Functional and molecular characterization of an anion exchanger in airway serous epithelial cells

J. Loffing1, B. D. Moyer1, D. Reynolds1, B. E. Shmukler2, S. L. Alper2, and B. A. Stanton1

1 Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire 03755; and 2 Molecular Medicine and Renal Units, Beth Israel Deaconess Medical Center, and Departments of Medicine and Cell Biology, Harvard Medical School, Boston, Massachusetts 02215


    ABSTRACT
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INTRODUCTION
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Serous cells secrete Cl- and HCO3- and play an important role in airway function. Recent studies suggest that a Cl-/HCO3- anion exchanger (AE) may contribute to Cl- secretion by airway epithelial cells. However, the molecular identity, the cellular location, and the contribution of AEs to Cl- secretion in serous epithelial cells in tracheal submucosal glands are unknown. The goal of the present study was to determine the molecular identity, the cellular location, and the role of AEs in the function of serous epithelial cells. To this end, Calu-3 cells, a human airway cell line with a serous-cell phenotype, were studied by RT-PCR, immunoblot, and electrophysiological analysis to examine the role of AEs in Cl- secretion. In addition, the subcellular location of AE proteins was examined by immunofluorescence microscopy. Calu-3 cells expressed mRNA and protein for AE2 as determined by RT-PCR and Western blot analysis, respectively. Immunofluorescence microscopy identified AE2 in the basolateral membrane of Calu-3 cells in culture and rat tracheal serous cells in situ. In Cl-/HCO3-/Na+-containing media, the 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP)-stimulated short-circuit anion current (Isc) was reduced by basolateral but not by apical application of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (50 µM) and 4,4'-dinitrostilbene-2,2'-disulfonic acid [DNDS (500 µM)], inhibitors of AEs. In the absence of Na+ in the bath solutions, to eliminate the contributions of the Na+/HCO3- and Na+/K+/2Cl- cotransporters to Isc, CPT-cAMP stimulated a small DNDS-sensitive Isc. Taken together with previous studies, these observations suggest that a small component of cAMP-stimulated Isc across serous cells may be referable to Cl- secretion and that uptake of Cl- across the basolateral membrane may be mediated by AE2.

cystic fibrosis; anion exchanger 2; serous cell; submucosal gland; chloride transport; sodium bicarbonate cotransport


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INTRODUCTION
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AIRWAY EPITHELIAL CELLS REGULATE the volume and composition of airway surface fluid by mediating salt and water absorption and secretion (16, 32, 33). Pulmonary mucocillary clearance is dependent on the thickness of the airway surface fluid and on the ability of cilia to propel mucus out of the lungs. Adequate mucocillary clearance is essential for the removal of inhaled particulate matter from the airways and is vital for the prevention of airway tract infection and obstruction. Submucosal glands play a major role in the secretion of airway surface fluid (9, 21, 44). In particular, serous cells in human airway submucosal glands express high levels of cystic fibrosis transmembrane conductance regulator (CFTR), secrete Cl- and HCO3-, and fluid rich in antibiotic defensin and maganin peptides (8, 9, 15, 44). Moreover, dysregulation of CFTR-mediated Cl- secretion in serous cells is thought to contribute to the pathophysiology of cystic fibrosis lung disease (9, 45). However, the cellular mechanisms of fluid and electrolyte transport by human serous cells are incompletely understood. Recent studies on Calu-3 cells, a human airway cell line with a serous-cell phenotype, demonstrated that serous cells secrete Cl- and HCO3- by electrogenic processes (12, 17, 20, 24, 30, 36, 37). HCO3- secretion is a two-step process: uptake across the basolateral membrane is mediated by a Na+/HCO3- cotransporter (NBC), and exit from the cell is mediated by CFTR channels that are permeable to Cl- and HCO3- (12, 20, 24). Cl- secretion is also a two-step process: uptake across the basolateral membrane is mediated by a Na+/K+/2Cl- cotransporter (NKCC1), and exit from the cell is mediated by CFTR Cl- channels (12, 17, 20, 26, 27, 30, 37).

In many epithelia that secrete Cl-, the uptake of Cl- across the basolateral membrane is also mediated in part by Cl-/HCO3- exchange (1, 2, 13, 43). The anion exchanger (AE, i.e., Cl-/HCO3- exchanger) gene family includes three structurally and functionally related anion exchangers: AE1, AE2, and AE3 (1, 2). AE1 is expressed at highest levels in red blood cells and renal type A intercalated cells, AE2 is expressed broadly in epithelia, and AE3 is expressed in brain and heart, as well as in other excitable and some epithelial tissues. Recently, AE2 and bAE3 (brain AE3) mRNA were detected in human airways; however, the cell type and the subcellular location of AE2 and bAE3 in the airway is unknown (13). Accordingly, the aim of the present study was to test the hypothesis that AEs are expressed in submucosal glands, particularly in serous cells, and that AEs contribute to transepithelial Cl- secretion. To this end, we studied Calu-3 cells, a human airway cell line with a serous-cell phenotype (17, 20, 27, 30, 36, 37), and characterized the possible role of AEs in Cl- secretion by RT-PCR, Western blot analysis, and electrophysiological analysis. In addition, we examined the cellular location of AE proteins in Calu-3 and rat serous cells by immunofluoresence microscopy. We report that a small component of 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP)-stimulated short-circuit anion current (Isc) across Calu-3 cells may be referable to Cl- secretion and that Cl- uptake across the basolateral membrane may be mediated in part by AE2.


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Cell culture. Calu-3 cells were obtained at pass 17 from the American Type Culture Collection (HTB-55; Rockville, MD) and grown in air-interface culture (i.e., cell culture media added only to the basolateral side of cells grown on Millicell PCF filters) as described in detail previously (26, 27).

Measurement of Isc. Isc was measured by placing monolayers grown on Millicell polycarbonate (PCF) filters for 14-28 days in culture into an Ussing-type chamber (Jim's Instrument, Iowa City, IA) as described previously (26, 27). Bath solutions were maintained at 37°C and were stirred by bubbling with 5% CO2 air (CO2/HCO3--containing solutions) or room air (CO2/HCO3--free solutions).

In Ussing chamber studies, cells were bathed in either a control solution containing (in mM) 116 NaCl, 24 NaHCO3, 3 KCl, 2 MgCl2, 0.5 CaCl2, 3.6 NaHEPES and 4.4 HHEPES (pH 7.4) or a 0Na+ solution containing (in mM) 116 NMDGCl, 24 choline HCO3, 3 KCl, 2 MgCl2, and 0.5 CaCl2 (pH 7.4). These solutions were gassed with 5% CO2 balance air. In some studies, monolayers were bathed in a nominally Na+ and CO2/HCO3--free solution containing (in mM) 140 NMDGCl, 3 KCl, 2 MgCl2, 0.5 CaCl2, 3.6 NaHEPES, and 4.4 HHEPES (pH 7.4), gassed with room air, and including acetazolamide (100 µM) to inhibit the endogenous production of HCO3-.

Western blot analysis. Cell monolayers grown on Millicell filters and/or tissue culture flasks were solubilized in lysis buffer [50 mM Tris · HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, and containing Complete Protease Inhibitor cocktail (Boehringer Mannheim, Indianapolis, IN)] for 60 min at 4°C and spun at 14,000 g for 4 min to pellet insoluble material. Supernatants were separated on 4-20% Tris · HCl gradient gels (Bio-Rad) and transferred to polyvinylidene difluoride Immobilon membranes (Millipore, Bedford, MA). Membranes were blocked overnight at 4°C in 5% nonfat dry milk in Tris-buffered saline/0.02% Tween 20 and incubated with an affinity-purified polyclonal antibody (SA6) against the COOH-terminal 12 amino acids (1,224-1,237) of mouse AE2 (165 kDa). This antibody cross-reacts with another member of the AE anion exchanger family, AE1 (100-115 kDa), but does not recognize AE3 (at a 1:10,000 dilution) (3, 5, 6). After incubation with the primary antibody, blots were incubated with an anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:5,000; Amersham, Arlington Heights, IL). Blots were developed by enhanced chemiluminescence using Hyperfilm ECL (Amersham). Other primary antibodies used to detect AEs included affinity-purified polyclonal antibodies against the COOH terminus of AE3 (SA8) and the cytoplasmic terminus of cardiac AE3 (cAE3; antibody no. SA17) (7).

Laser scanning confocal fluorescence microscopy. Calu-3 cells were grown on Millicell PCF filters and were fixed and processed for laser scanning confocal fluorescence microscopy as previously described (26, 27). Briefly, cells were fixed with 3% paraformaldehyde for 15 min at room temperature and subsequently embedded into cryoembedding compound (Microm, Walldorf, Germany). Rat tracheas were harvested from four adult male Wistar rats (BRL, Füllinsdorf, Switzerland) that weighed ~180 g. The rats were anaesthetized with 100 mg/kg body wt of thiopental (Pentothal; Abbott, Cham, Switzerland), and the tracheas were fixed by intravascular perfusion through the abdominal aorta. The fixative solution consisted of 3% paraformaldehyde and 0.05% picric acid in a 6:4 mixture of 0.1 M cacodylate buffer (pH 7.4, adjusted to 300 mosmol/kgH2O with sucrose) and 10% hydroxyethyl starch in saline (HAES steril; Fresenius, Stans, Switzerland). After 5 min of fixation, the fixative was washed out for 5 min with the cacodylate-buffered solution. Tracheas were removed, cut into ~2-mm-thick slices horizontal to the long axis, and embedded into cryoembedding compound. Embedded Calu-3 cells and tracheas were frozen in liquid propane and stored at -80°C. Six-micrometer-thick sections of frozen Calu-3 cells and rat tracheas were cut in a cryostat and placed on chrome alum gelatin-coated glass slides. After SDS-antigen retrieval [i.e., preincubation of the sections with 1% SDS for 10 min (11)], nonspecific binding sites were blocked with 10% normal goat serum (DAKO, Glostrup, Denmark) in PBS. Subsequently, sections were incubated with 1:500-1:1,000 dilutions of polyclonal AE antibodies (described above) in PBS with 0.5% BSA (PBS-BSA) for 1 h at room temperature. After repeated washings with PBS, binding sites of the secondary antibody were revealed with a FITC-conjugated swine-anti-rabbit IgG (DAKO) diluted 1:40 in PBS-BSA. To identify cell nuclei, nucleic acids were stained with propidium iodide (2.5 µg/ml). Sections were mounted in DAKO-Glycergel (DAKO) that contained 2.5% 1,4-diazabicyclo[2.2.2]octane to retard fading. Fluorescent images were acquired using a Zeiss Axioskop microscope (Thornwood, NY) equipped with a laser scanning confocal unit (model MRC-1024; Bio-Rad Labs, Hercules, CA), a 15-mW krypton-argon laser, and a ×63 PlanApochromat/1.4 numerical aperture (NA) or ×40 PlanNeofluor/1.3 NA oil-immersion objective. FITC fluorescence was excited using the 488-nm laser line and collected using a standard FITC filter set (530 ± 30 nm). Propidium iodide fluorescence was excited using the 568-nm laser line and collected using a standard Texas Red filter set (605 ± 32 nm). Acquired images were imported into Adobe Photoshop version 3.0 for image processing and printing.

RT-PCR. mRNA was prepared from cells grown to confluency on Millicell PCF filters using the FastTrack 2.0 kit (Invitrogen, Carlsbad, CA). Reverse transcription was performed with the First Strand DNA synthesis kit from Ambion. Each synthesis was loaded with 40 ng mRNA (estimated to correspond to 1 µg total RNA), and one-twentieth aliquots of the RT reaction were used in PCR reactions of 50 µl final volume. PCR was performed by the hot start procedure using Taq DNA polymerase (Promega) in the supplier's recommended buffer.

PCR mixes lacking only primers were preheated at 82°C for 1 min, and then gene-specific primers (Table 1) were injected into the mix through oil. The complete reaction mixes were denatured for 3 min at 95°C and then cycled through these conditions: denaturation for 45 s at 94°C, annealing for 2 min at 60°C, and elongation for 2 min at 72°C. Final extension of 10 min at 72°C was terminated by rapid cooling to 4°C after 35 cycles. PCR products were separated and analyzed in 1% agarose gels and sequenced to confirm their identities as specific AE cDNA fragments (ABI 373; Applied Biosystems, Foster City, CA).

                              
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Table 1.   Oligonucleotide sequences of RT-PCR primers to detect AE mRNA in Calu-3 cells

Statistical analysis. Differences between means were compared by either unpaired Student's t-tests or by analysis of variance followed by Bonferroni multiple-comparisons test as appropriate. All analyses were performed with the InStat statistical software package (Graphpad, San Diego, CA). Data are expressed as the means ± SE. P < 0.05 was considered significant.


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Molecular identification of AE isoforms in Calu-3 cells. By RT-PCR, we determined that Calu-3 cells express mRNA for AE2 and low levels of mRNA for bAE3. AE1 and cAE3 transcripts could not be detected in Calu-3 cells (Fig. 1). Negative controls for RT-PCR included omission of cDNA from the reaction and yielded no product (data not shown). Positive controls for AE1 and cAE3, including rat kidney and reticulocyte (AE1) and heart and placenta (cAE3), were positive (Fig. 1). Thus Calu-3 cells express mRNA for AE2 and bAE3.


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Fig. 1.   RT-PCR analysis of mRNA in Calu-3 cells compared with positive control human tissues including kidney and reticulocytes (Retic). Calu-3 cells expressed anion exchanger (AE2) mRNA and low levels of brain AE3 (bAE3) mRNA. AE1 and cardiac AE3 (cAE3) transcripts, however, were undetectable in Calu-3 cells.

Western blot analysis of AE isoforms in Calu-3 cells. Three independent preparations of Calu-3 cells were assessed by immunoblot for the presence of AE1, AE2, and AE3 polypeptides. As shown in Fig. 2, a 165-kDa polypeptide was detected by an antibody to the COOH terminus of AE2 (SA6). The 165-kDa protein corresponding to AE2 was not detected when antibody incubations were conducted in the presence of the immunizing peptide antigen. As expected, on the basis of our RT-PCR studies, a polypeptide of the appropriate molecular mass (100-115 kDa) could not be detected with a mouse monoclonal antibody to rat AE1 (data not shown). Although mRNA for bAE3 was identified, the expression of AE3 polypeptide could not be detected by immunoblot analysis using two different polyclonal antibodies (data not shown). Thus either Calu-3 cells do not express AE3 polypeptide, or the level of AE3 expression was below the sensitivity of our immunoblot analysis. Taken together, our RT-PCR and Western blot studies demonstrate that Calu-3 cells express AE2 mRNA and AE2 polypeptide.


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Fig. 2.   Representative Western blots using anti-AE2 polyclonal antibody (SA6: 1,000 dilution). A single 165-kDa band was observed with SA6 but not when the immunizing peptide (1:500 dilution from a stock of 1.2 mg/ml) was included in the incubation buffer. AE proteins were not detected when the blots were probed with antibodies (Ab) that recognize AE3 (SA8 and SA17) or AE1 (negative blots not shown). Equal amounts of protein (150 µg/lane) were added to each well of the gel. See METHODS for details on the experimental protocol.

Immunolocalization of AE isoforms in Calu-3 and rat tracheal epithelia. Immunocytochemical localization of AE isoforms in Calu-3 cells and rat trachea was performed using the same anti-AE antibodies that were used for Western blot analysis. All Calu-3 cells and serous acinar cells of rat submucosal glands examined had detectable AE2 polypeptide that was localized to the basolateral plasma membrane (Fig. 3, E and F). Furthermore, AE2 was seen in the basolateral membrane of some ciliated surface epithelial cells but not in the epithelial cells lining the ducts of submucosal glands (Fig. 3, A-D). AE2 was not detected when antibody incubations were conducted in the presence of the immunizing peptide. Consistent with our RT-PCR and Western blot studies, AE1 polypeptide was not detected in Calu-3 cells or rat submucosal glands using a monoclonal antibody specific for AE1 (14). However, as a positive control, the AE1 antibody did stain intercalated cells in rat kidney as demonstrated previously (Ref. 3; results not shown). Thus Calu-3 and rat submucosal gland cells express AE2 polypeptide in the basolateral but not the apical plasma membrane.


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Fig. 3.   Indirect immunofluoresence localization of AE2 using the anti-AE2 antibody SA6 in rat trachea (A-E) and Calu-3 cells (F). AE2 is shown in green, and cell nuclei, stained with propidium iodide, are shown in red. A: overview of the mucosa and submucosa of rat trachea. The asterisk identifies the lumen of the trachea. AE2 is seen in the acini (a) of submucosal glands and in single cells of the tracheal surface epithelium. Other cells in the surface epithelium and epithelial cells lining the ducts of submucosal glands (d) are negative for AE2. The bright fluorescent discs in the submucosa are erythrocytes stained with the AE2 antibody due to its cross-reactivity with erythrocyte AE1. The punctate fluorescence dots below the surface epithelium represent nonspecific staining of elastic fibers that are highly abundant in the tracheas of rodents. B-D: higher magnifications of a submucosal gland. B: AE2 is seen in the serous cells of the acinus (a) but not in the mucous cells (arrows) in an adjacent mucous tubule. AE2 is shown in green. C: same image as in B, however, cell nuclei, stained with propidium iodide, are shown in red. D: merge of B and C demonstrating that AE2 is only expressed in serous cells. E: high magnification of an acinus. AE2 is localized in the basolateral plasma membrane (arrowhead) of the acinar cells; L is the lumen of the acinus. F: high magnification of Calu-3 cells; AE2 is localized in the basolateral plasma membrane (arrowhead). Scale bar in A is 20 µm. Scale bars in B and E are 10 µm.

Electrophysiological studies: an AE in the apical membrane does not contribute to Isc in Calu-3 cells. To determine if AEs contribute to anion secretion by Calu-3 cells, monolayers were mounted in an Ussing chamber, and the CPT-cAMP-stimulated Isc was measured. In Calu-3 cells, Isc is referable to electrogenic Cl- and/or HCO3- secretion (12, 17, 20, 26, 27, 30, 36, 37). Our immunolocalization studies suggest that AE2 is expressed in the basolateral but not the apical membrane; however, it is possible that an AE is present in the apical membrane, but it cannot be detected by immunofluorescence microscopy. For example, a functionally defined AE has been detected in the apical membrane of beta -intercalated cells in the renal cortical collecting duct, but this AE cannot be detected with antibodies to AE1 or AE2 (3, 6, 40). Thus to explore the possibility that functional AEs are present in the apical membrane, we determined whether the anion exchange inhibitors 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), which inhibit Cl-/HCO3- exchange (1), and dibenzamidostilbene disulfonic acid (DBDS), which inhibits Cl-/OH- exchange (41), reduced Isc. Although AEs are electroneutral, inhibition of AE activity would be expected to alter Isc indirectly by changing the intracellular activities of Cl- and HCO3-, and thus the electrochemical driving force for Cl- and HCO3- exit across the apical plasma membrane. Neither DIDS, DNDS, nor DBDS altered Isc when added to the apical bath solution (Table 2). Thus we could not detect stilbene-sensitive AEs in the apical membrane of Calu-3 cells by indirect immunofluoresence microscopy or by electrophysiological techniques.

                              
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Table 2.   Apical addition of AE inhibitors DIDS, DNDS, and DBDS had no effect on CPT-cAMP-stimulated Isc

Electrophysiological studies: an AE in the basolateral membrane contributes to Isc in Calu-3 cells. Ussing chamber studies were conducted to provide functional evidence for an AE in the basolateral membrane of Calu-3 cells. We examined the ability of the AE inhibitors DIDS and DNDS to inhibit Isc, which is defined as an anion current attributed to Cl- and/or HCO3- secretion. CPT-cAMP-increased Isc from 24.5 ± 3.0 µA/cm2 to 69.0 ± 4.2 µA/cm2 (Figs. 4 and 5A). DIDS (50 µM) and DNDS (500 µM), when added separately to the basolateral bath solution, reduced CPT-cAMP-stimulated Isc (Figs. 4 and 5A). DIDS (50 µM) completely inhibited the CPT-cAMP-stimulated Isc (Fig. 4). DNDS (500 µM) also inhibited CPT-cAMP-stimulated Isc but, unlike DIDS, did not reduce Isc to basal levels (Figs. 4 and 5A). DIDS inhibits AE and the Na+/HCO3- cotransporter (i.e., NBC). The EC50 for DIDS inhibition of AE2 is 3-15 µM (14, 18), whereas DIDS inhibition of NBC requires significantly higher concentrations (i.e., 500 µM) (10, 35). Accordingly, the observation that 50 µM DIDS completely inhibited CPT-cAMP-stimulated Isc is consistent with the view that an AE in the basolateral membrane contributes to CPT-cAMP-stimulated Isc. However, we cannot formally exclude the possibility that DIDS also reduced Isc, in part, by inhibiting NBC in the basolateral membrane. Thus additional studies were conducted to determine whether a Na+-independent AE (i.e., Cl-/HCO3- exchanger) is present in the basolateral membrane of Calu-3 cells (12).


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Fig. 4.   Effects of AE inhibitors 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) on 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP)-stimulated short-circuit anion current (Isc; y-axis in µA/cm2). Basal represents the Isc before addition of CPT-cAMP (100 µM) to the apical and basolateral solutions. DIDS (50 µM) and DNDS (500 µM) were added to the basolateral solution after CPT-cAMP treatment. Higher concentrations of DNDS (1 and 3 mM) had no additional inhibitory effect on CPT-cAMP-stimulated Isc [69.0 ± 4.2 µA/cm2 (no DNDS), 49.9 ± 2.1 µA/cm2 (500 µM DNDS), 48.5 ± 2.8 µA/cm2 (1 mM DNDS), and 49.5 ± 6.0 µA/cm2 (3 mM DNDS); n = 6 monolayers per group]. *P < 0.05 vs. CPT-cAMP alone. **P < 0.05 vs. basal. As demonstrated previously (26, 27), the CPT-cAMP-stimulated Isc reached a peak value after ~2 min.



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Fig. 5.   Representative Isc records illustrating the effect of Na+ removal on basal and CPT-cAMP-stimulated Isc. CPT-cAMP (100 µM) was added to both apical and basolateral bathing solutions at the time indicated by the arrows. DNDS (500 µM) was added to the basolateral bathing solution at the time indicated by the arrows. A: monolayer was bathed in Cl-/HCO3-/Na+-containing media. B: monolayer was bathed in a Na+-free medium to inhibit Na+/HCO3- cotransporter (NBC) and Na+/K+/2Cl- cotransporter activity. Note the difference in the Isc scale between A and B.

To provide additional support for the view that AE2 in the basolateral membrane contributes to CPT-cAMP-stimulated Isc, we removed Na+ from the apical and basolateral bath solutions to eliminate the possible contribution of NBC and NKCC1 to Isc. In the absence of Na+ in the bath solutions, CPT-cAMP increased Isc from 3.1 ± 0.6 µA/cm2 to 10.6 ± 1.2 µA/cm2 (P < 0.01; n = 4 monolayers per group; Fig. 5B). DNDS (500 µM), an AE inhibitor, completely blocked the CPT-cAMP-stimulated increase in Isc [Isc was 3.1 ± 0.6 µA/cm2 before addition of CPT-cAMP and 2.6 ± 0.6 µA/cm2 (P < 0.01) after addition of CPT-cAMP; n = 4 monolayers per group; Fig. 5B]. If the CPT-cAMP-stimulated Isc is mediated by AE2 located in the basolateral membrane, removal of HCO3- from the 0Na+ bath solutions should eliminate the contribution of AE2 to the CPT-cAMP-stimulated Isc. When monolayers were bathed in Na+- and HCO3--free solutions, CPT-cAMP failed to increase Isc (Isc was 2.3 ± 0.4 µA/cm2 before CPT-cAMP and 3.7 ± 0.8 µA/cm2 after CPT-cAMP; n = 4 monolayers per group). Taken together, these observations support the view that a small component of CPT-cAMP-stimulated Isc across Calu-3 cells may be referable to Cl- secretion and that Cl- uptake across the basolateral membrane may be mediated in part by AE2.


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The major new findings in this report are that Calu-3 and serous cells in rat submucosal glands express AE2 in the basolateral membrane and that AE2 may contribute to electrogenic anion secretion across Calu-3 cells.

AE2 is expressed in the basolateral membrane of Calu-3 and serous cells in rat submucosal glands. Five lines of evidence in this report support the view that AE2 is expressed in Calu-3 cells: 1) abundant AE2 mRNA was detected by RT-PCR; 2) AE2 polypeptide was detected by Western blot analysis; 3) AE2 was localized to the basolateral membrane by indirect immunofluoresence; 4) in the absence of Na+, to eliminate the contribution of the NBC and NKCC1 to Isc, CPT-cAMP stimulated DNDS-sensitive Isc, and 5) DIDS, at a concentration well above the EC50 for AE2 (i.e., 4 µM) (14, 18, 19) and AE3 (23), inhibited the CPT-cAMP-stimulated Isc. In addition, AE2 was identified in the basolateral membrane of rat serous cells in airway submucosal glands. Thus AE2 was identified in the basolateral membrane of rat serous cells in vivo and in Calu-3 cells, a human airway cell line with a serous-cell phenotype (12, 17, 20, 30, 36, 37).

Neither pharmacological nor indirect immunofluoresence techniques identified AE2 in the apical membrane of Calu-3 or rat serous cells, in agreement with a recent study (37). Moreover, we did not find any evidence for AE1 expression in Calu-3 or rat serous cells. cAE3 mRNA was not expressed in Calu-3 cells; however, low levels of bAE3 mRNA were detected by RT-PCR. These observations are in agreement with a recent study in which AE2 and bAE3 mRNA was detected in human airways; however, the cell type expressing these AEs and the subcellular location of AE polypeptide was not determined (13). Thus our results extend these observations by localizing AE2 to the basolateral membrane of serous cells in the submucosal gland. Although mRNA for bAE3 was identified in Calu-3 cells, the expression of AE3 polypeptide could not be detected by immunoblot analysis using two different polyclonal antibodies. Thus Calu-3 cells may not express AE3 polypeptide. Alternatively, the level of AE3 polypeptide expression in the Calu-3 cell line may be below the sensitivity of our immunoblot analysis.

AE2 has been localized to the basolateral membrane, but not the apical membrane, of numerous polarized epithelial cells including epididymis, stomach, small and large intestines, salivary glands, cochlea, vestibular apparatus, and alveolar type II cells (2, 5, 6, 11, 31, 39, 40, 47). bAE3 has been identified in the basolateral membranes of renal tubular cells (7) and has been observed on both basolateral and apical membranes of ileal and jejunal enterocytes (Stuart-Tilley, Wilhelm, and Alper, unpublished observations). cAE3 is expressed in the apical and basolateral membranes of renal epithelial cells, gut, and choroid plexus epithelia (Stuart-Tilley, Wilhelm, and Alper, unpublished observations). Thus although there is precedent for AE expression in apical membranes, our studies did not support the presence of an AE in the apical membrane of Calu-3 cells or in rat serous cells in situ.

cAMP-stimulated Isc in Calu-3 cells. In a previous study on Calu-3 cells, it was deduced from ion substitution studies that forskolin-stimulated (i.e., cAMP-stimulated) Isc was referable to electrogenic HCO3- secretion driven by a DNDS-sensitive, Na+- and HCO3--dependent cotransporter (i.e., NBC) located in the basolateral membrane (12). Removal of HCO3- or Na+ from the bath solutions dramatically reduced the forskolin-stimulated Isc. Data in the present study confirm the conclusion that the majority of cAMP-stimulated Isc is referable to NBC-driven HCO3- secretion in Calu-3 cells (12). We observed that removal of Na+ from the bath solutions dramatically reduced the CPT-cAMP-stimulated increase in Isc from 44.5 to 7.5 µA/cm2. Our data are also consistent with the view that a small component of CPT-cAMP-stimulated Isc (~15% or 7.5 µA/cm2) is referable to Cl- secretion. In the absence of Na+ in the bath solutions, to eliminate the contribution of NBC and NKCC1 to Isc, CPT-cAMP elicited a small increase in the DNDS-sensitive Isc (Fig. 5B). This DNDS-sensitive current was dependent on HCO3-. These observations are most consistent with the view that a small component (~15%) of CPT-cAMP-stimulated Isc is mediated by Cl- secretion, at least in the absence of Na+. Several other laboratories have demonstrated that cAMP stimulates Cl36 efflux from Calu-3 cells and that the cAMP-stimulated increase in Isc is equivalent to the increase in Cl36 flux from the serosal to the mucosal solution across Calu-3 cells (22, 25, 36). Moreover, in a recent study, removal of Cl- from the bath solutions reduced forskolin-stimulated Isc across Calu-3 cells (from 66 to 46 µA/cm2), an observation consistent with the view that cAMP stimulates some Cl- secretion across Calu-3 cells (12). On the other hand, some laboratories report that cAMP does not stimulate Cl- secretion across Calu-3 cells (12, 24). Thus the effects of cAMP on Cl- secretion across Calu-3 cells are controversial.

Cell model of electrogenic anion secretion by serous cells. Figure 6 is a cellular model illustrating the cellular location of transport proteins identified in Calu-3 and serous cells that are relevant to Cl- and HCO3- transport. The location of these transport proteins can account for Cl- and HCO3- secretion across serous cells in vivo (8). CFTR Cl- channels are located in the apical membrane, and NKCC1, Na+-K+-ATPase, NBC, and K+ channels are expressed in the basolateral membrane (12, 17, 20, 26, 27, 30, 37). In the present study, we have demonstrated that AE2 is also expressed in the basolateral membrane. According to this model, cAMP-stimulated HCO3- secretion is a two-step process: uptake across the basolateral membrane is mediated by NBC, and CFTR channels in the apical membrane mediate HCO3- exit from the cell (12, 20, 24). Cl- secretion is also a two-step process: uptake across the basolateral membrane is mediated by NKCC1, and Cl- exit from the cell is mediated by CFTR Cl- channels (12, 17, 20, 26, 27, 30, 37). In addition, the data in this manuscript are consistent with the conclusion that a small fraction of the cAMP-stimulated increase in Isc may be referable to Cl- secretion. We propose that Cl- uptake across the basolateral membrane is mediated by AE2, and Cl- exits the cell through CFTR Cl- channels in the apical membrane. We cannot exclude the possibility that AE2 may also contribute to the regulation of intracellular pH. The relative importance of each transport pathway to electrogenic anion secretion is uncertain and may be dependent on the agonist stimulating the secretion. However, this model is consistent with observations in native tissue that cAMP stimulates Cl- and HCO3- secretion by submucosal glands (8, 38).


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Fig. 6.   Cell model of electrogenic Cl- and HCO3- secretion by serous cells. Cystic fibrosis transmembrane conductance regulator (CFTR) in the apical plasma membrane is conductive to Cl- and HCO3-. The relative rates of Cl- vs. HCO3- secretion depend on the conductance of the channel to Cl- and HCO3-, to the electrochemical gradient for each ion across the apical membrane, the rate of Cl- and HCO3- uptake across the basolateral membrane, and the agonist stimulating anion transport (12). Although the stochiometry of Na+ and HCO3- for NBC in Calu-3 cells has not been determined, it is likely to operate in a 1Na+/2HCO3- mode that would favor Na+/HCO3- entry into the cell across the basolateral membrane (12, 34). Given reasonable estimates of the Cl- and HCO3- gradients across the basolateral membrane, it can be predicted that AE2 mediates Cl- uptake into the cells and HCO3- efflux into the basolateral solution. We know of no physiological condition in which an AE mediates Cl- efflux and HCO3- influx. The role of AE2 is uncertain. AE2 may mediate some Cl- secretion and/or play a role in intracellular pH (pHi) regulation. NKCC1, Na+/K+/2Cl- cotransporter.


    ACKNOWLEDGEMENTS

We gratefully acknowledge Alice Givan and Ken Orndorff for assistance with confocal microscopy.


    FOOTNOTES

These studies were supported by the National Institutes of Health (NIH) Grant DK-45881 and the Cystic Fibrosis Foundation (to B. A. Stanton), by NIH Grants DK-43495 and DK-34854 (Harvard Digestive Diseases Center, to S. L. Alper), and by National Research Service Award HL-09853 (to B. E. Shmukler). Confocal microscopy was performed at Dartmouth Medical School in the Herbert C. Englert Cell Analysis Laboratory, which was established by a grant from the Fannie E. Rippel Foundation and is supported in part by the Core Grant of the Norris Cotton Cancer Center (CA-23108).

J. Loffing was supported by a fellowship from the Swiss National Science Foundation. B. D. Moyer was supported by a predoctoral fellowship from the Dolores Zohrab Liebmann Foundation.

Address for reprint requests and other correspondence: B. A. Stanton, Dept. of Physiology, 615 Remsen Bldg., Dartmouth Medical School, Hanover, NH 03755 (E-mail: Bruce.A.Stanton{at}Dartmouth.edu).

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.

Received 7 December 1999; accepted in final form 27 April 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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