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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
Departments of 1Medicine and Physiology, Cardiovascular Research Institute, and 2Department of Pediatrics, University of California, San Francisco, California
Submitted 27 July 2005 ; accepted in final form 28 September 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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cystic fibrosis transmembrane conductance regulator; airway; fluorescence microscopy; pH regulation
Defective functioning of submucosal glands in the airways is thought to contribute to the pathogenesis of airway disease in CF (40, 42), acting perhaps in synergy with abnormalities in airway surface epithelial function producing fluid hyperabsorption and ASL dehydration (30). CFTR is expressed in fluid-secreting serous epithelium in airway submucosal glands (10, 19), which make up the majority of surface area of epithelium lining glands (11, 28, 42), as well as in the ciliated epithelium near gland orifices (24). Studies in CF gland cell cultures (8, 22, 27), in CF human bronchi (20, 23), and in pig airways using CFTR inhibitors (38), support the conclusion that CFTR is an important determinant of gland fluid secretion. Recently, we reported reduced and hyperviscous gland fluid secretions in nasal biopsies from pediatric CF subjects having minimal lung disease (35), suggesting that defective submucosal gland function is a primary defect in CF. Because it is likely that submucosal gland fluid secretions are a major determinant of ASL composition and physical properties, we proposed that reduced and hyperviscous gland fluid secretions in CF may contribute to ASL dehydration and hyperviscosity in CF.
CFTR is able to transport both chloride and bicarbonate (33, 41), as evidenced, for example, in CFTR-dependent alkaline pancreatic fluid secretions (13, 34). It has thus been postulated that defective CFTR-facilitated bicarbonate transport in the airways might produce an abnormally acidic ASL, which could promote airway infection in CF by reducing mucociliary clearance and antimicrobial action, and increasing the viscosity of pH-sensitive mucous glycoproteins (1, 6, 14, 30). We found previously that the thiazolidinone CFTR inhibitor CFTRinh-172 mildly reduced the pH of fluid secreted onto the pig tracheal surface by submucosal glands (38), although it was not possible to determine whether the hyperacidity was the consequence of abnormalities in airway surface vs. gland function.
There is evidence that ASL and/or gland fluid pH might be regulated by cell membrane ion transporters such as CFTR, anion and cation exchangers, and H+ pumps. In primary cell cultures of the airway surface epithelium, Coakley et al. (7) reported a relatively acidic ASL in CF cell cultures, with cAMP-induced alkalinization in normal but not CF cells. They found evidence for ouabain-sensitive H+-K+-ATPase activity that could acidify ASL pH; however, its activity was similar in normal and CF cells. Krouse et al. (25) also reported ouabain-sensitive H+-K+-ATPase activity in Calu-3 cells, a model of submucosal gland cells that secrete bicarbonate in response to forskolin. Fischer et al. (12) reported greater H+ secretion in human tracheal epithelium cells than in Calu-3 cells, and inhibition of H+ secretion by mucosal ZnCl2. In contrast, Inglis et al. (18) reported that luminal acidification in pig distal airways could be inhibited by bafilomycin A1, an inhibitor of the vacuolar-type H+ ATPase. Thus, although the evidence is somewhat conflicting, it appears that multiple H+/HCO3– transporters, including CFTR, are involved in establishing pH in primary gland fluid secretions and in maintaining/regulating airway surface fluid pH.
The main purpose of this study was to determine whether pH in secreted gland fluid is abnormal in CF, testing the hypothesis that reduced bicarbonate secretion by the glandular serous epithelium in CF produces hyperacidic gland fluid secretions. Comparing pH in secreted fluid from submucosal glands in nasal biopsies of CF vs. non-CF pediatric subjects with minimal lung disease, we found significant hyperacidity in secreted fluid from CF specimens. Studies in pig and human airways using CFTR inhibitors, microneedle gland fluid collections, and pH changes in fluid droplets deposited on the airway surface, supported the conclusion that gland fluid hyperacidity is produced by defective CFTR function in the submucosal glandular secretory epithelium.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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4 mm fragments of mucosa and underlying tissue from the anterior-inferior turbinate bilaterally. All biopsy sites were confirmed to be healed at a 3-wk postoperative follow-up visit. All procedures were approved by the University of California San Francisco Committee on Human Research, and informed consent was obtained.
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Measurements of gland fluid pH.
Gland fluid droplets under oil were imaged by light microscopy with side illumination using a Nikon SMZ1500 stereomicroscope. Gland fluid pH was measured by ratio imaging of the membrane-impermeant pH-sensitive fluorescence indicator BCECF-dextran (40 kDa, Molecular Probes), which has a pKa of 7.0. Gland fluid droplets of volume 150–200 nl were microinjected with 2–4 nl of a 10 mg/ml solution of BCECF-dextran in water using a glass microneedle and nanoinjector, resulting in a <2% dilution of gland fluid. BCECF fluorescence was measured at 525 nm with excitation wavelengths of 490 and 440 nm for computation of a pH-dependent ratio signal (F490/F440). Fluorescence images (500-ms acquisition per image) were acquired using a x1.6 objective lens (numerical aperture 0.21, working distance 24 mm) and cooled charge-coupled device camera (model CH250, Photometrics), with custom filter sets (Chroma) for BCECF. Calibration of F490/F440 vs. pH was done using HEPES-buffered saline titrated to different pH, in which 300-nl droplets containing BCECF-dextran were placed at the bottom of a petri dish covered with mineral oil. The F490/F440 vs. pH calibration was not significantly influenced by the mineral oil, fluid droplet size, droplet geometry, or solution composition.
Buffer capacity measurements.
Buffer capacity was measured in secreted gland fluid and in solutions that were deposited on airway surface. After pilocarpine stimulation, gland fluid droplets were collected with microcapillary tubing and a total of 10 µl fluid (under oil) was deposited in a BCECF-precoated petri dish. Microtitrations were done with small quantities of acid (HCl, 0.1 N) or base (NaOH, 1 N) that were added by microinjection, with pH measured by BCECF ratio imaging. Buffer capacity was computed from the acid/base added to change pH by 1 unit pH 7.
Pig airways.
Pig airways were obtained from a nearby farm (Pork Power, Fresno, CA). After the pig was euthanized, the trachea and large bronchi were transported to the laboratory in a plastic bag kept on ice. Generally, experiments were done within 6–12 h after death. We found that ciliary beating and gland fluid secretion were maintained for 24 h when the trachea was kept on ice before the experiments.
Human airways. Human tracheas were obtained from the Northern California Lung Transplantation Donation Network as lungs that were harvested but not used for transplantation. The tracheas were received in a plastic bag on ice containing organ preservative solution. Ciliary beating and gland secretion were checked to verify viability.
Measurement of gland fluid pH in microcannulated gland orifices in pig and human bronchi.
A capillary microcannulation method was developed to sample freshly secreted gland fluid that has not contacted with the airway surface. Capillary tubing (catalog no. 3-000-203-G/X; Drummond Scientific) was pulled to tip size 100 µm. The tip was broken by forceps to give a flat end, then dipped into an ethanol solution of BCECF acid (1 mg/ml, Molecular Probes), and dried at room temperature for 1 h. This procedure produced a uniform coating of the interior glass surface with BCECF near the pipette tip. A calibration curve of F490/F440 vs. pH was generated by 50-nl samples of fluid that were drawn up at different pH. Solutions of different buffer capacities and volumes were used to confirm that the calibration was not affected by the glass capillary.
As described previously, the bronchial mucosa was isolated and mounted on a silicone (Sylgard)-embedded petri dish with medium on the serosal side (35). The medium was either Krebs or bicarbonate-free Krebs buffer at pH 7.4, sometimes containing inhibitors composed of (in µM) 100 amiloride, 100 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), 20 CFTRinh-172, GlyH-101, or 200 DIDS. After the mucosa was bathed in the same solution at 37°C for 30 min, the mucosal surface was cleaned, dried with air, and covered with mineral oil. The silicone plate was then transferred to a 37°C open-perfusion microincubator (model PDMI-2, Harvard Apparatus) on the microscope stage. A Saran wrap enclosure was constructed around the sample and microincubator, in which the atmosphere was controlled by blowing humidified air (for nonbicarbonate studies) or humidified 5% CO2-95% O2 at 37°C. The medium used for tissue incubation was then replaced with fresh medium containing pilocarpine (50 µM) or forskolin (20 µM) to stimulate gland fluid secretion. Gland fluid droplets accumulating under the mineral oil were visualized in 10–15 min. The secreted droplet over a gland orifice was removed using capillary tubing. A BCECF-precoated glass microcapillary tube was then inserted by micromanipulator into the gland orifice at a nearly vertical orientation. Freshly secreted fluid was collected into the capillary tubing and was stained uniformly with BCECF. After 30–45 s, the microcapillary containing up to 30 nl of fluid was withdrawn and tip fluorescence was imaged for determination of F490/F440 for pH computation.
Kinetics of pH change in fluid droplets deposited onto the airway surface. The pig tracheal mucosa was isolated, mounted on a silicone-embedded petri dish, and bathed in Krebs or bicarbonate-free Krebs buffer (with or without inhibitors) for 30 min as described above. If the tissue was bathed in Krebs buffer, the droplets deposited on the surface were also from the Krebs buffer, in which pH was altered and/or test compounds were added, and the sample was surrounded by 5% CO2-95% O2. If the tissue was bathed in bicarbonate-free Krebs buffer, the deposited droplets were also from the bicarbonate-free solution and the sample was surrounded by air. The medium was then replaced with fresh solution, and the mucosal surface was dried with air and covered with water-saturated mineral oil. The tissue plate was then transferred to the 37°C microincubator with humidified tent blowing air or 95% O2-5% CO2. After 10 min, 300 nl of a solution containing BCECF-dextran (0.2 mg/ml) with or without forskolin (20 µM) and/or inhibitors composed of (in µM) 100 or 500 amiloride, 100 NPPB, 20 GlyH-101, 20 CFTRinh-172, or 200 DIDS at pH 6.4 or 7.6 was deposited onto the airway surface (see Table 2 for solution compositions). Controls were incubated with same amount of vehicle-DMSO. An area without gland secretion and far from gland openings was selected for droplets deposition. The characteristic morphology of the pig airway mucosa allowed accurate prediction of the location of gland orifices. If an inhibitor was used, the droplet deposited onto the airway surface also contained inhibitor. For bicarbonate-free solutions (in mM: 140 NaCl, 0.8 K2HPO4, 3.3 KH2PO4, 1.2 CaCl2, 1.2 MgCl2, 5 HEPES, and 10 glucose), the pH was adjusted to 6.4 with HCl, or 7.6 with NaOH. For bicarbonate-containing solutions, the solution was bubbled with 5% CO2-95% O2 and appropriate quantities of NaHCO3 were added to adjust the pH to 6.4 or 7.6 (see Table 2). Serial fluorescence images at 440 and 490 nm excitation wavelengths were obtained to follow pH changes. Background fluorescence, measured at both wavelengths from the average fluorescence in three regions around each droplet, was subtracted for computation of F490/F440. Data were fitted to monoexponential functions by nonlinear least-squares regression to compute initial rates of pH change. In some experiments, the involvement of H+ pumps was studied by inclusion of ATP (100 µM) and histamine (100 µM) in the droplets (to stimulate H+ secretion) with/without various inhibitors (500 µM ouabain, 100 nM bafilomycin A1, and 10 µM Sch28080).
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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F508 gene, except for one subject, whose genotype was G542X/unknown. The freshly obtained nasal biopsy specimens were oriented under a dissection microscope, immobilized in agarose with mucosa facing upward, as described in METHODS, and covered with oil to visualize individual gland fluid droplets. Generally, three or more well-demarcated droplets appeared by 10–15 min at 37°C. A very small volume (1–2 nl) of solution containing the fluorescent pH indicator BCECF-dextran (10 mg/ml) was microinjected into each droplet, which after a few minutes stained the droplet uniformly. Figure 1A shows representative light and fluorescence micrographs of stained fluid droplets at the mucosal surface of a nasal biopsy. The pseudocolored ratio image, computed from BCECF-fluorescence images obtained at 490 nm (pH-sensitive wavelength) and 440 nm (pH-insensitive wavelength), showed uniform pH throughout the droplet.
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Gland fluid hyperacidity in microcannulated bronchi treated with CFTR inhibitors. The limited availability and small size of nasal biopsy specimens precluded a more detailed characterization of gland fluid pH, such as measurements in response to cAMP vs. cholinergic agonists, and measurements of pH immediately after fluid exit from gland orifices. A potential concern in the interpretation of gland fluid hyperacidity in CF in terms of an intrinsic defect in the glandular secretory epithelium is possible modification of fluid pH by the airway surface epithelium where it makes contact for up to tens of minutes before pH measurements. To address this concern, we developed a microcannulation method to measure pH in fluid exiting gland orifices without allowing contact with airway surface epithelium. As described in METHODS, the approach involved identification of gland orifices from the location of fluid droplets, followed by cannulation of orifices (after removal of the surface droplet) with a glass microneedle, whose interior was precoated with BCECF. The BCECF promptly dissolved in the gland fluid for measurement of pH by ratio imaging.
Figure 2A diagrams the microcannulation method. The two panels at the right show the gland opening and gland orifice cannulation with a precoated capillary tip. Figure 2B shows light and fluorescence micrographs of microcapillary tips, which contained up to 30 nl of freshly secreted gland fluid. The pseudocolored ratio image shows uniform fluid pH. Figure 2C shows the calibration of F490/F440 vs. pH obtained using solution standards in the glass microcapillaries, which was used to compute pH from fluorescence images obtained at 490 and 440 nm excitation wavelengths. Figure 2D summarizes gland fluid pH after stimulation with pilocarpine. Whereas amiloride did not significantly affect gland fluid pH, the CFTR inhibitors NPPB, GlyH-101, and CFTRinh-172 produced a significantly acidic gland fluid. A limited data set for human bronchi is also included. Figure 2E summarizes gland fluid pH after stimulation with forskolin, again showing significantly lower pH with CFTR inhibitors. Gland fluid pH was slightly greater with forskolin than with pilocarpine stimulation. These results support the conclusion that the gland fluid hyperacidity seen in the nasal biopsy experiments is the consequence of altered function of the glandular secretory epithelium rather than modifications by contact with surface epithelium.
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10 min. H+/HCO3– transport mechanisms were studied from the effect of ion substitutions and transport inhibitors on the kinetics of pH equilibration. Figure 3A shows light and fluorescence micrographs of 300 nl fluid droplets deposited on the airway surface, along with a pseudocolored ratio image of pH. Generally, there was little change in the size or shape of fluid droplets over the 10-min course of the pH measurements (Fig. 3B). The first set of experiments was done under nominally bicarbonate-free conditions. Deposited fluid buffer capacity was measured to be 3.7 ± 0.3 mM/pH unit. Figure 3C shows the kinetics of pH after deposition of droplets at pH 6.4 or 7.6. Approximately exponential pH equilibration kinetics was observed in most experiments. Initial rates of pH change, deduced by exponential regression of the time-course data, are summarized in Table 3. After fluid was deposited at pH 6.4, droplet fluid pH equilibrated over several minutes, reaching in most cases, a steady-state pH of 6.9–7.0, except in the NPPB-, CFTRinh-172-, and GlyH-101-treated samples, in which the steady-state pH was lower. The CFTR inhibitors slowed to various extents the pH increase, yet they did not affect the pH decrease after deposition of fluid at pH 7.6. Amiloride did not affect the kinetics of pH equilibration, nor did Na+ replacement, providing evidence against Na+/H+ exchange. Also, there was no significant effect of forskolin.
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6.72 at 10 min. pH was slightly reduced when the droplets contained forskolin. However, as summarized in Fig. 5B, none of the H+ pump inhibitors caused significant inhibition of ATP/histamine-induced fluid acidification. ZnCl2 could not be studied because it was found to quench BCECF-dextran fluorescence.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Submucosal glands in the airways consist of a serous epithelium, where it is thought that the majority of salt, water, and antimicrobial peptides are secreted (2, 42). The serous secretions then pass through mucous tubules, where viscous glycoproteins are added, and then through a ciliated collecting duct onto the airway surface. We used a microcannulation approach to determine whether the reduced gland fluid pH measured in the CF nasal biopsies might be caused by modification of fluid pH by the surface epithelium in contact with freshly secreted fluid droplets. Direct microcannulation avoided contact of freshly secreted gland fluid with the airway surface epithelium. The pH of fluid collected with glass microneedles was measured by ratio imaging of BCECF, which was introduced into the fluid by precoating the interior tip surface of the microneedles. We found that various CFTR inhibitors, including the thiazolidinone CFTRinh-172 (29) and the glycine hydrazide GlyH-101 (31), reduced the pH of gland fluid secretions. These results provide evidence for CFTR as an important determinant of the pH of secreted gland fluid, and support the conclusion that the reduced gland fluid pH in CFTR deficiency is the consequence of an abnormality in the glandular secretory epithelium rather than modification of secreted fluid by the airway surface epithelium.
Several studies report reduced ASL pH compared with plasma pH. Kyle et al. (26) reported an ASL pH of 6.85 using pH microelectrodes in the in vitro ferret trachea. Microelectrode measurements in human airway cultures gave a pH of 6.9 (7). Using a pH-stat titration method, Fischer et al. (12) reported an ASL pH of 6.85 in primary cultured human airway epithelium. Our laboratory found that ASL pH in bovine airway cell cultures was 6.98 in the absence and 6.81 in the presence of HCO3–/CO2 using BCECF-dextran deposited onto the ASL (21). There are multiple determinants of ASL pH and ionic composition, including the activities of various transporters on the airway surface epithelium, electrochemical driving forces, and effects of gland fluid secretion and convective fluid removal. It is thus not possible to easily predict, for example, that the presence of CFTR on surface epithelium would produce a relatively acidic ASL pH in CF.
The mechanisms for generation of a mildly acidic ASL are not completely understood. In addition to CFTR-mediated HCO3– transport, involvement in ASL pH regulation has been proposed for gastric and nongastric forms of the H+-K+-ATPase, a vacuolar-type H+ pump, and Na+/H+ and Cl–/HCO3– exchangers (2, 6, 16). The expression pattern of these transporters may be species dependent (6, 12, 16). Coakley et al. (7) identified a nongastric form of the H+-K+-ATPase inhibitable by ouabain in human airway epithelium, whereas Fischer et al. (12) reported a Zn2+-sensitive H+ pump in the same system, and Inglis et al. (16) reported bafilomycin A1 inhibitable H+ secretion in pig distal airways. There is also evidence for the expression of Na+/H+ and possibly Cl–/HCO3– exchangers in the basolateral membrane in the human airway epithelium (2).
A series of experiments was done to test for functioning of these various transporters and pumps in the surface epithelium of pig trachea with regard to modifying the pH of surface fluid. Our approach involved measurements of the kinetics of pH equilibration or pH change in small fluid droplets deposited onto the airway surface. The droplets contained BCECF as the pH indicator, and various agonists, inhibitors, and ionic substitutions to test for specific transporters. Experiments were done with pH of the deposited fluid droplet of 6.4, 6.9, or 7.4. We did not detect functional Na+/H+ or Cl–/HCO3– exchange based on the lack of effect of Na+ and Cl– substitutions, and the inhibitors amiloride and DIDS. The CFTR inhibitors NPPB, GlyH-101, and CFTRinh-172 significantly slowed alkalization after depositing acidic fluid onto the airway surface, suggesting CFTR-mediated HCO3– secretion. The CFTR inhibitors also reduced fluid droplet pH after a 10 min equilibration. Interestingly, the impaired alkalinization with CFTR inhibitors was found both in the presence or absence of forskolin, suggesting significant basal CFTR activity for HCO3– transport. With regard to active H+ secretion, there was little change in pH when fluid at pH 6.89 was deposited onto the airway surface in the absence of agonists. The agonists ATP and histamine further acidified fluid droplets, possibly by one or more H+ pump mechanisms; however, none of the H+ pump inhibitors tested significantly blocked fluid droplet acidification.
We previously measured ASL pH in primary cultures of bovine tracheal epithelial cells in response to changes in pH in solutions bathing the basolateral surface, or additions of acid/alkali to the ASL (21). NPPB but not glibenclamide reduced the rate of ASL pH recovery in response to an ASL acid challenge, with neither compound affecting steady-state ASL pH (21). The current study shows reduced ASL pH recovery from an acid challenge in the presence of NPPB, CFTRinh-172, or GlyH-101, which supports the presence of functional CFTR in pig surface airway epithelium. However, as mentioned above, it is difficult to predict the consequences of surface epithelial CFTR expression on steady-state ASL pH.
There are several mechanisms by which reduced gland fluid and airway surface pH might contribute to the development and progression of lung disease in CF. There is evidence for pH-dependent changes in the rheological properties of secreted mucus, with low pH increasing hydrogen bond cross-linking between mucins (14, 15). The addition of acid to sputum to drop pH by one unit increased dynamic viscosity two to threefold (15). An acidic airway surface fluid could induce neutrophil activation by enhanced release of H2O2 and myeloperoxidase (39). Extracellular acidification also delays neutrophil apoptosis and prolongs their lifespan, promoting damage to the airway epithelium (41). There is also evidence that ASL acidity impairs mucociliary clearance and intrinsic antimicrobial function. A reduction in extracellular pH by 0.5 pH unit reduced mucociliary beat frequency by 22% in bronchi and 16% in bronchioles (5). Finally, an acidic extracellular fluid could promote bacterial colonization and invasion by inhibition of neutrophil and macrophage cell function (1, 3), as well as inhibition of 
-defensin-1 (32).
In summary, our data provide evidence for CFTR-mediated HCO3– transport in submucosal gland fluid secretion, and for an intrinsic acidification defect in gland fluid secretion in CF. These findings thus support the possibility of correction of gland fluid and airway surface hyperacidity as a new mechanism-based therapy for reducing lung disease in CF.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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  C. S. Rogers, W. M. Abraham, K. A. Brogden, J. F. Engelhardt, J. T. Fisher, P. B. McCray Jr., G. McLennan, D. K. Meyerholz, E. Namati, L. S. Ostedgaard, et al. The porcine lung as a potential model for cystic fibrosis Am J Physiol Lung Cell Mol Physiol, August 1, 2008; 295(2): L240 - L263. [Abstract] [Full Text] [PDF]
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 R. J. Lee, J. M. Harlow, M. P. Limberis, J. M. Wilson, and J. K. Foskett HCO3- Secretion by Murine Nasal Submucosal Gland Serous Acinar Cells during Ca2+-stimulated Fluid Secretion J. Gen. Physiol., July 1, 2008; 132(1): 161 - 183. [Abstract] [Full Text] [PDF]
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 K. L. Palmer, L. M. Aye, and M. Whiteley Nutritional Cues Control Pseudomonas aeruginosa Multicellular Behavior in Cystic Fibrosis Sputum J. Bacteriol., November 15, 2007; 189(22): 8079 - 8087. [Abstract] [Full Text] [PDF]
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 X. Liu, M. Luo, L. Zhang, W. Ding, Z. Yan, and J. F. Engelhardt Bioelectric Properties of Chloride Channels in Human, Pig, Ferret, and Mouse Airway Epithelia Am. J. Respir. Cell Mol. Biol., March 1, 2007; 36(3): 313 - 323. [Abstract] [Full Text] [PDF]
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J. V. Wu, M. E. Krouse, and J. J. Wine Acinar origin of CFTR-dependent airway submucosal gland fluid secretion Am J Physiol Lung Cell Mol Physiol, January 1, 2007; 292(1): L304 - L311. [Abstract] [Full Text] [PDF]
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