 104 cells in three runs; AEBSF-treated hTE, n = 68 cells in three runs; * indicates significant difference from hTE.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Acid Secretion Is Driven by a H⁺ Gradient Generated by the Activity of NADPH Oxidase—Activation by intracellular acidity and membrane depolarization are typical characteristics of H⁺ conductances in various cell types (15). In our study, whole cell patch clamp measurements were performed with an inside-to-outside H⁺ gradient that was used both to activate the H⁺ conductance and to provide a driving force for H⁺ movement, and the membrane voltage was controlled. Under these conditions both airway cell types showed similar specific H⁺ conductances with common biophysical characteristics and zinc sensitivity, suggesting a similar type of H⁺ channel in the plasma membrane of hTE and Calu-3 (Fig. 1). However, in intact epithelial preparations, hTE showed zinc-sensitive H⁺ secretion, whereas Calu-3 did not (Fig. 3). These observations suggested that in Calu-3 epithelia either the H⁺ conductance was inactive or there was no proton-motive driving force across the apical membrane to support epithelial H⁺ secretion.

In intact cells several factors affect the electrochemical driving forces for H⁺ exit, such as the apical H⁺ gradient and the apical membrane potential (V_a), the latter of which in turn is determined by the transepithelial resistance (R_t), the relative resistances of the tight junctions and the cells, and the relative conductance of the apical membrane to Na⁺ and Cl^–. Compared with Calu-3 cells, hTE have larger R_t (569 versus 257 ·cm²), more negative V_t (–26 versus –5.6 mV), and a larger apical Na⁺ conductance (30, 38). Thus, compared with Calu-3, the hTE cultures can be expected to possess relatively depolarized V_a values. Consistent with the observed transepithelial differences in our report, the reported V_a for hTE was V_a =–26 mV (38) or V_a =–19 mV (39), and in Calu-3 cells V_a =–52 mV (40). Thus, one explanation for the lack of zinc-sensitive H⁺ secretion in Calu-3 is the hyperpolarized V_a, which is expected both to inactivate the H⁺ conductance (15) and to reduce the electrochemical driving force for H⁺ exit. The presence of conductive H⁺ secretion by hTE in the face of a negative V_a indicates an intra- to extracellular H⁺ gradient across the apical membrane (13). In alveolar type II cells it was shown that intracellular acidification and a H⁺ gradient were necessary both to activate the H⁺ conductance and to drive H⁺ currents (41). In neutrophils, activation of NADPH oxidase caused the cytosol to acidify from pH 7 to 6.2 when the Na⁺/H⁺ exchanger was blocked by Na⁺ free solutions (18). In airway epithelia the Na⁺/H⁺ exchanger was shown to be a major regulator of intracellular pH, however, it is present only in the basolateral membrane (42). Therefore, any H⁺ produced apically by NADPH oxidase may not be readily transported by the Na⁺/H⁺ exchanger but likely, instead, to cause a local acidification of the intracellular face of the apical membrane.

In analogy to acid production by NADPH oxidase in neutrophils, addition of amiloride (to block the Na⁺/H⁺ exchanger) caused an AEBSF-blockable acidification of hTE cells (Fig. 6). Basal pH_i was not affected by treatment with AEBSF (Fig. 6), indicating a tight control of pH_i by the Na⁺/H⁺ exchanger under control conditions. Thus, treatment with amiloride uncovered intracellular acid production by NADPH oxidase activity in hTE cells.

The effect of AEBSF on both the rates of intracellular acidification (Fig. 6C) and epithelial acid secretion by hTE (Fig. 3) is consistent with the notion that NADPH oxidase-generated intracellular H⁺ both activates the plasma membrane H⁺ conductance and provides a driving force for H⁺ exit across the apical membrane. When assuming that the pH_i measured in the presence of amiloride corresponds to the local pH at the cytosolic face of the apical membrane in untreated epithelia, then, with the measured pH gradient (6.89 to 7.4) and an average V_a of –22.5 mV (43, 44), an outwardly directed proton-motive force of 11.5 mV is predicted using the Nernst equation for conductive H⁺ secretion across the apical membrane. The critical role of intracellular acidification for H⁺ secretion is further supported by the data found in Calu-3 cells, which show little intracellular acid production and no evidence for an active, zinc-sensitive H⁺ secretion into the mucosal solution despite a significant plasma membrane H⁺ conductance.

Expression of NADPH Oxidase in Airway Epithelia—Duox1 and -2 were initially identified in thyroid epithelia where they were found to be expressed in the apical membrane (22, 45). Investigation of a panel of tissues showed a high expression of Duox1 and -2 transcripts in thyroid and of Duox1 in whole lung (23). In this report we show that in human tracheal surface epithelia both Duox1 and -2 are expressed at levels >1000-fold higher than other Nox isoforms (Fig. 4) and thus represent the major Nox isoforms in airways. In contrast, in Calu-3 cells Nox isoforms were generally expressed at low levels, and no transcript for Duox1 was found. The high expression of Duox1 in hTE, its apical localization, and the high rates of acid secretion, compared with the lack of expression of Duox1 in Calu-3, which also showed no zinc-sensitive acid secretion, suggest Duox1 as the isoform responsible for acid production and secretion in airways. The effects of block of NADPH oxidase by AEBSF on both H⁺ production and H⁺ secretion underline the correlation between these two functional parameters and Duox-expression.

Regulation and function of the NADPH oxidase of phagocytes greatly depends on the proper assembly of Nox2 with its subunits (46). Currently there is no known subunit that interacts with Duox. Interestingly, hTE express at high levels p22^phox, p40^phox, p47^phox, and p67^phox (Fig. 4). In contrast Calu-3 lacked the two subunits p40^phox and p47^phox. Although we currently have no evidence for a functional interaction between Duox and additional subunits of NADPH oxidase, our expression data support the notion of a fully functional NADPH oxidase complex in hTE that allows for acid production, whereas Calu-3 may not assemble a functional complex and do not generate H⁺ by this mechanism.

H⁺ Channels in Airways—Several small Nox isoforms have previously been suggested to function as H⁺ channels, including Nox2 (16), Nox5 (47), and a truncated form of Nox1 (48). However, DeCoursey and colleagues (34) challenged this conclusion owing to (i) the expression of the Nox constructs in cell systems that already expressed native H⁺ currents, (ii) a lack of H⁺ selectivity of the resulting currents, and (iii) the much shorter time constants of current activation recorded after Nox expression in these studies when compared with time constants of native H⁺ currents. Several additional studies then presented evidence that expression of Nox2 did not correlate with H⁺ channel function, but a parallel non-Nox H⁺ channel structure was suggested to be the H⁺ conductive site (summarized in Ref. 34). Thus the identity of the H⁺ channel and the H⁺ conductance of the NADPH oxidase is controversial. Because the putative H⁺ permeation pathway across Nox2 involves strictly conserved histidine residues (16, 49), the argument made for Nox2 can be extended to the other Nox family members, including Duox.

We found in airway cells a H⁺ conductance with characteristics very similar to the native H⁺ conductances found in white blood cells or alveolar type II cells: H⁺ currents activated slowly with time constants in the order of seconds, steady-state H⁺ currents were outwardly rectifying and showed H⁺ selectivity, and ZnSO₄ readily blocked the depolarization-activated currents (Figs. 1 and 2). Both hTE and Calu-3 expressed qualitatively and quantitatively similar H⁺ conductances suggesting the same type of H⁺ channel was active in both cell types. However, the high expression of Duox1 and -2 in hTE, and the total lack of Duox1 and low expression of Duox2 in Calu-3, suggest that the Duox isoforms do not function as H⁺ channels in airways, but a parallel non-Duox entity serves as a H⁺ channel. In analogy this argument may be extended to the other Nox family members based on the high homology between Duox and Nox in the histidine-containing transmembrane domains. However, our data cannot exclude the possibility that the lowly expressed isoforms Nox1, -2, and -5 may function as H⁺ channels, because they were found in both cell types. Thus a general conclusion about the channel characteristics of the Nox family from our data is precluded by the expression of multiple Nox isoforms in our cell system. Our data indicate that in airways a Duox-based NADPH oxidase generates intracellular H⁺ and a proton-motive force and H⁺ is conducted across the membrane through a parallel non-Duox-mediated pathway.

Two previous reports showed acid secretion of airway epithelia governed by different mechanisms. Coakley et al. (12) reported a ouabain-sensitive non-gastric K⁺/H⁺-ATPase in human primary cultures, and Inglis et al. (37) found a bafilomycin-sensitive vacuolar-type H⁺-ATPase in distal pig bronchi. Using the effects of ouabain and bafilomycin as indicators for these mechanisms, in this report we found their quantitative contributions to be 15 and 11%, respectively, of total acid secretion by hTE airway epithelia in pH-stat experiments. Although the quantitative contribution of these mechanisms is likely dependent on a number of factors, such as tissue origin, species, and experimental pH values, these observations suggest that the airway epithelium expresses additional mechanisms to acidify the airway surface liquid. Fig. 7 shows a model that displays the relation of acid transporting mechanisms and NADPH oxidase in the apical membrane of airway cells as derived from our study. See Fig. 7 legend for details.

View larger version (15K): [in this window] [in a new window]   FIG. 7. Model of acid release in the apical membrane of airway surface epithelial cells. In this model a Duox-based NADPH oxidase releases intracellular H+ from NADPH and acidifies pHi. Intracellular H+ exits across a Zn2+-sensitive H+ channel whose molecular identity is currently unclear. This mechanism is similar to the model proposed for NADPH oxidase function in phagocytes (34). In addition, airway cells release acid by way of a bafilomycin-sensitive H+ ATPase and an ouabain-sensitive K+/H+ ATPase. Percentages give relative contribution to total H+ secretion as determined from blocker experiments. Residual, non-blockable H+ secretion may be through a separate pathway, or may be due to incomplete inhibition of transporters by used blockers. Quantitative contribution of individual H+ secretory mechanisms may be different under experimental circumstances. Note that the source of H+ for the H+ channel is NADPH (which results in an acidification of pHi as observed in this study), whereas the ATPases release H+ from water resulting in an alkalinization of pHi in this model. For simplicity all transporters are shown located in one cell. Electron transport of NADPH oxidase is not shown in this model.

In contrast, for the airway epithelium, it appears prudent to assume a functional role of Duox during bacterial killing, which is the major function of the airways. The airway epithelium is the first line of defense against inhaled bacterial pathogens, and the epithelium secretes a number of antimicrobial factors, which act in combination with the mechanical movement of the ciliary escalator to keep the distal lungs sterile. Several antimicrobial factors have been isolated from airway secretions indicating the role of the airways in bacterial defense. Production of by airway cultures by NADPH oxidase has been shown (24). Acidification of the airway surface liquid during acid secretion by the epithelium is expected to support conversion of into H₂O₂ and HOCl, which show bactericidal activity only in an acidic environment (50). High activity of extracellular superoxide dismutase in airways (51) and the presence of significant lactoperoxidase activity in airway gland secretions suggest a rapid turnover of in support of bactericidal reaction products. Thus we suggest that the expression of NADPH oxidase in the airway epithelium and acid secretion represents an innate defense function of the airway epithelium.

	FOOTNOTES

 
^* This work was supported in part by National Institutes of Health Grants HL071829, DK51799, and 1P50HL60288 and a Pilot and Feasibility Grant by the Cystic Fibrosis Foundation (Grant FISCHE02I0). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Recipient of an Elizabeth Nash Memorial Fellowship from Cystic Fibrosis Research, Inc.

^|| To whom correspondence should be addressed: Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland CA 94609. Tel.: 510-450-7696; Fax: 510-450-7910; E-mail hfischer{at}chori.org.

¹ The abbreviations used are: ASL, airway surface liquid; hTE, human tracheal epithelial cells; DMEM, Dulbecco's modified Eagle's medium; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; MES, 2-(N-morpholino)ethanesulfonic acid; ANOVA, analysis of variance; pF, picofarad; pS, picosiemen; ZO1, zonula occludens 1.

² F. Miot, personal communication.

	ACKNOWLEDGMENTS

 
Thanks to Drs. Jonathan H. Widdicombe, University of California at Davis, and Robert Tarran, University of California at Berkeley, for their support in obtaining primary cultures; to Dr. Francoise Miot, Université Libre de Bruxelles, Belgium, for kindly providing the Duox antibody; to Dr. Gregory M. Dolganov, University of California at San Francisco for performing real-time PCR; and to Gordana Borcanski and Jacob Haight for technical assistance.

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