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Am J Physiol Cell Physiol 281: C1504–C1511, 2001. Airway surface liquid pH in well-differentiated airway epithelial cell cultures and mouse trachea SUJATHA JAYARAMAN, YUANLIN SONG, AND A. S. VERKMAN Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521 Received 23 April 2001; accepted in final form 6 July 2001 (ASL) is the thin layer of liquid at the air-facing epithelial surface in the upper and lower airways. The regulation of ASL volume, ionic composition, and pH is believed to be important in normal airway physiology and in the pathophysiology of genetic and acquired diseases of the airways such as cystic fibrosis and asthma (3, 10, 15, 19). Abnormalities of the ASL may induce bronchoconstriction and the cough reflex and interfere with epithelial cell ionic homeostasis and airway defense mechanisms such as antimicrobial activity and bacterial clearance (10, 17, 23, 26). The determination of ASL composition has posed a considerable challenge because of its small volume (2–3 l of ASL per square centimeter of mucosal surface). Invasive sample methods utilizing filter paper and microcapillary tubes have yielded a wide range of ionic concentrations (1, 5, 9, 13, 14, 23, 28) and have been criticized because of potential perturbation of the airway surface and sampling of intracellular and interstitial fluids by capillary suction (3, 6, 19, 25). We recently developed a minimally invasive in situ approach to measure ASL volume, ionic composition, and osmolality (12). The ASL is stained with ion-sensitive fluorescent indicators and viewed with a microscope equipped with z-scanning confocal optics and ratio image detection. The ASL in airway cell cultures and in the in vivo mouse trachea was approximately isotonic and not dependent on cystic fibrosis transmembrane conductance regulator (CFTR) Cl⫺ channels. Initial measurements of ASL pH in living anesthetized mice showed a pH ⬃7 (12). On the basis of evidence that CFTR may transport HCO3⫺ directly (2, 11, 20, 24) and/or modify the activities of HCO3⫺ exchangers (4), it has been proposed that ASL pH may be abnormal in cystic fibrosis. There is evidence that ASL acidity stimulates neurogenic reflexes in asthma (26), and it has been postulated that lung damage in cystic fibrosis occurs because the airways are unable to effectively neutralize acid in repeated occurrences of subclinical gastric acid aspiration (7). The ASL is a unique compartment in terms of its low volume, exposure to an air interface, and contact with a large surface area of beating cilia. The ASL in vivo is exposed to air with time-varying CO2, O2, and moisture content during inspiration and expiration. Theoretically, the determinants of ASL pH include the composition of air at the tracheal mucosal surface and of blood at the serosal surface, the activities of membrane transporters at the epithelial cell apical and basolateral membranes, and the permeability properties of the paracellular pathway; there may also be neuroendocrine regulatory mechanisms. Given the limited information about the transporting properties Address for reprint requests and other correspondence: A. S. Verkman, 1246 Health Sciences East Tower, Cardiovascular Research Institute, Univ. of California, San Francisco, San Francisco, CA 941430521 (E-mail: [email protected]; http://www.ucsf.edu/verklab). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. cystic fibrosis; acidification; trachea; fluorescence microscopy THE AIRWAY SURFACE LIQUID C1504 0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society http://www.ajpcell.org Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 14, 2017 Jayaraman, Sujatha, Yuanlin Song, and A. S. Verkman. Airway surface liquid pH in well-differentiated airway epithelial cell cultures and mouse trachea. Am J Physiol Cell Physiol 281: C1504–C1511, 2001.—Airway surface liquid (ASL) pH has been proposed to be important in the pathophysiology of cystic fibrosis, asthma, and cough. Ratio image analysis was used to measure pH in the ASL after staining with the fluorescent pH indicator 2⬘,7⬘-bis(2-carboxyethyl)5(6)-carboxyfluorescein (BCECF)-dextran. ASL pH in bovine airway cell cultures grown at an air-liquid interface was 6.98 ⫾ 0.06 in the absence and 6.81 ⫾ 0.04 in the presence of HCO3⫺/CO2. Steady-state ASL pH changed in parallel to changes in bath pH and was acidified by Na⫹ or Cl⫺ replacement but was not affected by the inhibitors amiloride, glibenclamide, or 4,4⬘-dinitrostilbene-2,2⬘-disulfonic acid. In response to sudden acidification or alkalization of the ASL by ⬃0.4 pH units by HCl/NaOH, ASL pH recovered to its initial value at a rate of 0.035 pH units/min (⫺HCO3⫺) and 0.060 pH units/min (⫹HCO3⫺); the pH recovery rate was reduced by amiloride and H2DIDS. In anesthetized mice in which the trachea was surgically exposed for measurement of BCECFdextran fluorescence through the translucent tracheal wall, ASL pH was 7.14 ⫾ 0.01. ASL pH was sensitive to changes in blood pH created by metabolic (HCl or NaHCO3 infusion) or respiratory (hyperventilation, hypoventilation) mechanisms. ASL pH is thus primarily determined by basolateral fluid pH, and H⫹/OH⫺ transport between the ASL and basolateral fluid involves amiloride-sensitive Na⫹/H⫹ exchange and stilbene-sensitive Cl⫺/HCO3⫺ exchange. The rapid response of ASL pH to changes in systemic acid-base status may contribute to airway hypersensitivity in asthma and other airway diseases. AIRWAY SURFACE LIQUID PH METHODS Cell culture experiments. Well-differentiated cultures of bovine tracheal cells were grown on collagen-coated 12-mmdiameter Costar snapwell inserts with polycarbonate semipermeable membranes at an air-liquid interface at 37°C in a 5% CO2-95% air atmosphere (27). Culture medium was changed every 2–4 days. Cultures were generally used 25–30 days after plating, at which time the electrical resistance was ⬎300 ⍀cm2, and the transepithelial potential difference was ⬎20 mV. Cell inserts were mounted (cells facing upward) in a stainless steel perfusion chamber in which the undersurface of the insert was perfused as described previously (12). The perfusate bathed the cell basolateral surface. The cell mucosal surface containing the ASL faced upward. The chamber was maintained at 37°C using a PDMI-2 microincubator (Harvard Apparatus) positioned on the stage of an upright epifluorescence microscope and enclosed in a 100% humidified air-5% CO2 tent maintained at 37°C. For pH measurements, the ASL was stained with the dual-excitation wavelength pH indicator 2⬘,7⬘-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) conjugated to dextran (40 kDa, Molecular Probes), dispersed in a low boiling point perfluorocarbon (Fluorinert FC-72, boiling point 56°C, 3M Company). Measurement protocols. The pH sensitivity of BCECFdextran in the ASL was calibrated by incubating the cell culture inserts with high-K⫹ perfusates (120 mM KCl, 20 mM NaCl, 1 mM CaCl2, 1 mM MgSO4, and 20 mM HEPES) containing nigericin (10 M), valinomycin (10 M), carbonyl cyanide m-chlorophenylhydrazone (5 M), and forskolin (10 M). Perfusates were titrated to specified pH (6.5–8.0) and equilibrated with cells for 4 h to set ASL pH, a time at which pH equilibration was found to be complete. For experiments involving HCO3⫺-free conditions, the perfusate consisted of HEPES buffer (124 mM NaCl, 5.8 mM KCl, 10 mM glucose, 1 mM CaCl2, 1 mM MgCl2, and 20 mM HEPES, pH 7.4), and the apical cell surface was exposed to a 100% humidified air atmosphere. For experiments in the presence of HCO3⫺, the perfusate consisted of 120 mM NaCl, 25 mM NaHCO3, 3.3 AJP-Cell Physiol • VOL mM KH2PO4, 0.8 mM K2HPO4, 1.2 mM MgCl2, 1.2 mM CaCl2, and 10 mM glucose, pH 7.4, and the apical surface was exposed to a humidified 5% CO2-95% air atmosphere. In some experiments, perfusate pH was adjusted to 6.0–8.0 by titration with HCl/NaOH (for HCO3⫺-free buffer) or NaH2PO4/Na2HPO4 (for HCO3⫺-containing buffer). For ion substitution experiments, perfusate Na⫹ was replaced by choline⫹ or Cl⫺ by gluconate⫺. In experiments involving recovery from acute ASL acidification or alkalization, 20–50 l of a perfluorocarbon suspension of HCl/NaOH (prepared by brief sonication) was added onto the ASL to change pH by 0.4–0.5 pH units. In some experiments, transport inhibitors were added to the perfusate or to both the perfusate and the ASL as described in RESULTS. Measurements in mouse trachea in vivo. Mice (25–35 g body wt) were anesthetized with ketamine (60 mg/kg body wt) and xylazine (8 mg/kg) 15 min after pretreatment with atropine (1 mg/kg intraperitoneal) to prevent secretions as discussed previously (12). A midline incision was made in the neck to expose the trachea for measurement of fluorescence through the translucent tracheal wall. Unless otherwise indicated, the ASL was stained by instillation of 5 l of the BCECF-dextran suspension in perfluorocarbon using a microcatheter passed through a feeding needle that was introduced via the mouth. The mouse was positioned on the microscope stage for fluorescence measurements as described below. Arterial blood (0.2–0.3 ml) was sampled through a PE-10 catheter inserted into the carotid artery, and blood pH and PCO2 were measured using a blood gas analyzer (Ciba Corning Diagnostic). After completion of the measurements, mice were euthanized by an overdose of pentobarbital (150 mg/kg). Animal protocols were approved by the University of California San Francisco Committee on Animal Research. In some experiments, amiloride (10 mg/kg of 1 mM solution) was injected intraperitoneally 30 min before anesthesia, and 2.7 g of amiloride (dispersed in perfluorocarbon) was instilled into trachea together with BCECF-dextran. ASL pH was measured 10 min after the perfluorocarbon instillation. Mice were treated with glibenclamide by intraperitoneal injection of 0.3 ml of a 1 mM glibenclamide solution and intratracheal instillation of 4.9 g of glibenclamide in perfluorocarbon. To create acute metabolic acidosis or alkalosis, HCl (0.5 meq H⫹) or NaHCO3 (0.3 meq) were injected intraperitoneally. ASL pH was measured after 20 min. To create respiratory acidosis or alkalosis, the upper trachea of anesthetized, paralyzed (pancuronium, 1 mg/kg intraperitoneal) mice was cannulated with PE-90 tubing, and mice were mechanically ventilated with room air (tidal volume 8 ml/kg, respiratory rate 90 respirations/min) using a mouse constant volume ventilator (Harvard Apparatus). Acute hyperventilation was produced by increasing the respiratory rate to 140 respirations/min for 10 min before ASL pH measurements and arterial blood gas analysis. Acute hypoventilation/hypercarbia was produced by decreasing respiratory rate to 90 respirations/min and addition of 5% CO2 (95% O2, to prevent hypoxia) to the inspired gas. Fluorescence microscopy. The chamber containing the cultured cells or the mouse was positioned on the stage of a Leitz upright fluorescence microscope with a Technical Instruments coaxial-confocal attachment. Fluorescence was detected using a Nikon ⫻50 extra-long working distance air objective (numerical aperture 0.55, working distance 8 mm) for ratiometric measurement of pH at 440- and 490-nm excitation wavelengths and a 535-nm emission wavelength. Background fluorescence (unstained cells or trachea) was ⬍1% of total fluorescence. 281 • NOVEMBER 2001 • www.ajpcell.org Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 14, 2017 of the airway epithelium and the complexity of the system, it is difficult to predict a priori the value and principal determinants of ASL pH and whether ASL pH is tightly regulated. The purpose of this study was to define the principal determinants of ASL pH. Measurements were done on well-differentiated primary cultures of bovine airway epithelial cells grown at an air-liquid interface and in the in vivo mouse trachea. The bovine airway cell culture model was chosen as a well-established system for which there exists a considerable body of data on ion transport mechanisms, including an analysis of intracellular pH regulation (21). The polarized cell culture system permitted measurements of ASL pH in response to transporter agonists/inhibitors, ion substitution, and imposed pH gradients. The mouse trachea was studied as an in vivo model that permitted the testing of transporter agonists/inhibitors as well as clinically important systemic acid-base disturbances. The data reported here establish empirically the major determinants of ASL pH and the transporting systems involved in transepithelial pH equilibration. An important unanticipated finding was the lack of a strict regulatory mechanism maintaining absolute ASL pH. C1505 C1506 AIRWAY SURFACE LIQUID PH RESULTS Fig. 1. Airway surface liquid (ASL) pH measurement in bovine tracheal epithelial cells using 2⬘,7⬘-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-dextran. A: calibration of the dependence of BCECF-dextran fluorescence excitation ratio (F490/F440) as a function of pH in solution (E) and ASL (‚) (left). Also shown is pH measured in the ASL in the absence (䊐) and presence (■) of CO2/HCO3⫺ (SE, n ⫽ 10–14 cultures). Right: pH measured in individual cultures. Each point represents the mean ⫾ SE of 4–8 measurements in different locations in each culture. B: ratio image analysis of ASL pH. Images are shown at 490-nm (left) and 440-nm (middle) excitation wavelengths along with pseudocolored ratio image (right). AJP-Cell Physiol • VOL 281 • NOVEMBER 2001 • www.ajpcell.org Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 14, 2017 Cell culture experiments. The ASL of tracheal cells cultured at an air-liquid interface was stained with the pH indicator BCECF-dextran by addition of microliter quantities of a low boiling point perfluorocarbon containing the dispersed indicator. The perfluorocarbon evaporated within a few seconds, permitting the BCECF microparticles to dissolve rapidly in the ASL. The cells on the porous support were mounted in a 37°C perfusion chamber in which the basolateral surface was perfused, and the apical surface was exposed to a 100% humidified atmosphere. Figure 1A shows an in situ calibration of the ratio of BCECF:fluorescence at 490- and 440-nm excitation wavelengths (F490/F440). ASL pH was set using perfusates containing high K⫹ and ionophores. The pKa of BCECF-dextran in the ASL was 7.05, not different from that in saline. Figure 1A also shows the averaged results from a series of measurements done in cells in the absence and presence of CO2/HCO3⫺ (in both perfusate- and atmosphere- bathing apical surface); the individual data (right) represent results obtained from different cultures with standard errors determined from measurements done at 4–8 different locations. Averaged ASL pH was 6.98 ⫾ 0.06 in the absence and 6.81 ⫾ 0.04 in the presence of CO2/HCO3⫺ (P ⬍ 0.01), lower than the perfusate pH of 7.40. Figure 1B shows images of the ASL surface recorded at excitation wavelengths of 490 nm (left) and 440 nm (middle) together with a computed ratio image (right) showing excellent pH uniformity throughout the ASL. The sensitivity of ASL pH to changes in pH of the basolateral surface perfusate was measured in the absence and presence of CO2/HCO3⫺. Figure 2A shows the ASL pH at 2 h after changing perfusate pH from 7.4 to 6.0 or 8.0. ASL pH changes paralleled changes in perfusate pH. The pH changes were reversible, and cells remained viable after incubation in the acidic and alkaline media as shown by transepithelial resistance (400–800 ⍀cm2) and trypan blue dye exclusion. Figure C1507 AIRWAY SURFACE LIQUID PH 2B shows the kinetics of ASL pH following changes in perfusate pH in the absence and presence of CO2/ HCO3⫺. The initial rates of pH change (in pH units/min) were ⫺0.030 ⫾ 0.003 (perfusate pH 6.0) and 0.023 ⫾ 0.005 (pH 8.0) in the absence of CO2/HCO3⫺ and ⫺0.027 ⫾ 0.003 (pH 6.0) and 0.025 ⫾ 0.007 (pH 8.0) in the presence of CO2/HCO3⫺. CO2/HCO3⫺ did not significantly affect the initial rate of pH change. The time course of ASL pH recovery was measured in response to sudden acidification or alkalization of the ASL by 0.3–0.5 pH units by addition of perfluorocarbon containing dispersed HCl or NaOH. Perfusate pH was maintained at 7.4. Measurements were done in the absence (Fig. 3A) and presence (Fig. 3B) of CO2/ HCO3⫺. The average ASL buffer capacity, computed from the decreased pH produced by addition of known molar quantities of H⫹, was 14 ⫾ 3 mM H⫹/pH unit, much less than that of cytoplasm (generally ⬎50 mM H⫹/pH unit) because of the relatively low protein content in ASL. ASL pH returned to its initial values over 5–10 min, with substantially more rapid pH equilibration in the presence of CO2/HCO3⫺, as summarized in Fig. 3C. The approximate twofold increase in the rate of pH recovery in the presence of HCO3⫺ suggests the involvement of an HCO3⫺-dependent pathway in ASL pH equilibration. The contribution of Na⫹-dependent transport to ASL pH regulation was studied using inhibitors and Na⫹ substitution. The inhibitors (1 mM each) amiloride (epithelial Na⫹ channel and Na⫹/H⫹ exchanger), furosemide (Na⫹-K⫹-2Cl⫺ cotransporter), omeprazole (K⫹/H⫹ exchanger), or 4,4⬘-dinitrostilbene-2,2⬘-disulfonic acid (Na⫹/3HCO3⫺ cotransporter) were added to the perfusate. Amiloride and omeprazole (dispersed in perAJP-Cell Physiol • VOL fluorocarbon) were also added to the ASL. Figure 4A shows ASL pH measured at 2 h after inhibitor addition. There was no significant effect of these compounds on steady-state ASL pH. Also shown is the acidified ASL following incubation of cells for 2 h with Na⫹-free medium (choline⫹ replacing Na⫹). Kinetic studies were done to identify functionally the Na⫹ transporter(s) involved in restoring ASL pH in response to a transepithelial pH gradient. Figure 4B shows the time course of ASL pH in response to rapid ASL acidification by HCl addition. Amiloride caused a remarkable inhibition of ASL alkalization in the absence of CO2/HCO3⫺, suggesting that H⫹/OH⫺ transport by Na⫹/H⫹ exchange is the principal mechanism of ASL pH equilibration in the absence of CO2/HCO3⫺. Figure 4C summarizes the initial rates of ASL alkalization in response to HCl addition in the absence or presence of CO2/HCO3⫺. There was significant inhibition of ASL alkalization by amiloride in the absence but not in the presence of HCO3⫺. Alkalization was also blocked by perfusate Na⫹ substitution. Similar experiments were done to investigate the role of Cl⫺ transporters in the regulation of ASL pH. The inhibitors included H2DIDS (Cl⫺/HCO3⫺ exchanger), glibenclamide, diphenylamine-2-carboxylate (DPC), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (Cl⫺ channel, CFTR), and acetazolamide (carbonic anhydrase), and the CFTR agonist forskolin was tested. All compounds were added to the perfusate, and the Cl⫺ transport inhibitors (dispersed in perfluorocarbon) were also added onto the ASL. Figure 5A shows that after a 2-h incubation, forskolin (20 M) and glibenclamide (500 M) had no significant effect on ASL pH, whereas NPPB (200 M), DPC (300 M), 281 • NOVEMBER 2001 • www.ajpcell.org Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 14, 2017 Fig. 2. Dependence of ASL pH on perfusate pH in tracheal cell cultures. A: ASL pH measured after incubating cultures for 2 h in perfusate at pH 6.0 and 8.0 in the absence (open bars) and presence (solid bars) of CO2/ HCO3⫺ (means ⫾ SE, n ⫽ 4–6 cultures). B: time course of ASL pH (means ⫾ SE, n ⫽ 4 cultures) in response to changes in perfusate pH to 8.0 (left) and 6.0 (right) in the presence (F) and absence (E) of CO2/HCO3⫺. C1508 AIRWAY SURFACE LIQUID PH H2DIDS (100 M), and acetazolamide (100 M) mildly acidified the ASL. Also shown is the acidified ASL following incubation of cells for 2 h with Cl⫺-free perfusate (gluconate⫺ replacing Cl⫺). Kinetic studies were done in which the time course of ASL alkalization was measured in response to addition of HCl to the ASL. Figure 5B shows the slowing of ASL alkalization in the presence of NPPB and H2DIDS. The averaged rates of Fig. 4. Role of Na⫹-dependent transport in ASL pH regulation. A: ASL pH (means ⫾ SE, 4–6 cultures) measured after 2-h incubations with furosemide (200 M), 4,4⬘-dinitrostilbene-2,2⬘-disulfonic acid (DNDS; 300 M), amiloride (1 mM), omeprazole (300 M), or after replacement of Na⫹ by choline⫹ (Na⫹-free buffer). *P ⬍ 0.02 compared with control. B: time course of ASL pH recovery after acidification of ASL by HCl in the presence of amiloride and absence (E) or presence (F) of CO2/HCO3⫺. C: averaged initial rates (means ⫾ SE) of ASL pH recovery for measurements done as in B for 4 sets of cultures. AJP-Cell Physiol • VOL 281 • NOVEMBER 2001 • www.ajpcell.org Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 14, 2017 Fig. 3. ASL pH regulation in response to sudden ASL acidification or alkalinization. Time course of ASL pH after alkalinization by direct addition of NaOH (top) or acidification by HCl (bottom) in the absence of CO2/ HCO3⫺ (A) and in the presence of CO2/HCO3⫺ (B). C: averaged initial rates (means ⫾ SE) of ASL pH recovery (shown as positive values) for measurements done as in A and B for 4 sets of cultures. *P ⬍ 0.02 comparing ⫾ CO2/HCO3⫺. C1509 AIRWAY SURFACE LIQUID PH ASL pH alkalization are summarized in Fig. 5C. The significant slowing of ASL pH recovery by H2DIDS suggests the involvement of Cl⫺-HCO3⫺ exchange. In vivo mouse trachea experiments. ASL pH was measured in mouse trachea after staining the tracheal lumen with BCECF-dextran. The perfluorocarbon suspension of BCECF-dextran was introduced into the trachea using a small feeding needle that was passed through the mouth and then promptly withdrawn to permit spontaneous breathing. Fluorescence was detected through the translucent tracheal wall after surgical exposure of the trachea by a midline neck incision. Figure 6 shows images of the trachea at excitation wavelengths of 440 nm (left) and 490 nm (middle) and the computed ratio image (right), showing a quite uniform pH distribution. The average ASL pH in anesthetized, spontaneously breathing mice was 7.14 ⫾ 0.01 (SE, n ⫽ 4 mice). Arterial blood gas analysis in these mice (room air) showed a PO2 of 107 ⫾ 23 mmHg, a PCO2 of 52 ⫾ 9, pH of 7.20 ⫾ 0.04, and a computed HCO3⫺ of 20 ⫾ 3 mM. ASL pH in mouse trachea was measured in response to the transport inhibitors amiloride and glibenclamide after introduction both systemically, by intraperitoneal injection, and directly onto the tracheal mucosa by instillation in a perfluorocarbon suspension. Figure 7 summarizes ASL pH along with arterial blood pH (A) and PCO2 (B). There was no significant effect of the inhibitors on ASL or blood pH. The effects of acute metabolic and respiratory acid-base disturbances were studied. As described in METHODS, metabolic acidosis and alkalosis were produced by intraperitoneal injection of HCl or NaHCO3; respiratory alkalosis and acidosis were produced in paralyzed, ventilated mice by hyperventilation or hypoventilation (with 5% CO2). Figure 7 shows that the maneuvers produced the predicted changes in blood pH and PCO2. Significant ASL acidification was produced by either HCl addition or Fig. 6. ASL pH in mouse trachea. After surgical exposure of the trachea in anesthetized mice, the tracheal mucosa was stained with BCECF-dextran and imaged as described in METHODS. Excitation ratio imaging of pH in mouse trachea. Fluorescence micrographs of BCECF-dextran stained ASL in mouse trachea (emission 535 nm) measured at 440-nm (left) and 490-nm (middle) excitation wavelengths and pseudocolored ratio image (right). See text for averaged pH values. Arrow points to unstained blood vessel. AJP-Cell Physiol • VOL 281 • NOVEMBER 2001 • www.ajpcell.org Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 14, 2017 Fig. 5. Role of Cl⫺-dependent transport in ASL pH regulation. Measurements were done in the presence of CO2/HCO3⫺. A: ASL pH (means ⫾ SE, 4–6 cultures) measured after 2-h incubations with acetazolamide (500 M), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 300 M), H2DIDS (100 M), diphenylamine-2-carboxylate (DPC; 500 M), glibenclamide (500 M), and forskolin (20 M), or after replacement of Cl⫺ by gluconate⫺ (Cl⫺-free buffer). B: time course of ASL pH recovery after ASL acidification by HCl in the presence of H2DIDS or NPPB (no inhibitor control from Fig. 3B shown for comparison). Control (no inhibitor) data (from Fig. 2B) is shown for comparison. C: averaged initial rates (means ⫾ SE) of ASL pH recovery for measurements done as in B for 4 sets of cultures. *P ⬍ 0.02 compared with control. C1510 AIRWAY SURFACE LIQUID PH hypoventilation/CO2 breathing. Although blood pH was increased comparably by NaHCO3 addition or hyperventilation/CO2, ASL pH was increased significantly only by acute respiratory alkalosis. DISCUSSION The purpose of this study was to characterize ASL pH using an established airway cell culture model and the in vivo mouse trachea. The complementary cell culture and in vivo systems were chosen to be able to test specific transporter agonists and inhibitors, perform ion substitution maneuvers, and examine the role of CO2/HCO3⫺, as well as to study the integrated physiological response of ASL pH to in vivo acid-base disturbances. The ratioable pH indicator BCECF-dextran was ideal for these measurements because of its excellent sensitivity to pH changes near pH 7.0 (7% change in fluorescence ratio for 0.1 unit pH change), permitting single measurements of ASL pH to better than 0.05 pH unit accuracy. We found that steady-state ASL pH is principally determined by serosal fluid/blood pH. Although specific Na⫹ and Cl⫺ transporters were found to be involved in the transient response to imposed pH gradients across the airway epithelium, ASL pH was not tightly regulated and thus subject to potentially large variations in response to changes in systemic acid-base status. Several lines of evidence have suggested a potentially important role for HCO3⫺ in the regulation of ASL pH. In human and bovine airway cell cultures, Smith and Welsh (24) reported that short-circuit current was HCO3⫺ dependent and inhibited by DPC and acetazolamide. In transfected fibroblasts, Poulsen et al. (20) AJP-Cell Physiol • VOL 281 • NOVEMBER 2001 • www.ajpcell.org Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 14, 2017 Fig. 7. Sensitivity of ASL pH to systemic acid-base status. Acute metabolic and respiratory acid-base disturbances were produced in anesthetized mice as described in METHODS. Averaged ASL pH is shown with arterial blood pH (A) and PCO2 (B; means ⫾ SE, n ⫽ 4–7 mice). *P ⬍ 0.02 compared with control mice. characterized Na⫹-independent, HCO3⫺-dependent pH regulation in cells expressing wild-type, but not ⌬F508, CFTR. These studies suggested that CFTR might be permeable to HCO3⫺. There is limited information on pH regulation in airway epithelial cells. In cultured human nasal epithelial cells, intracellular pH in the absence of HCO3⫺ was shown to involve amiloridesensitive Na⫹/H⫹ exchange (18). Poulsen and Machen (21) carried out a more extensive study of intracellular pH regulation in bovine tracheal epithelial cells. They found that in the absence of HCO3⫺, pH is regulated by the amiloride-sensitive Na⫹/H⫹ exchanger, but in the presence of HCO3⫺, the major pathway involved Na⫹and Cl⫺-independent, HCO3⫺-dependent transport, possibly the transport of HCO3⫺ by CFTR. They also reported evidence for an H2DIDS-inhibitable Cl⫺/ HCO3⫺ exchanger. Recently, Choi et al. (4) reported that CFTR mutants associated with pancreatic insufficiency do not support HCO3⫺ transport, suggesting a physiological role for CFTR-dependent HCO3⫺ transport. The cell culture studies here showed that although steady-state ASL pH is determined mainly by perfusate pH, transient responses to sudden changes in ASL or perfusate pH involved Na⫹ and Cl⫺ transporters. Ion substitution and inhibitor studies suggested the involvement of amiloride-sensitive Na⫹/H⫹ exchange and H2DIDS-sensitive Cl⫺/HCO3⫺ exchange. The recovery of ASL pH in response to sudden acidification of the ASL was strongly inhibited by amiloride or Na⫹ replacement in the absence of CO2/HCO3⫺, indicating that Na⫹/H⫹ exchange is the principal HCO3⫺-independent route for transepithelial H⫹/OH⫺ transport. In the presence of CO2/HCO3⫺, steady-state ASL pH was slightly increased (compared with no CO2/HCO3⫺), and the recovery from changes in ASL pH was accelerated approximately twofold in an H2DIDS-inhibitable manner. Steady-state ASL pH was mildly decreased by Cl⫺ replacement and by various Cl⫺ transport inhibitors. Activation of CFTR by forskolin did not affect ASL pH. Our results are best explained by Cl⫺/HCO3⫺ exchange as the principal route for transepithelial H⫹/OH⫺ transport in the presence of HCO3⫺, although a contribution of CFTR cannot be easily assessed because of the imperfect specificity of the Cl⫺ transport inhibitors and the complexity of the system. Assessment of the role of CFTR in HCO3⫺ will require comparative measurements in CFTR-expressing vs. cystic fibrosis cells as well as single cell/membrane experiments. ASL pH in the in vivo mouse trachea was measured using a minimally invasive procedure in which the trachea was exposed by a skin incision in the neck, and the ASL was stained with BCECF-dextran using a blunt feeding needle introduced via the mouth. Measurements were made without direct contact with the tracheal mucosa or invasion of the tracheal wall. We showed previously that measured ASL depth, salt content, and pH remained stable over time (12). Additionally, [Na⫹] and pH were not different as measured by direct dye addition through a tracheal window or by the less invasive procedure used here of dye addition AIRWAY SURFACE LIQUID PH This work was supported by National Institutes of Health Grants HL-60288, HL-59198, DK-35124, and DK-43840 and National Cystic Fibrosis Foundation Research and Development Grant R613. REFERENCES 1. Baconnais S, Tirouvanziam R, Zahm JM, Bentzmann S, Peault B, Balossier G, and Puchelle E. Ion composition and rheology of airway liquid from cystic fibrosis fetal tracheal xenografts. Am J Respir Cell Mol Biol 20: 605–611, 1999. 2. Ballard ST, Trout L, Bebok Z, Sorscher EJ, and Crews A. CFTR involvement in chloride, bicarbonate, and liquid secretion by airway submucosal glands. Am J Physiol Lung Cell Mol Physiol 277: L694–L699, 1999. 3. Boucher RC. Molecular insights into the physiology of the ‘thin film’ of airway surface liquid. J Physiol 516: 631–638, 1999. 4. Choi JY, Muallem D, Kiselyov K, Lee MG, Thomas PJ, and Muallem S. Aberrant CFTR-dependent HCO3⫺ transport in mutations associated with cystic fibrosis. Nature 410: 94–97, 2001. AJP-Cell Physiol • VOL 5. Cowley EA, Govindaraju K, Guilbault C, Radzioch D, and Eidelman DH. Airway surface liquid composition in mice. Am J Physiol Lung Cell Mol Physiol 278: L1213–L1220, 2000. 6. Erjefält I and Persson CG. On the use of absorbing discs to sample mucosal surface liquids. Clin Exp Allergy 20: 193–197, 1990. 7. Effros RM, Jacobs ER, Schapira RM, and Biller J. Response of the lung to aspiration. Am J Med 108, Suppl: 15s–19s, 2000. 8. Fine JM, Gordon T, Thompson JE, and Sheppard D. The role of titratable acidity in acid aerosol-induced bronchoconstriction. Am Rev Respir Dis 135: 826–830, 1987. 9. Govindaraju K, Cowley EA, Eidelman DH, and Lloyd DK. Microanalysis of lung airway surface fluid by capillary electrophoresis with conductivity detection. Anal Chem 69: 2793–2797, 1997. 10. Higenbottam T. The ionic composition of airway surface liquid and coughing. Bull Eur Physiopathol Respir 23, Suppl: 25s–27s, 1987. 11. Illek B, Yankaskas JR, and Machen TE. cAMP and genistein stimulate HCO3⫺ conductance through CFTR in human airway epithelium. Am J Physiol Lung Cell Mol Physiol 272: L752– L761, 1997. 12. Jayaraman S, Song Y, Vetrivel L, Shankar L, and Verkman AS. Noninvasive in vivo fluorescence measurement of airway-surface liquid depth, salt concentration, and pH. J Clin Invest 107: 317–324, 2001. 13. Joris L, Dab I, and Quinton PM. Elemental composition of human airway surface fluid in healthy and diseased airways. Am Rev Respir Dis 148: 1633–1637, 1993. 14. Knowles MR, Robinson JM, Wood RE, Pue CA, Mentz WM, Wager GC, Gatzy JT, and Boucher RC. Ion composition of airway surface liquid of patients with cystic fibrosis compared with normal and disease-control subjects. J Clin Invest 100: 2588–2595, 1997. 15. Kyle H, Ward JP, and Widdicombe JG. Control of pH of airway surface liquid of the ferret trachea in vitro. J Appl Physiol 68: 135–140, 1990. 16. Lowry RH, Wood AM, and Higenbottam TW. Effects of pH and osmolality on aerosol-induced cough in normal volunteers. Clin Sci (Lond) 74: 373–378, 1997. 17. Matsui H, Grubb BR, Tarran R, Randell SH, Gatzy JT, Davis CW, and Boucher RC. Evidence for periciliary liquid layer depletion, not abnormal ion composition in the pathogenesis of cystic fibrosis airway disease. Cell 95: 1005–1015, 1998. 18. Paradiso AM. Identification of Na⫹-H⫹ exchange in human normal and cystic fibrotic ciliated airway epithelium. Am J Physiol Lung Cell Mol Physiol 262: L757–L764, 1992. 19. Pilewski JM and Frizzell RA. Role of CFTR in airway disease. Physiol Rev 79: S215–S255, 1999. 20. Poulsen JH, Fischer H, Illek B, and Machen TE. Bicarbonate conductance and pH regulatory capability of cystic fibrosis transmembrane conductance regulator. Proc Natl Acad Sci USA 91: 5340–5344, 1994. 21. Poulsen JH and Machen TE. HCO3⫺-dependent pH regulation in tracheal epithelial cells. Pflügers Arch 432: 546–554, 1996. 22. Rao S and Verkman AS. Analysis of organ physiology in transgenic mice. Am J Physiol Cell Physiol 279: C1–C18, 2000. 23. Smith JJ, Travis SM, Greenberg EP, and Welsh MJ. Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid. Cell 85: 229–236, 1996. 24. Smith JJ and Welsh MJ. cAMP stimulated bicarbonate secretion across normal, but not cystic fibrosis, airway epithelia. J Clin Invest 89: 1148–1153, 1992. 25. Wine JJ. The genesis of cystic fibrosis lung disease. J Clin Invest 103: 309–312, 1999. 26. Wong CH, Matai R, and Morice AH. Cough induced by low pH. Respir Med 93: 58–61, 1999. 27. Wu DX, Lee CY, Uyekubo SN, Choi HK, Bastacky SJ, and Widdicombe JH. Regulation of the depth of surface liquid in bovine trachea. Am J Physiol Lung Cell Mol Physiol 274: L388– L395, 1998. 28. Zabner J, Smith JJ, Karp PH, Widdicombe JH, and Welsh MJ. Loss of CFTR chloride channels alters salt absorption by cystic fibrosis airway epithelia in vitro. Mol Cell 2: 397–403, 1998. 281 • NOVEMBER 2001 • www.ajpcell.org Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 14, 2017 through a feeding needle and measurement through the intact tracheal wall. ASL pH in mouse trachea was 7.14 when blood pH was 7.2. The mild systemic acidosis in control mice is probably related to the anesthesia, which was maintained at a minimal level using ketamine/xylazine. Mice are quite susceptible to hypoventilation during anesthesia (22), which probably accounts for the slightly lower ASL pH (6.9–7.0) in our preliminary measurements in mice anesthetized using pentobarbital (12). As summarized in Fig. 7, acute systemic acidbase disturbances produced substantial changes in ASL pH. Metabolic and respiratory acidosis resulted in decreased ASL pH. Mild metabolic alkalosis created by HCO3⫺ administration did not change ASL pH, whereas respiratory alkalosis (hyperventilation) producing comparable elevation in blood pH resulted in increased ASL pH. The principal conclusion from the mouse experiments is that ASL pH changes rapidly in response to systemic acid-base status. Because of the complex determinants of ASL pH in the in vivo model (e.g., changing PCO2 during inspiration/expiration, differential CO2 vs. HCO3⫺ permeabilities), it is difficult to establish more than an empirical relationship among changes in serum HCO3⫺, arterial blood PCO2, and ASL pH. The pH of ASL has been proposed to be important in the physiology of the cough reflex and airway reactivity. Wong et al. (26) reported that lowering airway pH by citric acid instillation into the human trachea induced cough, possibly by stimulation of stretch/irritant receptors (16). Our data in mouse trachea indicate that ASL pH is affected by serum pH and PCO2 and that ASL pH can change rapidly in response to changes in systemic acid-base status. These findings suggest that substantial changes in ASL pH are produced in clinically relevant situations such as lactic acidosis and acute changes in ventilation. Fine et al. (8) reported evidence of increased airway reactivity and bronchoconstriction after inhalation of buffered HCl or H2SO4. In severe asthma associated with CO2 retention, acute ASL acidosis might be an important exacerbating factor that causes further bronchoconstriction and impairment of ventilation. C1511