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J. Biol. Chem., Vol. 280, Issue 43, 35807-35814, October 28, 2005

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Rapid Effects of Dexamethasone on Intracellular pH and Na⁺/H⁺ Exchanger Activity in Human Bronchial Epithelial Cells^*

Valia A. Verrière, Darina Hynes, Sheila Faherty, James Devaney, Jean Bousquet, Brian J. Harvey, and Valérie Urbach1

From the INSERM U454, Centre Hospitalier Universitaire Arnaud de Villeneuve, 34295 Montpellier, France and Molecular Medicine Laboratories,Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Ireland

Received for publication, June 17, 2005 , and in revised form, July 12, 2005.

	ABSTRACT

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Glucocorticoids have been shown to produce rapid nongenomic responses in airway epithelia. By using an intracellular pH (pH_i) spectrofluorescence imaging system and the NH₄ Cl acid-loading technique, we have shown that the synthetic glucocorticoid,dexamethasone, accelerated intracellular pH recovery after an acid load in a human bronchial epithelial cell line (16HBE14o^– cells). Exposure to NH₄Cl (20 mM) elicited an intracellular acidification, followed by a pH_i recovery. Inhibition of the Na⁺/H⁺ exchanger decreased the steady-state pH_i and antagonized the dexamethasone stimulation of pH_i regulation. The rapid effect of dexamethasone on pH_i was neither affected by the inhibitor of transcription, cycloheximide, nor by the classical glucocorticoid and mineralocorticoid receptors antagonists, RU486 and spironolactone, respectively. The dexamethasone effect on pH_i regulation was reduced by inhibitors of adenylate cyclase, cAMP-dependent protein kinase and mitogenactivated protein kinase (ERK1/2). By using a PepTag assay system and Western blotting, we have shown that dexamethasone stimulated cAMP-dependent protein kinase and mitogen-activated protein kinase activities. Taken together our results provide evidence for the rapid stimulation of Na⁺/H⁺ exchange activity by glucocorticoids in bronchial epithelial cells via a nongenomic mechanism involving cAMP-dependent protein kinase and mitogen-activated protein kinase ERK1/2 pathways.

	INTRODUCTION

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Glucocorticoids are the most potent treatment of inflammatory airway disease such as asthma (1). However the cellular mechanisms involved in the response to glucocorticoids are not fully elucidated, and in particular, early cellular effects of corticotherapy are relatively unknown. Glucocorticoids are recognized to produce their pharmacological effects through a classical genomic pathway involving binding to a specific cytosolic receptor, translocation into the nucleus, and subsequent activation (transactivation) or repression (transrepression) of a target gene transcription. The anti-inflammatory properties of glucocorticoids are explained by the transrepression of genes coding for inflammatory molecules. In intestinal and renal epithelial cells, glucocorticoids stimulate the expression of mRNA coding for the Na⁺/H⁺ exchanger, one of the most important intracellular pH regulators in epithelial cells (2, 3). The genomic effects of steroids are characterized by a latency of onset lasting several hours.

In contrast, over the past decade, many steroid hormones have been reported to induce rapid responses (seconds to minutes) in various signal transduction pathways and ion transport. These fast responses are incompatible with the involvement of the classical genomic pathway for steroid hormone action. Rapid effects of glucocorticoids have been described in several tissues such as endometrial cells (4), vascular smooth muscle (5, 6), and renal cortical collecting duct (7). However, the cellular and molecular mechanisms involved are only partially described, and their physiological roles have not been elucidated (8, 9). In airway epithelial cells, dexamethasone produced a rapid decrease of intracellular [Ca²⁺] and inhibited Ca²⁺-dependent Cl^– secretion through a nongenomic mechanism (10). In vascular smooth muscle cells, glucocorticoids have been shown to stimulate rapidly the Na⁺/H⁺ exchanger activity through a mechanism independent of gene transcription and protein synthesis (6). In epithelia, the mineralocorticoid hormone, aldosterone, also stimulates the Na⁺/H⁺ exchanger activity through a nongenomic mechanism (11–13). A nongenomic effect of glucocorticoids on pH_i regulation in airway epithelium has not been described previously.

	MATERIALS AND METHODS

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Cell Culture—Epithelial cells were grown in Corning culture flasks coated with a collagen/fibronectin solution at 37 °C in a humidified 5% CO₂ atmosphere. The human bronchial epithelial cell line 16HBE14o^– was derived from the surface epithelium of mainstream, second generation bronchi. The 16HBE14o^– cells form polarized monolayers with intact tight junctions and retain chloride transport properties and other differentiated features characteristic of freshly isolated surface airway epithelial cells (14). The 16HBE14o^– cells were grown in Eagle's minimal essential medium supplemented with 10% fetal calf serum, 1% penicillin G, 1% streptomycin, and 1% L-glutamine. After reaching confluence, the cells were washed twice with a phosphate-buffered saline solution and isolated at 37 °C, using a trypsin solution (1% polyvinylpyrrolidone, 0.2% EGTA, and 0.25% trypsin containing 0.02% EDTA).

pH_i Measurement—For pH_i measurements, human bronchial epithelial cells were grown on collagen/fibronectin-coated glass bottom dishes (12 mm diameter) and loaded with the pH-sensitive dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein by incubating the cells in 5 µM of 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein-AM (Molecular Probes) for 60 min at 37 °C. The cells were bathed in a modified Krebs solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl₂,2mM MgSO₄,10mM HEPES, pH 7.4). All experiments were performed in the dark and at room temperature to minimize dye leakage. The glass bottom dishes were mounted on an inverted epi-fluorescence microscope (Diaphot 200, Nikon). The ultraviolet light from a xenon 100-watt lamp (Nikon) was filtered through alternating 440 and 505 nm interference filters using the OptoScan-CAIRN monochromator (CAIRN-DIPSI, Chatillon, France), which allowed an electronic adjustment of the duration of the exposure to each wavelength. The resultant fluorescence was filtered at 510 nm and collected using an intensified CCD camera system (CoolSNAP-fx, Roper Scientific-Princeton Instrument, Evry, France). Images were digitized and analyzed using Metafluor (Universal Imaging-Princeton Instrument, Evry, France). The ratio of fluorescent signals emitted at each excitation wavelength was calibrated using nigericin (15). The frequency of acquisition was 1 ratiometric image every 500 ms. The basal pH_i (steady-state pH_i) was calculated as the average of 1 min of acquisition under control conditions. The hormonal effect on pH_i regulation was investigated under intracellular acid loading conditions using the ammonium chloride (NH₄Cl = 20 mM) pre-pulse technique (16). Removal of NH₄⁺ resulted in the generation of intracellular H⁺, because of the dissociation of NH⁺₄ into H⁺ ions and NH₃, with subsequent efflux of NH₃. The resultant changes in pH_i (dpH_i) were used to estimate _i in single cells as follows: _i = [NH⁺₄]/dpH_i.

The acid load was carried out in the absence of extracellular Na⁺ in order to prevent acid extrusion. As shown in Fig. 1, the pH_i dependence of the intracellular buffering power was evaluated by plotting the _i values as a function of the pH_i measured at midpoint of the acid load (Fig. 1). The intrinsic buffering power of the two cell populations used to serve as control and to investigate the dexamethasone response was not significantly different (mean _i control is 15.01 ± 0.3, mean _i for dexamethasone experiment is 14.65 ± 0.182). Furthermore, the pH_i dependence of the buffering power was not significantly different for the two control and dexamethasone-treated cell populations (Fig. 1). The H⁺ flux after the acid load was calculated as the product of the buffering power, _i, and the initial rate of pH_i regulation (pH_i/dt) was measured over 60 s at any given pH_i: J_H = _i x pH_i/dt. For each experiment, 10–15 cells were imaged simultaneously.

Preparation of Cell Lysates—The 16HBE14o^– cells (5 x 10⁵/ml) were incubated under serum-free conditions overnight and then exposed to experimental drug treatment, if any, and stored on ice. The media were replaced with 1 ml of ice-cold phosphate-buffered saline for 1 min (pH 7.2). The phosphate-buffered saline wash was replaced with 150 µl of homogenization buffer (20 mM Tris, 1 mM EGTA, pH 7.4, 10 mg/ml bovine serum albumin, 100 mM phenylmethylsulfonyl fluoride, 1 mM 1,4-dithiothreitol, and 1 mM protease inhibitor mixture). Cells were lysed by Soniprep microprobe sonication for 15 s (Sanyo, Osaka Japan).

Detection of cAMP-dependent Protein Kinase Activity—cAMP-dependent protein kinase (PKA)² activity was assayed using the Promega PepTag® assay for nonradioactive detection of cAMP-dependent protein kinase (Promega, Madison, WI). Lysates were prepared as above and assayed immediately. All PepTag assay reaction components were combined on ice, and PKA activity was assayed at 30 °C for 20 min, in a final volume of 30 µl of the following mixture: 5 µlof5x reaction buffer, 5 µl of formyl-Leu-Arg-Arg-Ala-Ser-Leu-Gly (f-Kemptide) PepTag buffer, 5 µl of water, and 15 µl of sample homogenate (10 µg of protein) or 10 ng of commercial catalytic C subunit. The reaction was stopped after 20 min by incubating the tubes at 95 °C for 10 min. Before loading samples on previously prepared electrophoresis gel (0.8% agarose in 50 mM Tris buffer, pH 8.0), 2 µl of 80% glycerol was added to the sample. Electrophoresis was run at 110 V for 30 min and was imaged under UV light by a GeneSnap® Program.

Detection of MAP Kinase Activity by Western Blotting—Cells were pretreated with inhibitors H89 (10 µM) and PD98059 (25 µM) for 40 min and then exposed to dexamethasone (1 nM) or methanol for 20 min, and lysates were prepared as above. Proteins were separated on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Membranes were incubated with rabbit phospho-p44/42 MAP kinase (Thr-202/Tyr-204) primary antibody at a dilution of 1:2,000 (Cell Signaling, 9101S), washed, and incubated with an anti-rabbit IgG horseradish peroxidase-conjugated secondary (Sigma A9169) at a dilution of 1:4,000. For loading controls, membranes were washed and incubated with mouse IgG anti--actin primary antibody (1:2000 Sigma A5316), washed, and incubated with an anti-mouse IgG horseradish peroxidase-conjugated secondary (Sigma A9169) at a dilution of 1:20,000. Detection was carried out using the ECL Plus Western blotting system (Amer-sham Biosciences). Quantitative gel banding densitometry was performed using Gene Tools (SynGene, Cambridge, UK).

View larger version (10K): [in this window] [in a new window]   FIGURE 1. The pHi dependence of intracellular buffering. The intracellular buffering power (i) was evaluated at different pHi values in cells pre-pulsed with ammonium chloride (20 mM) in two populations of cells serving as either controls without exposure to steroid (closed circles) or cells or for dexamethasone (1 nM) treatment (open circles). Each data point represents the average of eight individual i values measured at a given pHi value corresponding to the midpoint of each acid load.

View larger version (16K): [in this window] [in a new window]   FIGURE 2. Role of Na+/H+ exchange in pHi regulation following an acid load. The pHi regulation during and following an acid load induced by exposure of 16HBE14o– cell monolayers to NH4Cl (20 µM) in control Krebs solution and during Na+/H+ exchanger inhibition. A, typical pHi recovery rates after an acid load in normal Krebs solution or in the presence of EIPA (0.1 µM). B, mean pHi recovery rates under control (cont) conditions, in the presence of EIPA (0.1 µM)orinNa+-free medium (NMDG substitution). **, p < 0.01.

View larger version (21K): [in this window] [in a new window]   FIGURE 3. Dexamethasone effect on the pHirecovery. A, typical effect of dexamethasone (dexa.) (1 nM) on pHi recovery rate (after an acid load with or without EIPA) (0.1 µM). B, concentration-response relationship of the dexamethasone stimulation of the mean pHi recovery from an acid load. C, mean pHi recovery rates after an acid load in the presence of three different concentrations of dexamethasone (dex.) (1 nM and 10 and 1 µM) and in the presence or absence of EIPA (0.1 µM). **, p < 0.01. D, relationship between the EIPA-sensitive H+ flux (mM/min) and the intracellular pHi recorded after an acid load in individual cells treated with dexamethasone (1 nM) and untreated.

	RESULTS

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pH_i Changes during an Acid Load—The regulation of intracellular pH in human bronchial epithelial cells (16HBE14o^– cell line) was studied using the acid-loading NH₄Cl pre-pulse method (16). In control Krebs solution, the steady-state pH_i was7.47 ± 0.01 (n = 120). As shown in Fig. 2, exposure of 16HBE14o^– cells to NH₄Cl (20 mM) elicited a large pH_i increase to 8.40 ± 0.04 (n = 36) followed by a plateau-phase acidification. Prior to the NH₄Cl exposure, ethylisopropylamiloride (EIPA), used as a specific inhibitor of the Na⁺/H⁺ exchanger, decreased the steady-state pH_i by 0.14 ± 0.01 pH units (n = 120), indicating the contribution of the Na⁺/H⁺ exchanger to the maintenance of the steady-state pH_i. The acid load was produced upon removal of NH₄Cl after 10 min of treatment. In order to amplify the acid loading after NH₄ Cl exposure, the cells were exposed to a Na⁺ -free and HCO^–₃-free Krebs solution in which Na⁺ was replaced by N-methyl-D-glucamine (NMDG) in a HEPES-buffered solution. Under these conditions, the pH_i decreased to 6.46 ± 0.03 (= n 36) within 1.67 ± 0.04 min. Following the acid load, the 16HBE14o^– cells spontaneously regulated their pH_i when superfused in normal Krebs solution (containing Na⁺). The pH_i increased at an initial rate of 0.046 ± 0.009 pH units per min (J_H = 0.67 ± 0.02 mM/min) up to a plateau value of pH_i = 6.9 ± 0.04, which was reached within 19.8 ± 1.2 min after the start of the acid load (n = 36). The EIPA exposure did not significantly affect the buffering power of the cells calculated after 20 mM NH₄Cl exposure. The buffering power obtained under control conditions (without EIPA) was 15.0 ± 0.4 mM/pH units (n = 120) and 14.2 ± 0.3 mM/pH units after EIPA treatment (n = 120, p > 0.05).

Role of the Na⁺/H⁺ Exchanger in pH_i Recovery Following an Acid Load—In order to investigate the role of Na⁺/H⁺ exchanger in the regulation of pH_i following an acid load, we tested the Na⁺ dependence of the pH_i recovery rate. After an acid load, the rate of pH_i recovery was significantly reduced to 0.016 ± 0.001 pH_i units/min (n = 38) under Na⁺-free conditions (NMDG substitution) compared with 0.048 ± 0.003 pH_i units/min (n = 38) under control conditions in the presence of Na⁺ (p < 0.001). EIPA was also tested (17). As shown in the Fig. 2, the pH_i recovery rate was reduced to 0.002 ± 0.001 pH units/min (n = 95) in the presence of EIPA (100 µM). This rate is over 20-fold slower than the control pH_i recovery rate (pH_i/dt control = 0.047 ± 0.002 pH units/min, n = 95, p < 0.001). These results indicate that the pH_i recovery after an acid load is mainly dependent on Na⁺/H⁺ exchanger activity.

Dexamethasone Effect on the pH_i Recovery from an Acid Load—The steady-state pH_i value was not significantly affected by adding the synthetic glucocorticoid, dexamethasone (1 nM), to the Krebs bathing solution (pH_i from control steady-state value = 0.0003 ± 0.0002 pH units/min, n = 80). However, dexamethasone (1 nM) enhanced the recovery of pH_i in 16HBE14o^– cells following an acid load. When the average pH_i reached after the acid load was 6.4 ± 0.04, the initial pH increased from a mean rate of 0.046 ± 0.021 pH units/min under control conditions to a mean rate of 0.106 ± 0.003 pH units/min (Fig. 3). In addition, the pH_i reached a mean plateau value of 7.57 ± 0.05 (n = 57, p < 0.05) within a delay of 10.23 ± 0.03 min. Thus, the pH_i value reached after recovery from an acid load in the presence of dexamethasone (1 nM)was significantly more alkaline than the final steady-state pH_i level reached without hormone treatment. The rapid dexamethasone effect on the pH_i recovery displayed a bell-shaped concentration dependence with a maximum effect on pH_i recovery rate produced at 1nM dexamethasone (Fig. 3B). The dexamethasone solvent (methanol) did not affect the pH_i recovery after an acid load (pH = 0.02 ± 0.001 pH units/min).

Role of Na⁺/H⁺ Exchanger Activity in the Dexamethasone-stimulated pH_i Recovery—The role of the Na⁺/H⁺ exchanger activity in pH_i recovery from an acid load under dexamethasone stimulation was tested by removal of external Na⁺ and by addition of the Na⁺/H⁺ exchanger inhibitor EIPA. As shown on Fig. 3, A and C, when cells were exposed to EIPA after the acid load, the dexamethasone stimulation of the pH_i recovery was inhibited (p < 0.001). When Na⁺ was absent from the bathing solution (NMDG substitution) after the acid load, the dexamethasone effect on pH_i recovery was also inhibited. In bronchial epithelial cells bathed with NMDG solution, the pH_i obtained after the acid load in the presence of dexamethasone (1 nM) was 0.021 ± 0.003 pH units/min (n = 54). This contrasts with the high rate of pH_i recovery stimulated by dexamethasone (1 nM) in the presence of extracellular Na⁺ (pH_i = 0.114 ± 0.004 pH_i units/min, n = 57, p < 0.001). These experiments indicate the complete Na⁺ dependence and the major role of the Na⁺/H⁺ exchanger in the dexamethasone-induced stimulation of pH_i recovery following an acid load.

In order to further investigate the effect of dexamethasone on the Na⁺/H⁺ exchanger activity, we compared the J_H at different starting pH_i values of the pH regulation phase with or without dexamethasone. The J_H has been calculated for each single cell (using the pH/dt recorded during 1 min) and expressed as a function of the lowest pH_i measured in each cell at that time (Fig. 3D). We determined the EIPA-sensitive component of the pH_i recovery from an acid load at each of the various pH_i starting values during the recovery phase. The contribution of the Na⁺/H⁺ exchanger to the pH_i recovery (pH/dt because of the Na⁺/H⁺ exchanger) has been calculated by subtracting the EIPA-insensitive pH/dt at a given pH_i from the total pH/dt measured at the corresponding control pH_i (without EIPA). The pH_i dependence of the exchanger was computed as the product of the pH_i/dt and the buffering power and plotted as a function of the initial pH_i (Fig. 3D).

Fluticasone and Aldosterone Effects on the pH_i Recovery—In order to test steroid specificity, the effects on pH_i regulation of the mineralocorticoid, aldosterone and the glucocorticoid, fluticasone were also investigated. As shown in Fig. 4A, fluticasone at 1 nM produced a faster pH_i recovery rate than dexamethasone used at the same concentration. Fluticasone stimulated a pH_i recovery rate of 0.173 ± 0.005 pH_i units/min (n = 52). On the other hand, aldosterone (1 nM) did not significantly increase the pH_i recovery rate after an acid load. The pH_i recovery rate in the presence of aldosterone treatment was 0.051 ± 0.002 pH_i units/min (n = 51), which was not significantly different from the pH_i recovery obtained under control conditions in the absence of the mineralocorticoid (p > 0.1).

View larger version (24K): [in this window] [in a new window]   FIGURE 4. Comparative steroid responses and effects of RU486, spironolactone, and cycloheximide on pHi. A, comparison of the pHi recovery rate under control conditions and in the presence of 1 nM of the following steroids: dexamethasone (dex.), aldosterone (aldo.), and fluticasone (fluti.). B, effect of RU486 (1 µM) and spironolactone (10 µM) on pHi recovery stimulated by dexamethasone at concentrations of 1 nM and 1 µM. C, effect of cycloheximide (1 µM) pretreatment on pHi recovery under control conditions and following stimulation by dexamethasone. Not significant (NS), p > 0.05.

View larger version (28K): [in this window] [in a new window]   FIGURE 5. Role of protein kinase A and MAP kinase in pHiregulation. A, effects of (Rp)-cAMP (Rp) (1 µM) and the myristoylated PKA inhibitor 14-22 amide (10–5M) on pHi recovery after an acid load under control conditions and following dexamethasone (dex.) (1 nM). B, effects of the MAPK inhibitor PD98059 (PD98) (25 µM) on pHi recovery after an acid load under control conditions and following dexamethasone (1 nM). C, effect of simultaneous treatment of cells with PD98059 (25 µM) and PKA inhibitors ((Rp)-cAMP, 1 µM, PKAI 14-22, 10 µM)onpHi recovery after an acid load under control conditions and in the presence of dexamethasone (1 nM). **, p < 0.01, not significant (NS), p > 0.05)

Role of cAMP-dependent Protein Kinase in the pH_i Response to Dexamethasone—A possible role for the PKA signaling pathway in the rapid steroid response was tested using (R_p)-cAMP, a competitive inhibitor of PKA activity and the PKA inhibitor 14-22 amide myristoylated (PKAI). Pretreatment with (R_p)-cAMP did not affect the control pH_i regulation following an acid load; however, PKAI partially inhibited pH_i recovery under control conditions. In contrast, the dexamethasoneinduced pH_i regulation was affected by the PKA antagonists. The pH_i recovery rate in the presence of dexamethasone was 0.056 ± 0.002 pH_i units/min (n = 57) in the presence of (R_p)-cAMP and 0.046 ± 0.001 pH_i units/min (n = 56) with PKAI. These recovery rates were significantly reduced compared with the pH_i recovery rate of 0.101 ± 0.004 (n = 59) obtained with dexamethasone alone (Fig. 5A).

View larger version (13K): [in this window] [in a new window]   FIGURE 6. Role of Gi protein in pHi regulation. Effect of pertussis toxin (PT) treatment (2 µg/ml) for 2 h on the pHi recovery after an acid load under control (cont) conditions and following dexamethasone (dex)(1nM).

Role of Pertussis Toxin-sensitive G Protein-coupled Receptors in the pH_i Response to Dexamethasone—The role of G protein-coupled receptors in the rapid response to dexamethasone was investigated using pertussis toxin (2 µg/ml). As shown in Fig. 6, pertussis toxin did not affect the basal pH_i recovery rate after an acid load under control conditions (pH_i = 0.056 ± 0.003 pH_i units/min, n = 21) but significantly inhibited the pH_i recovery stimulated by dexamethasone (10^–9 M) (pH_i = 0.074 ± 0.003 pH_i units/min, n = 52, p < 0.001).

Dexamethasone Effect on cAMP-dependent Protein Kinase Activity—As shown in Fig. 7, PKA activity was up-regulated in less than 5 min by 40% over control (p < 0.01) following 1 nM dexamethasone treatment. The G_i protein inhibitor, pertussis toxin (10 µg/ml), inhibited the activation of PKA by dexamethasone. The cells pretreated with pertussis toxin before dexamethasone did not show a significantly increased phosphorylation compared with untreated control cells (p > 0.1). In contrast, the ERK1/2 inhibitor, PD98059 (50 µM), had no significant effect on PKA activation by the steroid (p > 0.1). In these experiments, the adenylate cyclase activator, forskolin, and the PKA antagonist, (R_p)-cAMP, were used as positive and negative controls for PKA stimulation, respectively. The PKA phosphorylation was not significantly increased with methanol (dexamethasone solvent) compared with the negative control (p > 0.1).

Dexamethasone Effect on the MAP Kinase Activity—As shown in Fig. 8, dexamethasone (1 nM) stimulated MAP kinases (Erk1 and Erk2) phosphorylation in 16HBE14o^– cells (p < 0.05) in less than 5 min (lane 3). The rapid activation of MAPK by dexamethasone was significantly inhibited (p < 0.05) by the PKA inhibitor H89 (Fig. 8, lane 4) and the MAP kinase inhibitor PD98059. The basal MAP kinase activity was completely abolished by the PD98059 (Fig. 8, lane 7) but not by H89 (lane 6). Neither forskolin nor methanol affected MAPK activity (Fig. 8, lanes 1 and 2). These results indicated that the basal MAP kinase phosphorylation in 16HBE14o^– did not require PKA activity, whereas dexamethasone induced a MAP kinase activation depending on upstream PKA activity.

View larger version (27K): [in this window] [in a new window]   FIGURE 7. Dexamethasone effect on phosphorylation of PKA. A, UV-illuminated agarose gel of the products of reactions run with f-Kemptide and HBE cell homogenate. PKA activity phosphorylated the PepTag peptide (f-Kemptide) changing its net charge from +1 to –1. This allows the phosphorylated (–) and nonphosphorylated (+) forms of the substrate to be rapidly separated on an agarose gel. A, lane 1, forskolin (20 µM/5 min); lane 2, untreated; lane 3, vehicle control (methanol (meth) 0.01% v/v); lane 4, (Rp) (Rp)-cAMP(S) (prior treatment 20 µM/40 min) and dexamethasone (dex) (1 nM/5min); lane 5, dexamethasone (1 nM/5 min). B, lane 1, forskolin (20 µM/5 min); lane 2, dexamethasone (dex) (1 nM/5 min); lane 3, PD98059 (PD98) (prior treatment 50 µM/40 min) and dexamethasone (1 nM/5 min); lane 4, pertussis toxin (PT) (prior treatment 2 µg/liter, 20 min) and dexamethasone (1 nM/5 min); lane 5, untreated.

View larger version (44K): [in this window] [in a new window]   FIGURE 8. Dexamethasone effect on ERK1/2 phosphorylation. A, 16HBE14o– cells were treated with 20 µM forskolin (lane 1), methanol 0.001% (lane 2), dexamethasone (Dexa)1nM (lane 3), dexamethasone (1 nM) after pretreatment with H89 (10 µM)(lane 4), dexamethasone (1 nM) after pretreatment with PD98059 (25 µM)(lane 5). Effect of MeOH on inhibitor pretreatments of H89 (lane 6) and PD98059 (lane 7). B, -actin loading control. C, relative densitometric units of the ERK P44 bands (n = 3).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The basal pH_i values measured in human bronchial epithelial cells (16HBE14o^– cells) are consistent with pH_i values obtained in other epithelial cell types (16), including human nasal epithelial cells (18, 19). The regulation of pH_i in airway epithelial cells has been shown to be dependent on a variety of acid extruders such as the Na⁺/H⁺ exchanger (18) and the H⁺-ATPase pump (20) and on alkali extrusion via Cl^–/HCO^–₃ exchanger (21, 22). However, in our study, all the experiments were performed in HEPES-buffered and HCO^–-free medium, which reduces the contribution of Cl^–3 /HCO^–₃ exchange to pH_i regulation. In addition, we showed that EIPA (100 µM) produced a rapid and significant decrease of the basal pH_i, indicating a contribution from the Na⁺/H⁺ exchanger activity to maintenance of the steady-state pH_i. This result is consistent with previous observations on the role of Na⁺/H⁺ exchanger activity in maintaining the steady-state pH_i in a wide variety of epithelia such as renal cells (23–25), retinal pigment epithelial cells (26–28), corneal epithelium (29), human esophageal epithelial cells (30), nasal epithelial cells (18), and lung epithelial cells (31–35).

In this study, we have shown the synthetic glucocorticoid dexamethasone stimulated a rapid pH_i recovery in bronchial epithelial cells following an acid load. The dependence on external Na⁺ and the sensitivity to the Na⁺/H⁺ exchanger inhibitor EIPA of the dexamethasone effect on the pH_i recovery strongly suggests that this response is mediated by stimulation of the Na⁺/H⁺ exchange. Stimulation of the Na⁺/H⁺ exchanger may reflect an increase in the turnover rate (V_max) and/or a change in its activation set point, which is regulated by H⁺ allosteric modulation (61, 62). The set point of the exchanger is the pH_i value at which the exchanger ceases to produce a net extrusion of H⁺. In our experiments, the pH_i dependence of the EIPA-sensitive H⁺ flux measured with or without dexamethasone (Fig. 3) indicates that dexamethasone increased the V_max of the Na⁺/H⁺ exchange. Dexamethasone did not affect the basal steady-state pH_i. However, after an acid load a higher plateau value of pH_i was reached with dexamethasone, suggesting that dexamethasone causes a shift in the set point of the exchanger to more alkaline pH_i values. The lack of effect of the steroid on basal pH_i may indicate an intracellular acidification is first required for dexamethasone activation of the exchanger or the set point is close to the basal pH_i level.

Perfusion with NMDG (Na⁺ substitution) did not completely inhibit the pH_i recovery after an acid load, whereas EIPA completely abolished it. This difference is compatible with Na⁺/H⁺ activation at the early phase of the pH_i recovery (initial rate). EIPA rapidly and completely abolished Na⁺/H⁺ activity, whereas the effect of the ionic substitution of Na⁺ by NMDG depends on the perfusion rate and might not be completed at the early stage of the recovery.

The effects of glucocorticoids on pH_i regulation and Na⁺/H⁺ exchange have been widely reported in epithelial tissues. The increased expression of the NHE3 isoform by glucocorticoids has been reported in rat renal brush border (36), opossum kidney cells (37), proximal tubule cells (3, 38, 39), rabbit ileal brush border (2), and the NHE1 isoform in intra-hepatic biliary epithelium (40). In contrast, our study showed that dexamethasone stimulated pH_i recovery through a rapid cycloheximide-insensitive mechanism that is not compatible with a genomic action. A nongenomic response to dexamethasone is consistent with previous reports of the rapid effects of mineralocorticoid and glucocorticoid hormones on Na⁺/H⁺ exchanger activity in other epithelia (11–13, 41, 42). Rapid nongenomic effects of glucocorticoids have been described in tissues such as endometrial cells (4), vascular smooth muscle (5, 6), human folliculostellate cells (43), renal cortical collecting ducts (7), and airway epithelium (10). Recently, a nongenomic effect of glucocorticoids has been reported in the allergic asthma reaction (44). The rapid pH_i responses to glucocorticoid in human airway epithelium raise the possibility of new therapeutic strategies for inflammatory airway disease associated with an airway acidification (asthma and cystic fibrosis).

The cellular and molecular mechanisms involved in rapid glucocorticoid responses are only partially described, and their physiological role has not yet been elucidated (8, 9). Here we have investigated the cellular mechanisms involved in the rapid response to dexamethasone on pH_i in airway epithelial cells. A role for the nuclear glucocorticoid receptor has been reported for some nongenomic actions of steroids (4, 43, 45, 46). However, in our study, the insensitivity of the dexamethasone-induced pH_i response to glucocorticoid and mineralocorticoid receptor antagonists indicates the response is not mediated by the classical steroid receptors. However, the higher potency of fluticasone compared with aldosterone on pH_i regulation after an acid load suggests the involvement of a receptor selective for glucocorticoids but distinct from the nuclear glucocorticoid receptor responsible for the genomic action of glucocorticoids. These results are consistent with other rapid responses to glucocorticoids that do not involve the "classical" nuclear receptor (6, 10, 47). The sensitivity of the response to pertussis toxin may indicate the involvement of a novel membrane-associated receptor, and the identification of membrane-binding sites for glucocorticoids supports this hypothesis (48–50). The rapid dexamethasone effect on pH_i recovery displayed a bell-shaped concentration dependence that can be explained by desensitization of the receptor at higher steroid concentrations as already reported from other studies of the nongenomic effects of steroid hormones (58, 59).

Because activation of the Na⁺/H⁺ exchanger in a wide variety of tissues has been shown to occur through protein phosphorylation (41, 43), we tested the possible involvement of several kinases in the rapid response to dexamethasone. Our data showed a sensitivity of the rapid dexamethasone effects on pH_i regulation to PKA and ERK1/2 MAP kinase inhibitors. In addition, because our results also demonstrate that PKA and MAPK activities are up-regulated in response to dexamethasone in less than 5 min, we conclude that dexamethasone stimulates Na⁺/H⁺ activity through PKA and ERK1/2 MAP kinase activation. A role for PKA in Na⁺/H⁺ activation has been reported in renal and intestinal epithelium (51–53). The stimulation of the Na⁺/H⁺ exchanger by other kinases has also been described. In endothelial and renal epithelial cells, ERK1/2 MAPK was shown to be involved in Na⁺/H⁺ activation (54, 55). Steroids such as 1,25-OH-D₃, 17-estradiol, and aldosterone have been reported to produce a nongenomic response on calcium signaling through PKA stimulation (56–60) and MAPK activation (60). In our study, the simultaneous exposure of the cells to both inhibitors did not produce an additive inhibitory effect on the dexamethasone-induced pH_i response, suggesting that PKA and ERK1/2 MAPK lie within common convergent signaling cascade. In addition, the inhibition of ERK1/2 phosphorylation had no effect on PKA phosphorylation by dexamethasone, whereas the PKA antagonist inhibited ERK1/2 activation by dexamethasone. These latter results indicate that dexamethasone stimulation of ERK1/2 is dependent on PKA activation. The pertussis toxin inhibition of the dexamethasone effects on PKA activation and on pH_i recovery after the acid load suggests a role for a G_i protein-coupled receptor in transducing the rapid response to dexamethasone.

In conclusion, we show that dexamethasone stimulates Na⁺/H⁺ exchanger activity through a rapid nongenomic pathway that does not implicate the classical nuclear receptors for glucorticoids or mineralocorticoids but implicates a novel signaling cascade involving sequentially a G_i protein-coupled receptor, adenylate cyclase, cAMP, PKA, and ERK1/2 activation.

	FOOTNOTES

 
^* This work was supported by INSERM, Vaincre la Mucoviscidose, the Regional Council of Languedoc-Roussillon (France), Wellcome Trust Grant 055695/Z/98/Z/CH/TG/JF, and the Higher Education Authority of Ireland PRTLI fund. 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.

¹ To whom correspondence should be addressed: INSERM U454, CHU A. de Villeneuve, 34295, Montpellier Cedex 05, France. Tel.: 04-67-33-59-31; Fax: 04-67-63-28-55; E-mail: urbach{at}montp.inserm.fr.

² The abbreviations used are: PKA, cAMP-dependent protein kinase; MAP, mitogen-activated protein; MAPK, MAP kinase; NMDG, N-methyl-D-glucamine; EIPA, ethylisopropylamiloride; PKAI, PKA inhibitor 14-22 amide myristoylated; ERK, extracellular signalregulated kinase.

	ACKNOWLEDGMENTS

 
The 16HBE14o–cell line was obtained as a gift from D. C. Gruenert (Human Molecular Genetics, Department of Medicine, University of Vermont, Burlington, VT).

	REFERENCES

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