CFTR induces the expression of DRA along with Clminus /HCO3minus exchange activity in tracheal epithelial cells

Valerie J. Wheat1, Holli Shumaker1, Charles Burnham1, Gary E. Shull2, James R. Yankaskas3, and Manoocher Soleimani1

Departments of 1 Medicine and 2 Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati School of Medicine, Cincinnati, Ohio 45267; and 3 Department of Medicine, University of North Carolina, Chapel Hill North Carolina 27599


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Thickening of airway mucus and lung dysfunction in cystic fibrosis (CF) results, at least in part, from abnormal secretion of Cl- and HCO3- across the tracheal epithelium. The mechanism of the defect in HCO3- secretion is ill defined; however, a lack of apical Cl-/HCO3- exchange may exist in CF. To test this hypothesis, we examined the expression of Cl-/HCO3- exchangers in tracheal epithelial cells exhibiting physiological features prototypical of cystic fibrosis [CFT-1 cells, lacking a functional cystic fibrosis transmembrane conductance regulator (CFTR)] or normal trachea (CFT-1 cells transfected with functional wild-type CFTR, termed CFT-WT). Cells were grown on coverslips and were loaded with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and intracellular pH was monitored. Cl-/HCO3- exchange activity increased by ~300% in cells transfected with functional CFTR, with activities increasing from 0.034 pH/min in CFT-1 cells to 0.11 in CFT-WT cells (P < 0.001, n = 8). This activity was significantly inhibited by DIDS. The mRNA expression of the ubiquitous basolateral AE-2 Cl-/HCO3- exchanger remained unchanged. However, mRNA encoding DRA, recently shown to be a Cl-/HCO3- exchanger (Melvin JE, Park K, Richardson L, Schultheis PJ, and Shull GE. J Biol Chem 274: 22855-22861, 1999.) was abundantly expressed in cells expressing functional CFTR but not in cells that lacked CFTR or that expressed mutant CFTR. In conclusion, CFTR induces the mRNA expression of "downregulated in adenoma" (DRA) and, as a result, upregulates the apical Cl-/HCO3- exchanger activity in tracheal cells. We propose that the tracheal HCO3- secretion defect in patients with CF is partly due to the downregulation of the apical Cl-/HCO3- exchange activity mediated by DRA.

cystic fibrosis transmembrane conductance regulator; cystic fibrosis; trachea; bicarbonate secretion; downregulated in adenoma; chloride ion/bicarbonate exchange


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CYSTIC FIBROSIS (CF), an autosomal recessive disease, presents with defective fluid and electrolyte secretion in secretory epithelia (24, 25, 37). CF results from mutational inactivation of a cAMP-sensitive Cl- channel (cystic fibrosis transmembrane conductance regulator, CFTR) with resultant functional impairments in the respiratory, pancreatic, hepatobiliary, and genitourinary systems (21, 26). With respect to the lung, respiratory dysfunction is thought to result primarily from the thickening of airway mucus, leading to increased infection and respiratory failure (27). This thickened mucus may be a result of abnormal secretion of Cl- and HCO3- across the tracheal epithelium, altering the ion composition and, as a result, the viscosity of the airway surface liquid (5, 15, 33).

Although gene therapy has been regarded as an appealing potential therapeutic option in the treatment of the CF defect, it has not been completely successful in reversing the defect (1). An alternative hope is that a better understanding of the physiological role(s) of CFTR in various organs will lead to novel strategies capable of correcting the functional defect in the absence of functional CFTR.

The currently accepted model of tracheal HCO3- secretion suggests that 1) intracellular HCO3- accumulates due to basolateral diffusion of CO2 and subsequent action of carbonic anhydrase, and 2) G protein-coupled receptors activate cAMP-sensitive CFTR, which then secretes HCO3- (34). According to this model, CFTR or a highly homologous transporter directly mediates HCO3- secretion into the tracheal lumen. Whether CFTR carries HCO3- directly or regulates other HCO3- transporting processes remains speculative (13, 23).

In addition to the currently recognized isoforms of anion exchangers (AEs) that have Cl-/HCO3- exchange activity (16), the "downregulated in adenoma" (DRA) protein, which has been shown to mediate sulfate, oxalate, and Cl- transport in Xenopus oocytes (32), was recently found to have Cl-/HCO3- exchange activity (19). DRA was originally cloned via subtractive hybridization in a colon cDNA library and was found to be expressed in normal colon but not in adenocarcinomas (28). Its function was not known at the time of its cloning. Subsequently, it was found that patients with congenital Cl- diarrhea, who lack apical Cl-/HCO3- exchange activity, had null mutations in DRA (11, 12). Taken together with recent functional studies (19), it is clear that DRA mediates Cl-/HCO3- exchanger activity.

To characterize the functional and molecular mechanism(s) of HCO3- secretion across the tracheal epithelium, an attempt was made to characterize HCO3- transporters in tracheal epithelial cells. We specifically tested the possibility that a Cl-/HCO3- exchanger is expressed in tracheal epithelial cells and may be regulated by CFTR.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell lines. CFT-1 cells are derived from a primary culture of tracheal epithelial cells isolated from a patient with CF (homozygous F508 mutation) and were cultured as previously described (38). Stably transfected CFT-1 cells bearing functional CFTR (termed CFT-WT) were cultured in a similar fashion (20). CFT-1 cells transfected with Delta F508 CFTR cDNA (termed CFT-MT) were used as a control for cells transfected with the functional CFTR to determine the role of the transfection procedure per se on the expression of ion transporters.

Cell pH measurement. Changes in intracellular pH (pHi) were monitored using the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF; see Refs. 2 and 3). Cells were grown to confluence on a glass coverslip, incubated in the presence of 5 µM BCECF in a solution consisting of 115 mM tetraethylammonium (TMA), 25 mM KHCO3 or choline HCO3, and 10 mM HEPES at pH 7.4, and gassed with 5% CO2-95% O2. The monolayer was then perfused with the appropriate solutions in a thermostatically controlled holding chamber (37°C) in a Delta Scan dual excitation spectrofluorometer (PTI, South Brunswick, NJ). The Cl- and HCO3--containing solution contained (in mM) 115 TMA-Cl, 25 mM KHCO3, 0.8 K2HPO4, 0.2 KH2PO4, 1 CaCl2, 1 MgCl2, and 10 HEPES. For HCO3-free solution, KHCO3 was replaced with an isosmolar concentration of TMA-Cl. The Cl--free, HCO3--containing solution contained (in mM) 115 mM N-methyl-D-glucamine (NMDG)-gluconate, 25 mM KHCO3, 0.8 K2HPO4, 0.2 KH2PO4, 1 calcium gluconate, 1 magnesium gluconate, and 10 HEPES. For HCO3-free solution, KHCO3 was replaced with an isosmolar concentration of NMDG-gluconate. The fluorescence ratio at excitation wavelengths of 500 and 450 nm was used to determine pHi values. Calibration curves were established by the KCl/nigericin technique. HCO3--free or HCO3--containing solutions were used to determine the HCO3- dependence of the transporter. To examine the Cl-/HCO3- exchanger activity, cells were switched to a Cl--free medium. This maneuver results in cell alkalinization due to reversal of Cl-/HCO3- exchanger. Upon pHi stabilization in Cl-free medium, cells were switched back to the Cl--containing solution. This resulted in rapid cell acidification back to baseline due to activation of the Cl-/HCO3- exchanger. The initial rate of cell acidification was used as the rate of Cl-/HCO3- exchanger activity. This rate of change in pHi was used to compile the summary data.

RNA isolation and Northern blot hybridization. Total cellular RNA was extracted from CFT-1, CFT-WT, and CFT-MT cells according to the established methods, quantitated spectrophotometrically, and stored at -80°C. Total RNA samples (30 µg/lane) were fractionated on a 1.2% agarose-formaldehyde gel, transferred to Magna NT nylon membranes, cross-linked by ultraviolet light, and baked (36). Hybridization was performed according to Church and Gilbert (7). The membranes were washed, blotted dry, and exposed to a PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA). A 32P-labeled specific 400-bp cDNA (EcoR I-EcoR I fragment) from the mouse DRA cDNA was used as a specific probe. For AE2, a 1.6-kb rat cDNA (codons 456-1002) was used.

Materials. [32P]dCTP was purchased from New England Nuclear (Boston, MA). Nitrocellulose filters and other chemicals were purchased from Sigma Chemical (St. Louis, MO). The RadPrime DNA labeling kit was purchased from GIBCO-BRL. BCECF was from Molecular Probes (Eugene, Oregon).

Statistical analyses. Values are expressed as means ± SE. The significance of difference between mean values was examined using ANOVA. P < 0.05 was considered statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the first series of experiments, we examined the presence of Cl-/HCO3- exchanger activity in CFT-1 cells in the presence or absence of HCO3-. Representative tracings are shown in Fig. 1A and demonstrate that, in the presence of HCO3-, CFT-1 cells mildly alkalinized upon removal of Cl- and acidified back to baseline upon switching to the Cl--containing solution. A summary of multiple experiments is shown in Fig. 1B. These results indicate that low levels of Cl-/HCO3- exchange activity are present in CFT-1 cells.


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Fig. 1.   Cl-/HCO3- exchanger activity in CFT-1 cells in the presence and absence of HCO3-. A: representative tracings demonstrating Cl-/HCO3- exchanger activity in CFT-1 cells. CFT-1 cells grown on glass coverslips were loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and were perfused with solutions as indicated (see MATERIALS AND METHODS for details). Brackets denote concentration; pHi, intracellular pH. B: summary of multiple experiments demonstrating the presence of Cl-/HCO3- exchanger activity in CFT-1 cells. The initial rate of intracellular acidification was used to compile the summary data (with HCO3-, n = 6; no HCO3-, n = 6).

In the next series of experiments, the presence of Cl-/HCO3- exchange activity in CFT-WT cells was examined in the presence or absence of HCO3-. Representative tracings in Fig. 2A demonstrate successive cell alkalinization and acidification upon removal and addition of Cl-, respectively. A summary of multiple experiments, shown in Fig. 2B, indicates the presence of an active Cl-/HCO3- exchanger in CFT-WT cells. Interestingly, the CFT-WT cells also showed significant cell alkalinization (upon removal of Cl-) and cell acidification (upon switching back to the Cl--containing solution) in the absence of HCO3- in the media (Fig. 2). These data are consistent with the presence of Cl-/base (i.e., Cl-/OH-) exchange.


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Fig. 2.   Cl-/HCO3- exchanger activity in CFT-1 cells transfected with wild-type cystic fibrosis transmembrane conductance regulator (CFTR; CFT-WT) in the presence and absence of HCO3-. A: representative tracings demonstrating presence of Cl-/HCO3- exchanger activity in CFT-WT cells. CFT-WT cells grown on glass coverslips were loaded with BCECF and were perfused with solutions as indicated (see MATERIALS AND METHODS for details). B: summary of multiple experiments demonstrating presence of Cl-/HCO3- exchanger activity in CFT-WT cells. The initial rate of intracellular acidification was used to compile the summary data (with HCO3-, n = 6; no HCO3-, n = 6).

Comparison of the results in Figs. 1 and 2 indicates that Cl-/HCO3- exchange activity is enhanced in cells transfected with functional CFTR. The experiments in CFT-1 and CFT-WT cells were performed on separate days. To verify the reproducibility of the results, Cl-/HCO3- exchange activity was compared in CFT-1 and CFT-WT cells on the same day. Representative tracings are shown in Fig. 3A and demonstrate enhanced Cl-/HCO3- exchange in cells transfected with functional CFTR. A summary of the results of multiple experiments, Fig. 3B, demonstrates an approximately threefold increase in Cl-/HCO3- exchange in CFT-WT cells.


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Fig. 3.   Comparison of Cl-/HCO3- exchanger activity in CFT-1 and CFT-WT cells. A: representative tracings demonstrating increase in activity in cells containing functional CFTR. Cells were grown to confluence on glass coverslips, loaded with BCECF, and perfused with appropriate solutions. Tracings were recorded from coverslips examined on the same day to directly compare Cl-/HCO3- exchanger activity from each cell type under similar conditions and confluency. B: summary of the results of multiple experiments demonstrating statistically significant increase in Cl-/HCO3- exchanger activity in functional CFTR-bearing cells (CFT-1 cells, n = 8; CFT-WT cells, n = 8).

The inhibitory effect of the disulfonic stilbene DIDS on Cl-/HCO3- exchange activity was examined in CFT-WT cells. The results of several experiments are summarized in Fig. 4A and indicate that Cl-/HCO3- exchange is inhibited by ~52% in the presence of 500 µM DIDS in CFT-WT. The AE activity in CFT-1 cells showed 62% inhibition by 300 µM DIDS (Fig. 4B). The lack of complete inhibition of Cl-/HCO3- exchange activity by DIDS could be due to the possibility that the tracheal AE is not as sensitive as the known AEs to the inhibitory effect of disulfonic stilbenes.


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Fig. 4.   Effect of DIDS on Cl-/HCO3- exchanger activity. A: summary of the experiments demonstrating inhibition of Cl-/HCO3- exchanger activity by DIDS in cells expressing functional CFTR (CFT-WT cells). DIDS was added to the final Cl--containing solution to a final concentration of 300 µM to determine its effect on Cl-/HCO3- exchanger activity (without DIDS, n = 4; with DIDS, n = 4). B: summary of the experiments indicating inhibition of Cl-/HCO3- exchanger activity by DIDS in cells expressing mutant CFTR (CFT-1 cells). DIDS was added to the final Cl--containing solution to a final concentration of 300 µM to determine its effect on Cl-/HCO3- exchanger activity (without DIDS, n = 6; with DIDS, n = 6).

The effect of cAMP stimulation on Cl-/HCO3- exchange was examined in CFT-WT cells. Cells were exposed to 10 nM forskolin at 5 min before switching to the Cl--free media and were kept exposed for the duration of the experiment. As shown in Fig. 5A (representative tracings) and Fig. 5B (summary of multiple experiments), Cl-/HCO3- exchange was not stimulated by forskolin.


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Fig. 5.   Effect of cAMP activation on Cl-/HCO3- exchanger activity. A: representative tracings demonstrating lack of stimulation of activity of Cl-/HCO3- exchanger upon stimulation with forskolin. Cells were grown to confluence on glass coverslips, loaded with BCECF, and perfused with appropriate solutions. Forskolin was added to the last Cl--containing solution to a final concentration of 10 µM to determine its effect on Cl-/HCO3- exchanger activity. B: summary of the results of multiple experiments demonstrating lack of increase in Cl-/HCO3- exchanger activity in the presence of forskolin (no forskolin, n = 7; with forskolin, n = 5).

The purpose of the next series of experiments was to determine the molecular identity of the AE that is responsible for enhanced Cl-/HCO3- exchange in cells transfected with functional CFTR. In the first series of experiments, expression of the ubiquitous basolateral AE2 Cl-/HCO3- exchanger was examined by Northern hybridization. As shown in Fig. 6, AE2 mRNA expression was the same in cells with functional CFTR as in cells that lack a functional CFTR.


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Fig. 6.   Northern hybridization of AE-2 anion exchanger in tracheal epithelial cells. As shown, the presence of functional CFTR does not alter the expression of AE2 in tracheal epithelial cells. Each lane represents one separate sample. RNA (30 µg) was loaded per each lane. The expression of 28S rRNA is shown as constitutive control. Statistical analysis, based on AE-2 expression over 28S rRNA expression ratios, showed no significant difference between the two groups (P > 0.05).

Because AE2 mRNA levels remained unchanged in cells with functional CFTR, we entertained the possibility that other AE(s) may be responsible for enhanced Cl-/HCO3- exchange in cells with functional CFTR. Toward this end, the mRNA expression of DRA, an anion transporter with Cl-/HCO3- exchanger activity (4, 10, 19) that is defective in congenital Cl- diarrhea, was examined. Northern hybridization (Fig. 7) demonstrated that DRA mRNA is expressed abundantly in functional CFTR-bearing tracheal epithelial cells but could not be detected in either CFT-1 cells or in CFT-1 cells that are transfected with a mutant CFTR (CFT-MT cells). To determine whether DRA is expressed in native tracheal tissue, RNA was extracted from normal mouse trachea and was used for Northern hybridization. The results showed that mouse trachea expresses DRA mRNA (Fig. 8).


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Fig. 7.   Northern hybridization of "downregulated in adenoma" (DRA) mRNA in tracheal epithelial cells. As indicated, functional CFTR induces the expression of DRA in tracheal epithelial cells (CFT-WT cells). RNA (30 µg) was loaded per each lane. The expression of 28S rRNA is shown as constitutive control. CFT-MT are CFT-1 cells transfected with mutant CFTR.



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Fig. 8.   Expression of DRA in native mouse trachea. RNA was extracted from normal mouse trachea and heart and utilized for Northern hybridization. RNA (30 µg) was loaded per each lane. Representative Northern blots are shown. The expression of 28S rRNA is shown as constitutive control. DRA mRNA levels are more abundant in the mouse heart than in the trachea.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The functional and molecular expression of Cl-/HCO3- exchangers was examined in validated human tracheal epithelial cells that exhibit physiological features prototypical of CF (CFT-MT) and normal tracheal epithelium (CFT-WT). The results demonstrated the presence of Cl-/HCO3- exchanger activity in both CFT-1 and CFT-WT cells (Figs. 1 and 2). Cl-/HCO3- exchanger activity was significantly enhanced in tracheal epithelial cells transfected with functional CFTR (Fig. 3). Cl-/HCO3- exchange was inhibited by DIDS (Fig. 4) and was not stimulated by forskolin (Fig. 5). Northern hybridizations indicated the induction of DRA in the wild-type CFTR-bearing tracheal epithelial cells (Fig. 7), whereas the mRNA expression of AE2 remained unaffected (Fig. 6).

Patients with CF demonstrate a significant impairment of Cl- and HCO3- secretion in their trachea (5, 15, 33). The defective Cl- secretion is due to decreased activity of CFTR, which is a cAMP-sensitive Cl- channel. The cellular mechanism of the HCO3- secretion defect in the trachea of CF patients, however, is less well understood. The currently accepted model of tracheal HCO3- secretion (34) suggests that HCO3- is secreted via a cAMP-sensitive electrogenic transporter. This could be consistent with direct secretion of HCO3- via CFTR (34). The results of studies in cultured 3T3 cells transfected with functional CFTR, however, have been contradictory; HCO3- transport via CFTR was demonstrated by one group (22) but not the other (18). Studies in HEK293 cells transfected with wild-type CFTR failed to demonstrate any HCO3- transport by CFTR (18). Recent data from our laboratory indicate that CFTR does not carry HCO3- in cultured pancreatic duct cells (31). Taken together, these results indicate that CFTR does not play a significant role in the direct transport of HCO3-.

Enhanced Cl-/HCO3- exchange in tracheal epithelial cells stably expressing a functional CFTR is intriguing (Fig. 7). Functionally, these results are consistent with indirect but tight coupling between luminal Cl- (secreted via CFTR) and the apical Cl-/HCO3- exchanger and provide a basis for enhanced HCO3- secretion in tracheal epithelial cells expressing functional CFTR. These studies do not conflict with the results of earlier studies demonstrating increased HCO3- secretion in tracheal epithelial cells transfected with functional CFTR (34). Indeed, our studies extend those observations by indicating that CFTR indirectly increases HCO3- secretion by upregulating the apical Cl-/HCO3- exchanger. Our results are also in agreement with recent studies in cultured cells (18) and the duct fragments from the mouse submandibular gland (17) indicating enhancement of Cl-/HCO3- exchanger activity by functional CFTR. The molecular identity of the Cl-/HCO3- exchanger that was upregulated by CFTR, however, was not examined in those studies (17, 18).

The novel aspect of our finding is the induction of the AE DRA in tracheal epithelial cells expressing functional CFTR (Fig. 7). DRA was originally found to be expressed in colon, cecum, and small intestine (12, 28). It is localized to the apical domain of the colonic cell membrane (6). Although RNase protection assay failed to identify DRA expression in lung (32), very low levels of DRA mRNA in mouse lung were observed in our studies (unpublished results). It is plausible that the very weak DRA mRNA signal in the lung is originating from the tracheal epithelial cells, which constitute a minority of the cell population in the lung samples.

In addition to being a Cl- channel, CFTR is also a regulator of other ion transporters. Whether CFTR-mediated enhanced expression of Cl-/HCO3- exchanger DRA requires Cl- transport by CFTR remains unknown. One plausible, although not very likely, mechanism is that functional CFTR increases intracellular HCO3- by increasing the basolateral NBC-driven HCO3- uptake secondary to membrane depolarization (22, 31). The increased intracellular HCO3- concentration can then increase the expression and activity of DRA. Another plasusible mechanism is that CFTR increases the expression of DRA via direct interaction [i.e., via its nuclear-binding domains (NBD)]. As such, it would be the presence of CFTR in the plasma membrane, and not its Cl- channel activity, that regulates DRA. Studies are under way to test these hypotheses. Alternatively, it is possible that the downregulation of DRA in CF tracheal cells results from the upregulation of chemical mediators that are increased in CF cells. Studies have shown that cultured delta F508 CF human bronchial gland cells express elevated levels of proinflammatory cytokines compared with non-CF bronchial cells (35). Whether the upregulation of these cytokines could mediate the downregulation of DRA remains speculative. Patients with CF are known to express elevated levels of proinflammatory cytokines in their trachea (35). It is plausible that these cytokines can downregulate DRA.

Patients with CF show a HCO3- secretion defect in the intestine (9). Studies in the CF mouse duodenum indicate impaired apical HCO3- secretion (29), confirming the important role that CFTR plays in HCO3- secretion in the intestine. Although some investigators have proposed a likely direct role for CFTR in HCO3- secretion (29), this has not been demonstrated by others (17, 18, 31). Whether decreased HCO3- secretion in the intestine of CF patients or knockout animals is due to the downregulation of the apical Cl-/HCO3- exchanger DRA remains unclear. Northern hybridizations on the RNA isolated from CF and wild-type mice intestine showed decreased expression of DRA in the cecum, proximal colon, and distal colon (data not shown). Whether a similar process occurs in the small intestine needs further investigation. Using voltage-clamped Ussing chambers, Clarke and Harline (8) demonstrated that CFTR stimulation increases apical HCO3- secretion, along with Cl-, in mouse small intestine. Enhanced HCO3- secretion was associated with the generation of a current, suggesting that the bulk of HCO3- secretion was mediated via an electrogenic pathway. A luminal Cl-/HCO3- exchanger also contributed to HCO3- secretion (8). In mice with mutant CFTR, both mechanisms of HCO3- secretion were significantly diminished. It was proposed that HCO3- secretion in small intestine involves electrogenic secretion via CFTR HCO3- conductance and electroneutral secretion via a CFTR-dependent Cl-/HCO3- exchange process (8).

The lack of an effect by cAMP on Cl-/HCO3- exchange in tracheal epithelial cells needs further clarification. cAMP activates CFTR in tracheal epithelial cells, leading to increased Cl- secretion. The results of the current studies are consistent with the possibility that the Cl- that is secreted via CFTR is recycled back via the apical Cl-/HCO3- exchanger. Lack of stimulation of Cl-/HCO3- exchange by cAMP should not affect the coordinated actions of the apical Cl-/HCO3- exchanger and CFTR in mediating HCO3- secretion in tracheal epithelial cells. It is likely that DRA is a high-capacity transporter and, given its high level of expression in CFT-WT cells, it could easily recycle the Cl- that is secreted via CFTR in response to cAMP.

In addition to trachea, pancreatic duct cells also demonstrate impaired HCO3- secretion in CF. Recent investigations indicate that the mechanism of HCO3- transport in the pancreatic duct cells is distinct from the tracheal epithelial cells. Studies in the guinea pig pancreatic duct cells demonstrated that removal of luminal Cl- or addition of DIDS only partially inhibited secretin-stimulated HCO3- secretion (<25%; see Ref. 14), strongly suggesting that the apical Cl-/HCO3- exchanger does not play a major role in agonist-stimulated HCO3- secretion. Recent studies from our laboratory indicate that HCO3- uptake across the basolateral membranes of pancreatic duct cells is mediated via the NBC (30). We further observed that cAMP potentiates NBC activity through membrane depolarization that results from the activation of CFTR-mediated Cl- secretion (30). Based on those studies, we proposed that the defect in agonist-stimulated ductal HCO3- secretion in patients with CF is predominantly due to decreased NBC-driven HCO3- entry at the basolateral membrane secondary to the lack of a sufficient electrogenic driving force in the absence of functional CFTR (30). Interestingly, HCO3- uptake across the basolateral membrane of tracheal epithelial cells occurs predominantly via a Na+-HCO3- cotransporter distinct from NBC-1 (unpublished observations), which is the dominant basolateral transporter that mediates the uptake of HCO3- in pancreatic duct cells. The schematic diagram in Fig. 9 illustrates the interaction of CFTR with the Cl-/HCO3- exchanger in tracheal epithelial cells and demonstrates the proposed mechanism of HCO3- uptake across the basolateral membrane. HCO3- enters the cells via an NBC distinct from NBC-1 (unpublished results) and is secreted at the apical membrane via the Cl-/HCO3- exchanger.


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Fig. 9.   Speculative schematic diagram demonstrating HCO3- transport in human tracheal epithelial cells. NBC, Na+-nHCO3- cotransporter (n, number of HCO3- >1).

In conclusion, CFTR induces the expression of DRA along with Cl-/HCO3- exchanger activity in tracheal epithelial cells. The specific mechanism of this induction (direct interaction with CFTR or indirect interaction by alterations in the cell or luminal Cl- concentration) remains unknown. We propose that the tracheal HCO3- secretion defect in patients with CF is partly due to the downregulation of the apical Cl-/HCO3- exchanger activity mediated by DRA.


    ACKNOWLEDGEMENTS

These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-46789 and DK-54430 (to M. Soleimani) and DK-50594 (to G. E. Shull) and by a grant from the Dialysis Clinic Incorporated (to M. Soleimani).


    FOOTNOTES

Address for reprint requests and other correspondence: M. Soleimani, Dept. of Medicine, Univ. of Cincinnati, 231 Bethesda Ave., MSB 5502, Cincinnati, OH 45267-0585 (E-mail: Manoocher.Soleimani{at}uc.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. §1734 solely to indicate this fact.

Received 21 September 1999; accepted in final form 20 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alton, EW, Geddes DM, Gill DR, Higgins CF, Hyde SC, Innes JA, and Porteous DJ. Towards gene therapy for cystic fibrosis: a clinical progress report. Gene Ther 5: 291-292, 1998[ISI][Medline].

2.   Amlal, H, Wang Z, Burnham C, and Soleimani M. Functional characterization of a cloned human kidney Na+:HCO3- cotransporter. J Biol Chem 273: 16810-16815, 1998[Abstract/Free Full Text].

3.   Amlal, H, Wang Z, and Soleimani M. Functional upregulation of H+-ATPase by lethal acid stress in cultured inner medullary collecting duct cells. Am J Physiol Cell Physiol 273: C1194-C1205, 1997[Abstract/Free Full Text].

4.   Bissig, M, Hagenbuch B, Stieger B, Koller T, and Meier PJ. Functional expression cloning of the canalicular sulfate transport system of rat hepatocytes. J Biol Chem 269: 3017-3021, 1994[Abstract/Free Full Text].

5.   Boucher, RC. Human airway ion transport. Am J Respir Crit Care Med 150: 271-281, 1994[ISI][Medline].

6.   Byeon, MK, Westerman MA, Maroulakou IG, Henderson KW, Suster S, Zang X-K, Papas TS, Vesely J, Willingham MC, Green JE, and Schweinfest C. The down-regulated in adenoma (DRA) gene encodes an intestine-specific membrane glycoprotein. Oncogene 12: 387-396, 1996[ISI][Medline].

7.   Church, GM, and Gilbert W. Genomic sequencing. Proc Natl Acad Sci USA 81: 1991-1995, 1994.

8.   Clarke, LL, and Harline MC. Dual role of CFTR in cAMP-stimulated HCO3- secretion across murine duodenum. Am J Physiol Gastrointest Liver Physiol 274: G718-G726, 1998[Abstract/Free Full Text].

9.   Eggermont, E. Gastrointestinal manifestations in cystic fibrosis. Eur J Gastroenterol Hepatol 8: 731-738, 1996[ISI][Medline].

10.   Hastabacka, J, de la Chapelle A, Mahtani MM, Clines G, Reeve-Daly MP, Daly M, Hamilton BA, Kusumi K, Trivedi B, Weaver A, Coloma A, Lovett M, Buckler A, Kaitila I, and Lander ES. The diastrophic dysplasia gene encodes a novel sulfate transporter: positional cloning by fine-structure linkage disequilibrium mapping. Cell 78: 1073-1087, 1994[ISI][Medline].

11.   Hoglund, P, Auranen M, Socha J, Popinska K, Nazer H, Rajaram U, Al Sanie A, Al-Ghanim M, Holmberg C, de la Chapelle A, and Kere J. Genetic background of congenital chloride diarrhea in high-incidence populations: Finland, Poland, and Saudi Arabia and Kuwait. Am J Hum Genet 63: 760-768, 1998[ISI][Medline].

12.   Hoglund, P, Haila S, Socha J, Tomaszewski L, Saarialho-Kere U, Karjalainen-Lindsberg M-L, Airola K, Holmberg C, de la Chapelle A, and Kere J. Mutations of the down-regulated in adenoma (DRA) gene cause congenital chloride diarrhoea. Nat Genet 14: 316-319, 1996[ISI][Medline].

13.   Illek, B, Yankaskas JR, and Machen TE. cAMP and genistein stimulate HCO3- conductance through CFTR in human airway epithelia. Am J Physiol Lung Cell Mol Physiol 272: L752-L761, 1997[Abstract/Free Full Text].

14.   Ishiguro, H, Steward MC, Wilson RW, and Case RM. Bicarbonate secretion in interlobular ducts from guinea-pig pancreas. J Physiol (Lond) 495: 179-191, 1996[Abstract].

15.   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 as compared with normal and disease-control subjects. J Clin Invest 100: 2588-2595, 1997[Abstract/Free Full Text].

16.   Kopito, RR. Molecular biology of the anion exchanger gene family. Int Rev Cytol 123: 177-199, 1990[Medline].

17.   Lee, MG, Choi JY, Luo X, Strickland E, Thomas PJ, and Muallem S. Cystic fibrosis transmembrane conductance regulator regulates luminal Cl-/HCO3- exchange in mouse submandibular and pancreatic ducts. J Biol Chem 274: 14670-14677, 1999[Abstract/Free Full Text].

18.   Lee, MG, Wigley WC, Zeng W, Noel LE, Marino CR, Thomas PJ, and Muallem S. Regulation of Cl-/HCO3- exchange by cystic fibrosis transmembrane conductance regulator expressed in NIH 3T3 and HEK 293 cells. J Biol Chem 274: 3414-3421, 1999[Abstract/Free Full Text].

19.   Melvin, JE, Park K, Richardson L, Schultheis PJ, and Shull GE. Mouse down-regulated in adenoma (dra) is an intestinal Cl-/HCO3- exchanger and is upregulated in colon of mice lacking the NHE3 Na+/H+ exchanger. J Biol Chem 274: 22855-22861, 1999[Abstract/Free Full Text].

20.   Olsen, JC, Johnson LG, Stutts MJ, Sarkadi B, Yankaskas JR, Swanstrom R, and Boucher RC. Correction of the apical membrane chloride permeability defect in polarized cystic fibrosis airway epithelia following retrovial-mediated gene transfer. Hum Gene Ther 3: 253-266, 1992[ISI][Medline].

21.   Pilewski, JM, and Frizzell RA. Role of CFTR in airway disease. Physiol Rev 79: S215-S255, 1999[Medline].

22.   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[Abstract].

23.   Poulsen, JH, and Machen TE. HCO3-dependent pHI regulation in tracheal epithelial cells. Pflügers Arch 432: 546-554, 1996[ISI][Medline].

24.   Quinton, PM. Cystic fibrosis: a disease in electrolyte transport. FASEB J 4: 2709-2717, 1990[Abstract/Free Full Text].

25.   Quinton, PM. Physiological basis of cystic fibrosis: a historical perspective. Physiol Rev 79: S3-S22, 1999[Medline].

26.   Riordan, JR, Rommens JM, Kerem B-S, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou J-L, Drumm ML, Iannuzzi MC, Collins FS, and Tsui L-C. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245: 1066-1073, 1989[ISI][Medline].

27.   Rosenstein, BJ, and Zeitlin PL. Cystic fibrosis. Lancet 351: 277-282, 1998[ISI][Medline].

28.   Schweinfest, CW, Henderson KW, Suster S, Kondoh N, and Papas TS. Identification of a colon mucosa gene that is down-regulated in colon adenomas and adenocarcinomas. Proc Natl Acad Sci USA 90: 4166-4170, 1993[Abstract].

29.   Seidler, U, Blumenstein I, Kretz A, Viellard-Baron D, Rossmann H, Colledge WH, Evans M, Ratcliff R, and Gregor M. A functional CFTR protein is required for mouse intestinal cAMP-, cGMP- and Ca2+-dependent HCO3- secretion. J Physiol (Lond) 505: 411-423, 1997[Abstract].

30.   Shumaker, H, Amlal H, Frizzell R, Ulrich CD, and Soleimani M. CFTR drives Na+-nHCO3- cotransport in pancreatic duct cells: a basis for defective HCO3- secretion in CF. Am J Physiol Cell Physiol 276: C16-C25, 1999[Abstract/Free Full Text].

31.  Shumaker H, Frizzell R, Ulrich C, and Soleimani M. CFTR does not transport HCO3- in a prototypical wild-type CFTR-bearing pancreatic duct cell line. Ann Meet Am Gastroenterol Assoc, Orlando, FL 1999, p. A1161.

32.   Silberg, DG, Wang W, Moseley RH, and Traber PG. The down regulated in adenoma (dra) gene encodes an intestine-specific membrane sulfate transport protein. J Biol Chem 270: 11897-11902, 1995[Abstract/Free Full Text].

33.   Smith, JJ, Karp PH, and Welsh MJ. Defective fluid transport by cystic fibrosis airway epithelia. J Clin Invest 93: 1307-1311, 1994[ISI][Medline].

34.   Smith, JJ, and Welsh MJ. cAMP stimulates bicarbonate secretion across normal, but not cystic fibrosis airway epithelia. J Clin Invest 89: 1148-1153, 1992[ISI][Medline].

35.   Tabry, O, Zahm JM., Ilinnrasky J, Couetil JP, Cornillet P, Guenounou M, Gaillard D, Puchelle E, and Jacquot J. Selective up-regulation of chemokine IL-8 expression in cystic fibrosis bronchial gland cells in vivo and in vitro. Am J Pathol 153: 921-930, 1998[Abstract/Free Full Text].

36.   Thomas, PS. Hybridization of denatured RNA transferred or dotted nitrocellulose paper (Abstract). Methods Enzymol 100: 255, 1983[ISI][Medline].

37.   Welsh, MJ. Electrolyte transport by airway epithelia. Physiol Rev 67: 1143-1184, 1987[Free Full Text].

38.   Yankaskas, JR, Haizlip JE, Conrad M, Koval D, Lazarowski E, Paradiso AM, Rinehart CA, Sarkadi B, Schlegel R, and Boucher RC. Papilloma virus immortalized tracheal epithelial cells retain a well-differentiated phenotype. Am J Physiol Cell Physiol 264: C1219-C1230, 1993[Abstract/Free Full Text].


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