Chloride conductance of CFTR facilitates basal Cl/HCO3 exchange in the villous epithelium of intact murine duodenum

Janet E. Simpson,1,2,3 Lara R. Gawenis,1,3 Nancy M. Walker,3 Kathryn T. Boyle,3 and Lane L. Clarke1,3

Departments of 1Biomedical Sciences and 2Veterinary Pathobiology, University of Missouri, and 3Dalton Cardiovascular Research Center, Columbia, Missouri

Submitted 1 November 2004 ; accepted in final form 10 January 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Villi of the proximal duodenum are situated for direct exposure to gastric acid chyme. However, little is known about active bicarbonate secretion across villi that maintains the protective alkaline mucus barrier, a process that may be compromised in cystic fibrosis (CF), i.e., in the absence of a functional CF transmembrane conductance regulator (CFTR) anion channel. We investigated Cl/HCO3 exchange activity across the apical membrane of epithelial cells located at the midregion of villi in intact duodenal mucosa from wild-type (WT) and CF mice using the pH-sensitive dye BCECF. Under basal conditions, the Cl/HCO3 exchange rate was reduced by ~35% in CF compared with WT villous epithelium. Cl/HCO3 exchange in WT and CF villi responded similarly to inhibitors of anion exchange, and membrane depolarization enhanced rates of Clout/HCO3in exchange in both epithelia. In anion substitution studies, anionin/HCO3out exchange rates were greater in WT epithelium using Cl or NO3, but decreased to the level of the CF epithelium using the CFTR-impermeant anion, SO42–. Similarly, treatment of WT epithelium with the CFTR-selective blocker glybenclamide decreased the Cl/HCO3 exchange rate to the level of CF epithelium. The mRNA expression of Slc26a3 (downregulated in adenoma) and Slc26a6 (putative anion exchanger-1) was similar between WT and CF duodena. From these studies of murine duodenum, we conclude 1) characteristics of Cl/HCO3 exchange in the villous epithelium are most consistent with Slc26a6 activity, and 2) Cl channel activity of CFTR facilitates apical membrane Clin/HCO3out exchange by providing a Cl "leak" under basal conditions.

bicarbonate secretion; anion exchange; cystic fibrosis; Slc26a; Slc4; mouse


THE ALKALINE MUCUS BARRIER of the duodenum plays an important role in protecting the epithelium from damage by acid chyme entering from the stomach (18). Alkalinity of the surface mucus is sustained by transcellular processes of HCO3 secretion that involve coordination of the activities of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) and anion exchanger proteins (11, 47, 55). Under basal, nonstimulated conditions, pH stat studies of CF patients and CF mouse models indicate that bicarbonate secretion by anion exchange predominates in the duodenum (11, 12, 47, 49). Most studies (12, 47, 49) further show that basal HCO3 secretion is reduced in CF duodenum, but the pathophysiology of this deficiency has not been elucidated. In contrast to basal secretion, it is known that CFTR is essential to cAMP stimulation of HCO3 secretion, a consequence of increased prostanoid and neural activity induced by exposure of the duodenum to the gastric effluent (18). The mechanism of this process has been an area of intense investigation in recent years. Most controversy surrounds the question of whether CFTR, a cAMP-stimulated anion channel (3), provides a conductive pathway for HCO3 ions across the apical membrane of the epithelium. CFTR is permeable to HCO3 but at a reduced level relative to Cl (PHCO3 : PCl {approx} 0.25) (22, 44), and the permeability may be reduced further due to anomalous mole fraction behavior of the channel (57). However, studies of intact intestine from animal models are consistent with dominance of a HCO3 conductance in that 75–80% of cAMP-stimulated HCO3 secretion is preserved in the absence of luminal (apical) Cl, a condition that inhibits the activity of the apical membrane Cl/HCO3 exchange proteins (11, 55). Interpretation of these data must accommodate recent evidence that CFTR permeability to HCO3 is dynamically regulated both by intracellular factors (48) and, important to this discussion, by extracellular Cl concentration, i.e., HCO3 permeability increases in the absence of extracellular Cl (50).

A second model proposed for cAMP-stimulated secretion involves the interaction of CFTR with anion exchange proteins. Studies of pancreatic duct epithelium first postulated that a cAMP-stimulated Cl channel (CFTR) indirectly facilitates Cl/HCO3 exchange by recycling Cl, thus maintaining a favorable outside/inside concentration gradient for Cl (40). Subsequent investigations of other epithelia and heterologous expression systems demonstrated that CFTR activation stimulates Cl/HCO3 exchange activity (32, 33). In the duodenum, studies of brush-border membrane vesicles have shown that Cl/HCO3 exchange is increased by cAMP stimulation (15) and evidence of CFTR facilitation of Cl/HCO3 exchange in intestinal mucosa was provided by investigations of cAMP-stimulated murine duodenum during isolation of carbonic anhydrase activity (11) and inhibition of the NKCC1 Na+-K+-2Cl cotransporter (59). Recent studies (36, 43, 63) have immunolocalized several anion exchange proteins to the apical membrane of intestinal epithelium including at least two members of the sulfate permease family, i.e., Slc26a3 [known as the downregulated in adenoma gene (DRA)] and Slc26a6 [known as the putative anion transporter-1 (PAT-1) or chloride-formate exchanger], and one member of the anion exchanger family, Slc4a9 (i.e., AE4). The Slc26a anion exchangers and CFTR possess PSD-25/Disc-large/zonula occludens-1 (PDZ) domains that enable colocalization of these proteins on the binding sites of scaffolding proteins such as the Na+/H+ exchanger (NHE) regulatory factor (31, 35, 52). Further, studies of recombinant proteins indicate the possibility of direct intermolecular associations between the anion exchange proteins and CFTR. During cAMP stimulation, the STAS domain of Slc26a transporters has been shown to enhance CFTR channel activity, whereas the R domain of CFTR increases Cl/base exchange by the anion exchangers (30). Thus our model of the interactions between CFTR and the apical membrane anion exchangers during cAMP-stimulation must be modified to include molecular associations between proteins.

Situated for direct exposure to gastric acid effluent, the villi of the duodenal mucosa are presented with a unique challenge in maintaining a protective alkaline mucus barrier. Although it is well-documented that CFTR expression is greatest in the crypts, CFTR mRNA and protein expression extend to the villous tips and the greatest levels of expression occur in the duodenum (1, 56). Evidence of CFTR channel activity in the villous epithelium includes X-ray microanalysis of intestinal cryosections from normal and CF jejuna, indicating CFTR-dependent Cl secretion (41) and BCECF studies of intact duodenum from normal and CF mice demonstrating the presence of a CFTR-dependent HCO3 conductance (20). In addition to the presence of a functional CFTR, all three of the known apical membrane anion exchangers (DRA, PAT-1, and AE4) in the duodenum have been immunolocalized to the villous epithelial cells (36, 43, 63). Thus the basic components for HCO3 transport are present, but little is known about the physiology of HCO3 secretion across the abundant mucosal surface area of the villous epithelium.

In the present study, we investigate basal activity of anion exchange across the apical membrane of epithelial cells in the midregion of villi of intact duodenal mucosa from wild-type (WT) and CFTR-null (CF) mice using BCECF microspectrofluorimetry of intracellular pH (pHi). The duodenal mucosa was mounted in a horizontal Ussing-type chamber that allowed independent superfusion of the luminal and basolateral surfaces of the epithelium. With the use of this system, the characteristics of anion exchange were investigated in the presence or absence of CFTR activity in the villous epithelium.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. The experiments in this study used mice 8–16 wks old that were either homozygous for the S489X mutation of the murine homolog of CFTR (cftrtm1UNC) or homozygous for the mCFTR {Delta}F508 mutation (cftrtm1Kth) and maintained on a C57BL/6J background (54, 64). The mutant mice were identified by using a PCR-based analysis of tail snip DNA, as previously described (10). No significant differences in experimental parameters were noted between the two CF mouse models which were compared with WT littermate mice. All mice were maintained ad libitum on standard laboratory chow (Formulab 5008 rodent chow; Ralston Purina) and drinking water containing an oral osmotic laxative (Schwartz Pharma, Seymour, IN) with the following composition (in g/l): 60.00 polyethylene glycol 3350, 1.46 NaCl, 0.75 KCl, 1.68 NaHCO3, and 5.68 Na2SO4. The mice were housed singly in a temperature (22–26°C) and light (12:12-h light-dark cycle)-controlled room in the Association for Assessment and Accreditation of Laboratory Animal Care accredited animal facility at the Dalton Cardiovascular Research Center. The mice were fasted overnight before experimentation but were provided with water ad libitum. All experiments involving animals were approved by the University of Missouri Animal Care and Use Committee.

Fluorescence measurement of pHi and image analysis. The method used for imaging intact intestinal epithelium was based on techniques for imaging isolated murine duodenal villi as previously described (20). Animals were killed by asphyxiation in a 100% CO2 atmosphere followed by a surgically induced bilateral pneumothorax. The duodenum was removed via an abdominal incision and was immediately placed in an ice-cold, oxygenated Ringer's solution. The duodenal segment was opened along the mesenteric border using sharp dissection and pinned flat with mucosal side down in a Sylgard-filled petri dish. After scoring along one end of the preparation with a scalpel blade, the serosa and muscularis externa were removed along the longitudinal axis by blunt dissection using fine forceps. Dissection was confirmed by histological examination of representative preparations. The muscle-stripped intestinal preparations were mounted luminal (apical) side up on a horizontal bilateral perfusion chamber in which luminal and serosal surfaces were independently bathed. Throughout the experimental period, all duodenal preparations were treated with indomethacin (1 µM) and TTX (0.1 µM) to minimize the effect of endogenous prostaglandins and neural tone, respectively (6, 51). To immobilize villi for imaging, the tips of a small percentage (<1%) of the villi were trapped under strands of a loose nylon mesh that was placed over the mucosal surface of the preparation. The duodenal preparation was washed with a solution containing 100 µM DL-dithiothreitol and incubated with 16 µM of BCECF-AM for 10 min on the luminal side in a isethionate-bicarbonate Ringer solution (IBR) containing (in mM): 140.0 Na+, 55.0 Cl, 55.0 isethionate, 25.0 HCO3, 5.2 K+, 5.0 TES, 4.8 gluconate, 2.8 PO42–, 1.2 Ca2+, 1.2 Mg2+, 10.0 glucose, and 6.8 mannitol that was gassed with 95% O2-5% CO2 at 37°C (pH 7.4). As described previously (20, 24), the duodenal villous epithelium was loaded with BCECF-AM (Fig. 1A) and an area in the midregion of a single, immobilized villus (100–150 µm from the villous tip) was visualized (Fig. 1B). Approximately 10 epithelial cells were selected for ratiometric analysis using a x40 water immersion objective (Fig. 1C). Epithelial viability in representative preparations was evaluated by using trypan blue exclusion. Changes in pHi were measured by the dual excitation wavelength technique (440- and 495-nm) and the villi were imaged at a 535-nm emission wavelength. Autofluorescence (in the absence of BCECF) was measured in villous epithelia from six mice and the average emission at each wavelength was subtracted for background correction. Ratiometric images were acquired at 20-s intervals with a Sensi-Cam digital camera (Cooke, Auburn Heights, MI), and images were processed by using Axon Imaging Workbench 2.2 (Axon Instruments, Union City, CA). The 495-to- 440-nm ratios were converted to pHi using a standard curve generated by the K+/nigericin technique (5, 58).



View larger version (90K):
[in this window]
[in a new window]
 
Fig. 1. Images of BCECF-loaded villi from murine duodenum. A: low-power image of BCECF-loaded duodenal villi visualized by conventional fluorescent microscopy using an upright, water-immersion objective (x100, {lambda} 535 nm). B: high-power en face image of the upper midregion of a single, immobilized duodenal villus visualized by light microscopy using an upright, water-immersion objective (x400). C: enhanced high-power en face image of BCECF-loaded duodenal epithelium in the midregion of a single villus. White circles denote regions of interest within individual epithelial cells used for data acquisition.

 
Determination of buffering capacity and base flux. Intracellular buffering capacity ({beta}i) of WT and CF duodenal villous cells was estimated by the ammonium alkalization technique (60). Briefly, intact duodenal tissue was mounted in a horizontal bilateral perfusion chamber and loaded with BCECF-AM as described above. The tissue was superfused with a Na+ and HCO3-free ringer containing varying concentrations of NH4Cl. [NH4+]i was calculated from the Henderson-Hasselbalch equation, and {beta}i was determined as {Delta}[NH4+]i/{Delta}pHi. Consistent with previous observations (4, 46), no differences in {beta}i between WT and CF murine intestinal epithelial cells were apparent. Interestingly, the {beta}i of the villous epithelial cells in the intact mucosa (31.8 mM at pH 7.6) exceeded previous estimates of {beta}i in isolated murine colonic crypts (26.7 at pH 7.6) and isolated murine duodenocytes (6.4 mM at pH 7.6) (4, 46). The total buffering capacity ({beta}total) was calculated from the equation: {beta}total = {beta}i + {beta}HCO3 = {beta}i + 2.3 x [HCO3]i, where {beta}HCO3 is the buffering capacity of the HCO3/CO2 system and [HCO3]i is the intracellular concentration of HCO3. The rates of pHi change measured in these experiments were converted to transmembrane base flux J(B) using the equation J(B) = {Delta}pH/{Delta}t x {beta}total. The {beta}total values used in the determination of base flux were calculated from the above equation using the average pHi during the 90-s period of linear {Delta}pH/{Delta}t changes (see below).

Measurement of apical membrane anion exchange. Duodenal preparations were typically perfused with IBR on the luminal side. The basolateral superfusate consisted of a Cl-free IBR (Cl replaced with isethionate) gassed with 95% O2-5% CO2 at 37°C (pH 7.4) and contained 1 µM EIPA to block the activity of NHE isoform 1 (NHE1). For nominally HCO3-free solutions, NaHCO3 was replaced equimolar with NaTES and gassed with 100% O2. For solutions with high K+ concentration ([K+]), 75 mM NaCl and Na+-isethionate were replaced equimolar with KCl and K+-isethionate, respectively, in both luminal and basolateral superfusates. Experiments to measure anion exchange consisted of pHi alkalization induced by replacement of luminal Cl with isethionate on an equimolar basis. After a stable pHi was obtained (~2 min), pHi recovery was initiated by replacing isethionate with Cl. In some experiments, SO42– and NO3 were used to replace Cl on an equimolar basis during pHi recovery. Rates of anion exchange during alkalization and recovery ({Delta}pH/{Delta}t) were calculated from a linear regression of the values from the first 90 s of the initial pHi changes during Cl removal and replacement, respectively. For inhibitor studies, 1 mM DIDS was added to the luminal superfusate from a 10-mM stock solution in IBR or 100 µM niflumic acid (NFA) was added to the luminal superfusate from a 100-mM stock solution in DMSO. For glybenclamide studies, 100 µM glybenclamide was added to the luminal superfusate from a 250-mM stock solution in DMSO.

Northern blot analysis. Northern blot analysis of total mRNA was performed as previously described (9). Total RNA from murine duodenum was extracted by using TRI reagent (Molecular Research Center), according to manufacturer's instructions. RNA was mixed with Glyoxal sample buffer (BioWhittaker Molecular Applications, Rockland, ME), separated by 1% agarose gel electrophoresis and transferred to a Hybond-N+ nylon membrane (Amersham Biosciences, Piscataway, NJ). The blot was probed by using {alpha}-dCTP32P-labeled cDNA PCR product for murine DRA (Slc26a3, mDRA), murine PAT-1 (Slc26a6, mPAT-1), and the L32 ribosomal protein. For the mDRA probe, the RT-PCR product was obtained by using the sense and antisense oligonucleotide primers with the sequences 5'-GGTTTAGCATTTGCTCTGCTGG-3' and 5'-TTACAGTCATGATGAGTTCGATG-3'. For the mPAT-1 probe, the RT-PCR product was obtained by using the sense and antisense oligonucleotide primers with the sequences 5'-GCGACTCTCTGAAAGAGAAGTG-3' and 5'-TCAGAGTTTGGTGGCCAAAACA-3'. Radiographic density of bands was measured by using a Kodak Imaging Station 2000R and expression levels of mDRA and mPAT-1 were normalized to L32.

Materials. The fluorescent dye BCECF-AM was obtained from Molecular Probes (Eugene, OR). TTX was obtained from Biomol International (Plymouth Meeting, PA). All other materials were obtained from either Sigma Aldrich (St. Louis, MO) or Fisher Scientific (Springfield, NJ).

Statistics. All values are reported as means ± SE. Data between two treatment groups were compared by using a two-tailed unpaired Student's t-test assuming equal variances between groups. Data from multiple treatment groups were compared by using a one-way ANOVA with a post hoc Tukey's t-test. A probability value of P < 0.05 was considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cl/HCO3 exchange in villous epithelium of murine duodenum. Cl/HCO3 exchange function across the apical membrane of villous epithelial cells using BCECF microfluorimetry has not been previously demonstrated in intact duodenal mucosa. The horizontal Ussing chamber system used in this study allowed separate superfusion of the luminal (apical) and basolateral (serosal) sides of the mucosa. In the basolateral superfusate, Cl was replaced by isethionate to minimize the contribution of basolateral anion exchange activity and 1 µM EIPA was included in the solution to inhibit basolateral NHE1. A standard experimental paradigm (see Fig. 2A) to demonstrate Cl/base exchange activity involves the replacement of luminal superfusate Cl with the impermeant isethionate, resulting in rapid alkalization of the pHi as the exchange process runs in reverse mode (Clout/HCO3in). Return of luminal superfusate Cl rapidly acidifies (i.e., recovers) pHi to baseline values as the exchanger process runs in forward mode (Clin/HCO3out). To investigate the Cl dependence of the anion exchange process, Cl was not returned to the luminal superfusate after maximal alkalization by Cl removal. As shown in Fig. 2B, the pHi acidified very little for the duration of the experiment (>10 min). To determine whether the anion exchange process was dependent on HCO3, CO2/HCO3 was removed from the superfusates and the duodenum was treated with 100 µM methazolamide to inhibit endogenous carbonic anhydrase activity during the Cl substitution protocol. As shown in Fig. 2C, Cl-dependent alkalization and recovery was greatly diminished during removal of CO2/HCO3 from the superfusing solutions. Addition of CO2/HCO3 to the superfusates reestablished anion exchange activity. Thus the process of Cl-dependent alkalization and recovery in the duodenal villus epithelium is primarily dependent on the presence of HCO3, indicating dominance of Cl/HCO3 rather than Cl/OH exchange.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2. Anion exchange in duodenal villous epithelium exhibits Cl and HCO3 dependence. Intact duodenum was loaded with the pH-sensitive dye BCECF-AM. The intestinal preparation was initially superfused with a Cl-containing isethionate-bicarbonate Ringer (IBR) luminal solution (Ap) and a Cl-free IBR basolateral solution (BL). A: standard experimental paradigm demonstrating intracellular pH (pHi) change as a result of Cl/base exchange activity in duodenal villous epithelial cells during changes in extracellular Cl concentration (Clo). B: representative pHi trace of duodenal villous epithelial cells during luminal Cl removal (replacement with isethionate). After alkalization, pHi remains constant for the duration of the experiment (>10 min), demonstrating a dependence on Clo for recovery from alkalization. Trace is representative of n = 3 experiments. C: representative pHi trace of duodenal villous epithelial cells during luminal Cl removal and replacement in the presence and absence of HCO3/CO2. In the absence of HCO3/CO2, villous epithelial cells exhibit minimal Cl/base activity (~20% of the activity in the presence of HCO3/CO2). Trace is representative of n = 3 experiments.

 
Reduced Cl/HCO3 exchange activity in the villous epithelium of CF mouse duodenum. Under basal conditions, both WT and CF intestine exhibit a finite rate of transepithelial HCO3 secretion, which involves the activity of an apical membrane Cl/base exchange process (11, 12, 47, 49). Several studies show that the rate of HCO3 secretion across CF duodenum is reduced relative to the WT intestine (12, 47, 49). Therefore, we compared the rates of Cl/HCO3 exchange activity in the villous epithelium between CF and WT murine duodena. As shown by the experiment in Fig. 3A, the basal pHi of the villous epithelium is elevated and the rates of alkalization and recovery during Cl substitution/replacement are reduced in CF compared with WT duodenum. Cumulative data for several experiments on CF and WT epithelia show that the baseline pHi (Fig. 3B) is significantly more alkaline in the CF villi, and both the rates of HCO3 influx during Cl removal and HCO3 efflux during Cl replacement are reduced relative to the WT villi (37 and 30%, respectively; Fig. 3, C and D). No differences in Cl/HCO3 exchange activity were found between duodenal epithelium from the CFTR knockout (cftrtm1UNC)and {Delta}F508 mCFTR (cftrtm1Kth) mice, although it was anticipated that differences in the latter mouse model might exist due to the potential for exchanger interaction with expressed mCFTR mutant protein (29). However, unlike some {Delta}F508 mCFTR mouse models (19), mRNA expression of the {Delta}F508 mCFTR is severely reduced (75–80%) in cftrtm1Kth mice, resulting in intestinal transport properties that more closely resemble the mCFTR knockout intestine (64).



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 3. Comparison of pHi and Cl/HCO3 exchange in duodenal villous epithelium of wild-type (WT) and cystic fibrosis (CF) mice. A: representative pHi trace of WT and CF duodenal villous epithelial cells during luminal Cl removal and replacement. B: summary of baseline pHi (means ± SE) in duodenal villous epithelial cells of WT (n = 11) and CF (n = 11) mice. Cumulative data show a significantly elevated baseline pHi in CF compared with WT mice. C and D: summary of HCO3 influx (C) and HCO3 efflux rates (D) in duodenal villous epithelial cells from WT (n = 9) and CF (n = 8) mice (means ± SE). The cumulative data show reduced rates of Cl/HCO3 exchange after luminal Cl removal and replacement in CF compared with WT. *Significantly different from WT. J (mM B/min), flux (mM of base/min).

 
Properties of apical membrane Cl/HCO3 exchange are similar in WT and CF villous epithelia. To determine whether the characteristics of the Cl/HCO3 exchange are similar between CF and WT villi, we compared inhibitor sensitivity, transport electrogenicity and anion selectivity of the villous anion exchange process. In inhibitor studies, the effects of two inhibitors of anion exchange, the distilbene DIDS and NFA (8), on Cl/HCO3 exchange rate were examined in CF and WT villi (Fig. 4). DIDS, at a concentration (1 mM) known to inhibit activity of recombinant isoforms of both the Slc26a and Slc4 transport families, had very little effect on the rates of villous epithelial anion exchange when applied to the luminal superfusate in either WT or CF duodenum (<5%). In contrast, NFA significantly inhibited the rate of Cl/HCO3 exchange to a similar degree (60%) in both WT and CF intestine.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 4. Effect of anion exchange inhibitors on Cl/HCO3 exchange rates in WT and CF villous epithelium. Intact duodenum was superfused in the luminal bath for 5 min with 1 mM DIDS or 100 µM niflumic acid (NFA) before luminal Cl removal and replacement. In the presence of 1 mM DIDS, the rate of HCO3 efflux (means ± SE) was minimally inhibited in both WT and CF duodena (n = 4). In the presence of 100 µM NFA, HCO3 efflux (means ± SE) was reduced by ~60% in both WT (n = 5) and CF (n = 4) duodena.

 
Recent studies (29, 39, 62) have indicated that members of the Slc26a family of anion exchangers (i.e., DRA and PAT-1) possess electrogenic properties when examined in recombinant systems. To investigate electrogenic properties of the villous Cl/HCO3 exchange process, the K+ concentration in both the luminal and basolateral superfusate was increased to 80 mM to depolarize the membrane potential of the villous epithelial cells. As shown in Fig. 5, cell depolarization significantly increased the mean rate of HCO3 influx during Cl removal in both the WT and CF villous epithelium, whereas the mean rate of HCO3 efflux during Cl replacement was not significantly different in the high [K+] medium. Thus the qualitative effect of membrane depolarization on Cl/HCO3 exchange is similar between WT and CF duodenal villi. In the case of the CF intestine, where the CFTR conductive pathway in the apical membrane is not present, the stimulatory effect of depolarization on HCO3 influx suggests the dominance of an anion exchanger that hyperpolarizes the apical membrane upon Cl removal.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 5. Effect of cell depolarization on Cl/HCO3 exchange rates in WT (A) and CF (B) villous epithelia. Intact duodenum was superfused bilaterally with medium containing 80 mM K+ during luminal Cl removal and replacement. In the presence of the high K+ concentration ([K+]), the rate of HCO3 influx (means ± SE) during luminal Cl removal was significantly increased, whereas the rate of HCO3 efflux (means ± SE) during Cl replacement was unaffected in WT (n = 8) or CF (n = 7) duodenal villous epithelia. *Significantly different from WT (n = 9) or CF (n = 8) controls (i.e., normal IBR medium).

 
Investigation of reduced Cl/HCO3 exchange in the CF villous epithelium. The anion selectivity of CFTR has been well characterized (34), and it is known that members of the Slc26a anion exchangers transport a number of monovalent and divalent anions (39). Therefore, we investigated the ability of selected anions to support CFTR-facilitated anion exchange. CFTR is essentially impermeant to SO42– (25), whereas recombinant Slc26a anion exchangers, including DRA and PAT-1 (39), are capable of transporting SO42–, although the rate of SO42– transport by DRA may be very low (8). To compare SO42– transport between WT and CF villous epithelium, we measured the rates of SO42–-dependent HCO3 efflux (SO42–in/HCO3out) after pHi alkalization induced by luminal Cl removal. As shown in Fig. 6A, left, the SO42–-dependent HCO3 efflux in WT villous epithelium occurred at a significantly reduced rate compared with recovery induced by luminal Cl addition. In contrast, the SO42–-dependent HCO3 efflux after alkalization was not different from the rate of Cl-dependent HCO3 efflux in the CF villous epithelium (Fig. 6A, right). Note also that the mean rates of SO42–-dependent HCO3 efflux were nearly identical between WT and CF villous epithelium. Because SO42– does not permeate CFTR (25), these findings suggest that CFTR facilitates Cl/HCO3 exchange in the WT duodenal villous epithelial cells by providing a "leak" pathway for intracellular Cl, whereas this pathway is not available for SO42–, and thus the rates of SO42–/HCO3 exchange are similar to CF epithelial cells. As a test of this hypothesis, we measured the rate of NO3-dependent HCO3 efflux after epithelial alkalization. CFTR also conducts NO3 ions (GCl:GNO3 {cong} 0.8, Ref. 25) and, like SO42–, NO3 can support anion exchange by DRA and PAT-1 (14, 62). As shown in Fig. 6A, the rate of NO3-dependent HCO3 efflux was significantly increased relative to SO42–-dependent HCO3 efflux in WT villous epithelium. However, in CF epithelium, the rate of NO3-dependent HCO3 efflux was equivalent to that measured for Cl and SO42–. These data are consistent with CFTR facilitation of anion exchange by providing a NO3 leak pathway. As a further test of the hypothesis, we then measured the rates of Cl-dependent pHi alkalization and recovery during treatment of the intestine with the CFTR-selective channel blocker, glybenclamide (13). Preliminary studies using murine duodenum mounted in Ussing chambers indicated that 100 µM glybenclamide in the luminal bath inhibited the forskolin-induced short-circuit current by 78% (vehicle {Delta}Isc = 187.8 ± 44.1; glybenclamide {Delta}Isc = 41.2 ± 8.6 µA/cm2, n = 4, P < 0.05). As shown in Fig. 6B, the rate of Cl/HCO3 exchange in WT villous epithelium pretreated with glybenclamide was significantly reduced relative to the vehicle (DMSO)-treated epithelium, whereas no change in the rates of Cl/HCO3 exchange were apparent in the CF villous epithelium.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 6. Effects of anion substitution or glybenclamide on HCO3 efflux in WT and CF duodenal villous epithelia. A: comparison of HCO3 efflux rates during luminal superfusion with 55 mM Cl, SO42–, or NO3 after pHi alkalization induced by luminal Cl removal. In WT duodenal epithelium (left), HCO3 efflux rate was increased over CF duodenal epithelium only in the presence of luminal Cl or NO3, but not luminal SO42– (n = 9). In CF duodena (right), similar rates of HCO3 efflux were measured during luminal superfusion with 55 mM Cl, SO42–, or NO3 (n = 5–8). Bars represent the means ± SE. a,b,cMeans with the same letters are not statistically different within either the WT or CF group. *Significantly different from WT. B: intact duodenum was treated for 5 min with 100 µM glybenclamide (luminal superfusate) before luminal Cl removal and replacement. HCO3 efflux was significantly reduced in WT but unaffected in CF villous epithelium (n = 8 WT and 4 CF). Control WT and CF duodena were treated with 0.4% DMSO vehicle (n = 8 WT and 3 CF). Bars represent means ± SE. *Significantly different from control.

 
The above studies are consistent with the hypothesis that the CFTR facilitation of apical membrane Cl/HCO3 exchange requires the anion conductive properties of CFTR. However, an alternative hypothesis is that both SO42– and glybenclamide reduce the rate of HCO3 efflux by blocking a HCO3 current mediated by CFTR. Although this alternative hypothesis may be consistent with the action of glybenclamide, previous studies (17, 42) using extracellular SO42– have not demonstrated blockade of the CFTR channel pore. Because it is possible that intracellular SO42– may block the CFTR channel, we investigated whether the rate of Cl-dependent HCO3 efflux was reduced in WT epithelium after a period of SO42– uptake resulting from SO42–in/HCO3out exchange. As shown in Fig. 7, initial exposure of the epithelium to SO42– induced a finite rate of HCO3 efflux but replacement of SO42– with Cl rapidly increased the rate to levels typical of Cl-dependent HCO3 efflux. Because SO42– uptake did not alter the subsequent rate of Cl/HCO3 exchange in WT epithelium, this finding suggests that intracellular SO42– accumulation in the villous epithelium does not affect the anion conductive properties of CFTR.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 7. Effect of a preceding period of SO42– uptake on Cl/HCO3 exchange in WT duodenal villous epithelium. Left: representative pHi trace of WT duodenal villous epithelial cells. After alkalization secondary to luminal Cl removal, 55 mM SO42– was introduced in the luminal superfusate to allow SO42– uptake by SO42–in/HCO3out exchange. This period was immediately followed by equimolar replacement of luminal SO42– with Cl, resulting in a typical rate of Cl/HCO3 exchange (HCO3 efflux). Right: summary of HCO3 efflux rates (means ± SE) induced by luminal SO42– followed by luminal Cl in duodenal villous epithelial cells (n = 6 mice), indicating that a preceding period of SO42– uptake by the epithelium does not decrease the rate of Cl-dependent HCO3 efflux. *Significantly different from SO42–.

 
The anion substitution studies indicated the presence of apical membrane anion exchanger(s) that can readily perform SO42–in/HCO3out exchange. If recent studies (8) indicating very limited SO42– transport by human DRA (hDRA) are also true for murine DRA (mDRA), it is possible that a severe reduction in mDRA expression in the CF mouse intestine could explain the reduced rates of Cl/HCO3 exchange. In other words, if both mDRA and mPAT-1 contribute to Cl/HCO3 exchange but only mPAT-1 contributes to SO42–/HCO3 exchange, then loss of mDRA expression in the CF intestine would result in reduced Cl/HCO3 exchange and unchanged SO42–/HCO3 exchange relative to WT intestine. This possibility is strengthened by studies of recombinant proteins that show a positive correlation between the expression of CFTR and the anion exchangers, DRA and PAT-1 (23). Therefore, we investigated the mRNA expression of DRA and PAT-1 in the WT and CF proximal intestine. As shown in Fig. 8, Northern blot analysis indicated only a slight, nonsignificant decrease in DRA (~25%) and essentially no change in PAT-1 mRNA expression in the CF compared with the WT duodenum. Although measures of protein expression or functional studies in knockout mice will be necessary to confirm these findings, the present data do not indicate a major reduction in the expression of DRA in the CF duodenum.



View larger version (59K):
[in this window]
[in a new window]
 
Fig. 8. mRNA expression of Slc26a3 [murine downregulated in adenoma (mDRA)] and Slc26a6 [murine putative anion exchanger (mPAT-1)] in the small intestine of gender-matched littermate WT and CF mouse pairs. A: representative Northern blot of mDRA, mPAT-1, and L32 ribosomal protein (as a loading control) in the small intestine of 3 pairs of gender-matched WT and CF littermate mice. B: densitometric analysis of mRNA expression for mDRA and mPAT-1 normalized to the mRNA expression of L32 ribosomal protein (n = 3 mouse pairs).

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In the present study, in situ measurements of epithelial cells from the midregion of duodenal villi demonstrated robust activity of Cl/HCO3 exchange across the apical membrane. Dependence of anion exchange activity on luminal Cl was shown by sustained intracellular alkalization during substitution of Cl with the impermeant anion, isethionate. The process was also found to be primarily HCO3-dependent in studies in which CO2/HCO3 was removed from the luminal and basolateral bathing medium. The rates of alkalization and recovery in the absence of CO2/HCO3 (i.e., Cl/OH exchange) were less than 15 and 24%, respectively, of the rates in the presence of CO2/HCO3. An important observation was the finding that the Cl/HCO3 exchange activity in CF duodenal villous epithelial cells was reduced by approximately one-third relative to WT cells. Despite reduced activity, investigations of inhibitor sensitivity and responses during membrane depolarization did not reveal overt differences in the characteristics of the anion exchange process between WT and CF duodenum.

In the inhibitor studies, treatment with the distilbene DIDS reduced Cl/HCO3 exchange by less than 5% in both WT and CF villous epithelium. DIDS resistance is consistent with properties reported for recombinant mDRA (37) but not for recombinant mPAT-1 or rat AE4 proteins (27, 28, 62). In contrast to the effect of DIDS, NFA treatment substantially reduced villous Cl/HCO3 activity by an equivalent degree in both WT and CF duodenum. Studies of recombinant hDRA, human PAT-1 (hPAT-1), and mPAT-1 have demonstrated that these proteins are sensitive to NFA (Ref. 8 and Chernova MN, Alper SL, unpublished observations); however, we are unaware of similar studies performed on the mDRA ortholog. Membrane depolarization using high [K+] medium also produced similar effects on the Cl/HCO3 exchange process in WT and CF duodena. In both cases, depolarization enhanced the rate of HCO3 influx during luminal Cl removal. Recent studies (27–29, 62) of recombinant mDRA and hPAT-1, but not rat AE4, indicate the transport activity of these proteins exhibit electrogenic properties. In the case of hPAT-1, removal of extracellular Cl induces membrane hyperpolarization and has led to the proposal that the apparent stoichiometry of the protein is 1 Cl/≥2 HCO3 (29) In contrast, removal of extracellular Cl from cells expressing recombinant mDRA caused membrane depolarization, suggesting an apparent stoichiometry of ≥2 Cl/1 HCO3. It has been further proposed that equal expression of these two exchangers would result in electroneutral activity because of their opposing effects on membrane potential (29). However, this hypothesis was not supported in our studies of villous epithelium because membrane depolarization significantly increased the rate of HCO3 influx during luminal Cl substitution.

Previous studies of cAMP regulation of HCO3 secretion in pancreatic ducts led to the proposal that the presence of a chloride channel in the apical membrane facilitates Cl/HCO3 exchange by recycling or leaking Cl back to the extracellular milieu, thereby providing a favorable Cl concentration gradient for exchange activity. Several lines of evidence suggest that this model also explains the present observation that Cl/HCO3 exchange activity is greater in WT vs. CF villous epithelium under basal conditions. First, only anions that permeate CFTR, i.e., Cl and NO3 but not SO42–, produced greater rates of anion exchange in WT villous epithelium. In contrast, the rate of SO42–/HCO3 exchange in the WT cells was equivalent to that in the CF epithelium. Because neither SO42– nor NO3 reduced the rate of anion exchange in the CF duodenum, the results indicate that these anions support the activity of the anion exchanger(s). Secondly, the lower rate of SO42–-dependent HCO3 efflux in the WT epithelium did not result from SO42– blockade of a CFTR-mediated HCO3 conductance because the rate of Cl-dependent HCO3 efflux was not reduced by preceding the experiment with a period of SO42– uptake across the apical membrane. Third, treatment of the WT epithelium with the CFTR-selective channel blocker glybenclamide also reduced the rate of Cl/HCO3 exchange to a level equivalent with that in CF epithelium. Because the rate of Cl/HCO3 exchange was not reduced in the CF epithelium treated with glybenclamide, it is unlikely that the effect of the channel blocker in the WT epithelium was due to nonspecific effects of the compound. The above data support the conclusion that the Cl channel function of CFTR facilitates the activity of Cl/HCO3 exchange in the villous epithelium of the murine duodenum under basal conditions. However, it should be emphasized that this conclusion only applies to the basal, unstimulated condition and, specifically, to Clin/HCO3out exchange, a physiologically relevant transport mode in duodenal villous epithelium.

Although the transport studies support the hypothesis that CFTR Cl conduction supports Cl/HCO3 exchange in duodenal villi, the possibility was raised that reduced Cl/HCO3 exchange in the CF epithelium may result from decreased expression of an anion exchanger that does not transport SO42–, e.g., DRA (8). However, the mRNA expression of mDRA was not significantly or consistently reduced in the CF duodenum. These data support previous studies which found only slight or no decrease in DRA mRNA expression in the distal intestine of CFTR-null mice (8), but are contrary to the effect of recombinant CFTR expression on native DRA and PAT-1 expression in pancreatic duct cell lines (23). Although large changes in DRA expression sufficient to account for the differences in Cl/HCO3 exchange activity in the CF duodenum were not apparent from the Northern blot analysis, additional studies measuring surface protein levels will be necessary to fully evaluate the contribution of changes in DRA expression to the reduced rates of Cl/HCO3 exchange in the CF duodenum.

It was interesting that basal pHi in the CF duodenal epithelium was significantly more alkaline than WT under the conditions of our study. Previous reports of CF pancreatic duct epithelial cell lines have found pHi to be alkaline relative to CFTR-corrected cells, whereas no pHi difference was apparent in primary CF airway epithelial cells (16, 61). Important to the present discussion, no differences from WT were found in measurements of pHi of isolated enterocytes from both human and murine CF duodena (46, 47) nor in in vivo preparations of intact duodenum from CFTR-deficient mice (24). However, in the latter study, exposure of the murine duodenum to luminal acid revealed differences in the CO2/HCO3-dependent buffering power in the CF epithelium that was tentatively ascribed to the imbalance resulting from intact HCO3-loading processes and the absence of a CFTR-mediated HCO3-unloading mechanism. Similarly, in the present study, the condition of disabling basolateral "housekeeping" pHi regulation by NHE1 (EIPA treatment) and AE2 (basolateral Cl removal) potentially accentuated CO2/HCO3-dependent buffering power in the CF duodenal epithelium. HCO3-loading processes of NaHCO3 cotransport and intracellular carbonic anhydrase activity were left intact. Thus an imbalance resulting in increased pHi may have been created in the CF villous epithelium which lacked CFTR facilitation of basal Cl/HCO3 exchange activity.

Our studies indicate that the Cl channel activity of CFTR is necessary for increased rates of Clin/HCO3out exchange in WT epithelium under basal conditions. The efficiency of this interaction may require a close association between the Cl/HCO3 exchanger(s) and CFTR either by direct binding or through scaffolding protein colocalization (29–31, 35). Interestingly, Fig. 5 shows that even under membrane depolarizing conditions (high [K+]), the rate of HCO3 influx during luminal Cl removal remains greater in WT than CF epithelium (WT = 23.6 ± 0.9 vs. CF = 16.5 ± 2.0 mM/min, P < 0.05). These data suggest that CFTR also facilitates Clout/HCO3in exchange, a process not predicted to require a Cl leak pathway. Although this observation may be indirect evidence of a functional intermolecular interaction between the Cl/HCO3 exchanger(s) and CFTR, other explanations for increased alkalization in WT epithelium during luminal Cl removal can be offered, including additive effects of luminal Cl removal and high [K+] medium on membrane depolarization and increased HCO3 influx via CFTR in the nominal absence of luminal Cl (50). Nonetheless, given estimates of luminal ionic content (45), it seems unlikely that the luminal Cl concentration in the duodenum would decrease to levels sufficiently low for sustained Clout/HCO3in exchange activity in vivo.

The present data are sufficient to allow a tentative conclusion regarding the identity of the predominant apical membrane Cl/HCO3 exchanger in the duodenal villous epithelium. However, a major difficulty with this exercise is that the characteristics of the anion exchangers are almost exclusively based on studies of recombinant proteins expressed in heterologous cell systems. Although necessary to isolate the protein's activity, the knowledge derived from those investigations are limited by factors such as expression level, lack of native binding partners, experiments performed at nonphysiological temperatures, and the use of a single splice variant of the proteins. Given these limitations, some comparisons of recombinant protein studies can be drawn with characteristics of Cl/HCO3 exchange activity in the villous epithelium. The inhibitor studies indicated a transport process insensitive to DIDS but significantly inhibited by NFA. Although this characteristic is consistent with a report on recombinant murine DRA that showed <25% inhibition by DIDS (37), additional studies on this and other murine orthologs of anion exchangers may be necessary because the effect of DIDS on the human DRA has remained controversial despite five studies reporting experiments (7, 8, 37, 38, 53). Another identifying characteristic was shown by studies inducing membrane depolarization with high [K+] medium which increased the rate of HCO3 influx during luminal Cl removal. Membrane depolarization would favor the activity of murine PAT-1, which has been shown to hyperpolarize the cell during Cl removal (39). Because the rate of HCO3 influx was also increased by membrane depolarization in CF epithelium, the aforementioned switching effect of luminal Cl removal on CFTR permeability to HCO3 would not be a consideration. Finally, the high rate of SO42–/HCO3 exchange in the villous epithelium is also consistent with the activity of murine PAT-1. Studies of murine PAT-1 have shown robust SO42– transport (39). In contrast, although DRA has been reported as a sulfate transporter (38, 53), 35SO42– uptake studies of human recombinant DRA show transport rates that are at least three orders of magnitude less than the SO42–/HCO3 (or Cl/HCO3) exchange rates measured in the present study, which would be too low to appreciably alter pHi (Ref. 8; J. Simpson, unpublished observations). Thus on the basis of the effects of membrane depolarization and high rates of SO42– transport, the data suggest that mPAT-1 provides a major portion of the anion exchange activity in the villous epithelium of the murine duodenum. However, it should be emphasized that this conclusion only applies to the villous epithelium located in the upper one-half of murine duodenal villi. The contribution of villous PAT-1 Cl/HCO3 exchange to the total transepithelial HCO3 secretion and Cl absorption across murine duodenum is yet unknown. Previous studies (26) have indicated that DRA Cl/HCO3 exchange provides a major contribution to electroneutral HCO3 secretion in the duodenum of several species. Other members of the Slc26a family of anion transporters and AE4 also likely contribute to transepithelial anion transport. Thus the activity of these anion exchange proteins in the lower villous and crypt epithelia, where CFTR expression is greatest, may have a dominant role in transepithelial HCO3 secretion and Cl absorption in the duodenum. Additional studies of recombinant proteins and Slc26a anion exchanger knockout mice will be necessary to evaluate these hypotheses.

The results of the present study suggest that CFTR facilitates anion exchange activity in the villous epithelium by providing a Cl leak channel that enables sustained Clin/HCO3out exchange activity under basal (nonstimulated) conditions. Physiologically, this mechanism may be important in restoring the alkaline mucus barrier during interdigestive periods. The HCO3 secretory mechanism is compromised in the CF duodenum but the adverse effects of this deficiency may be compensated by increased CO2/HCO3-dependent intracellular buffering power which protects the epithelial cell from acid insult (24). In WT epithelium, the efficiency of Cl recycling for anion exchange is likely increased by colocalization of CFTR with the anion exchanger(s) at the apical membrane. However, it was unnecessary in these studies to postulate a direct regulatory interaction between CFTR and the anion exchangers to explain the passive role of CFTR in facilitating Clin/HCO3out exchange. Although CFTR may have a different, perhaps direct, regulatory relationship during the Clout/HCO3in transport mode, the reduced expression of CFTR in the villous epithelium (56) makes it difficult to envision a one-to-one molecular interaction with the anion exchanger(s). On the basis of the relative equality of macroscopic flux rates (10, 21), it would be anticipated that the number of CFTR channels which transport ~1 x 106 ions/s may be one to two orders of magnitude less than the number of anion exchangers which transport at 1–5 x 104 ions/s (37a). Thus the current model for interactions between CFTR and the Slc26a anion exchangers may require modification for epithelia in which low-levels of CFTR expression provide indirect regulation of Cl/HCO3 exchange activity.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study was supported by National Institutes of Health Grants T32-RR-07004 (to J. E. Simpson) and DK-48816 (to L. L. Clarke).


    ACKNOWLEDGMENTS
 
The authors acknowledge the expert technical assistance of Emily Bradford and Dr. Alison L. Woo (University of Cincinnati Medical Center). The authors also thank Dr. Marshall H. Montrose (University of Cincinnati, Cincinnati, OH) for advice regarding the BCECF microspectrofluorimetry experiments.

Present address of L. R. Gawenis: Dept. of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, ML524, Cincinnati, OH 45267.


    FOOTNOTES
 

Address for reprint requests and other correspondence: L. L. Clarke, 324D Dalton Cardiovascular Research Center, 134 Research Park Drive, Univ. of Missouri-Columbia, Columbia, MO 65211 (E-mail: clarkel{at}missouri.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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Ameen NA, Alexis J, and Salas P. Cellular localization of the cystic fibrosis transmembrane conductance regulator in mouse intestinal tract. Histochem Cell Biol 114: 69–75, 2000.[ISI][Medline]
  2. Anderson MP, Gregory RJ, Thompson S, Souza DW, Paul S, Mulligan RC, Smith AE, and Welsh MJ. Demonstration that CFTR is a chloride channel by alteration of its anion selectivity. Science 253: 202–205, 1991.[ISI][Medline]
  3. Bachmann O, Wuchner K, Rossmann H, Leipziger J, Osikowska B, Colledge WH, Ratcliff R, Evans MJ, Gregor M, and Seidler U. Expression and regulation of the Na+-K+-2Cl cotransporter NKCC1 in the normal and CFTR-deficient murine colon. J Physiol 549: 525–536, 2003.[Abstract/Free Full Text]
  4. Boyarsky G, Ganz MB, Sterzel RB, and Boron W. pH regulation in single glomerular mesangial cell. I. Acid extrusion in the absence and presence HCO3. Am J Physiol Cell Physiol 255: C844–C856, 1988.[Abstract/Free Full Text]
  5. Bukhave K and Rask-Madsen J. Saturation kinetics applied to in vitro effects of low prostaglandin E2 and F2{alpha} concentrations on ion transport across human jejunal mucosa. Gastroenterology 78: 32–42, 1980.[ISI][Medline]
  6. Byeon MK, Frankel A, Papas TS, Henderson KW, and Schweinfest CW. Human DRA functions as a sulfate transporter in Sf9 insect cells. Protein Expr Purif 12: 67–74, 1999.[ISI]
  7. Chernova MN, Jiang L, Shmukler BE, Schweinfest CW, Blanco P, Freedman SD, Stewart AK, and Alper SL. Acute regulation of the SLC26A3 congenital chloride diarrhoea anion exchanger (DRA) expressed in Xenopus oocytes. J Physiol 549: 3–19, 2003.[Abstract/Free Full Text]
  8. Clarke LL, Gawenis LR, Bradford EM, Judd LM, Boyle KT, Simpson JE, Shull GE, Tanabe H, Ouellette AJ, Franklin CL, and Walker NM. Abnormal Paneth cell granule dissolution and compromised resistance to bacterial colonization in the intestine of CF mice. Am J Physiol Gastrointest Liver Physiol 286: G1050–G1058, 2004.[Abstract/Free Full Text]
  9. Clarke LL and Harline MC. CFTR is required for cAMP inhibition of intestinal Na+ absorption in a cystic fibrosis mouse model. Am J Physiol Gastrointest Liver Physiol 270: G259–G267, 1996.[Abstract/Free Full Text]
  10. 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]
  11. Clarke LL, Stien X, and Walker NM. Intestinal bicarbonate secretion in cystic fibrosis mice. JOP 2, Suppl 4: 263–267, 2001.[Medline]
  12. Schultz BD, Singh AK, Devor DC, and Bridges RJ. Pharmacology of CFTR chloride channel activity. Physiol Rev 79, Suppl 1: S109–S144, 1999.[Medline]
  13. Du L, Xi L, Chu S, Kere J, and Montrose MH. Chloride/hydroxyl and nitrate/hydroxyl exchange by DRA/CLD protein (Abstract). FASEB J 17: A467, 2003.
  14. Dunk CR, Brown CDA, and Turnberg LA. Stimulation of Cl/HCO3 exchange in rat duodenal brush border membrane vesicles by cAMP. Pflügers Arch 414: 701–705, 1989.[CrossRef][ISI][Medline]
  15. Elgavish A. High intracellular pH in CFPAC: a pancreas cell line from a patient with cystic fibrosis is lowered by retrovirus-mediated CFTR gene transfer. Biochem Biophys Res Commun 180: 342–348, 1991.[CrossRef][ISI][Medline]
  16. Elgavish A and Meezan E. Altered sulfate transport via anion exchange in CFPAC is corrected by retrovirus-mediated CFTR gene transfer. Am J Physiol Cell Physiol 263: C176–C186, 1992.[Abstract/Free Full Text]
  17. Flemstrom G. Gastric and duodenal mucosal bicarbonate secretion. In: Physiology of the Gastrointestinal Tract, edited by Johnson LR. New York: Raven, 1987, p. 1011–1030.
  18. French PJ, van Doorninck JH, Peters RH, Verbeek E, Ameen NA, Marino CR, De Jonge HR, Bijman J, and Scholte BJ. A delta F508 mutation in mouse cystic fibrosis transmembrane conductance regulator results in a temperature-sensitive processing defect in vivo. J Clin Invest 98: 1304–1312, 1996.[Abstract/Free Full Text]
  19. Gawenis LR, Franklin CL, Simpson JE, Palmer BA, Walker NM, Wiggins TM, and Clarke LL. cAMP inhibition of murine intestinal Na+/H+ exchange requires CFTR-mediated cell shrinkage of villus epithelium. Gastroenterology 125: 1148–1163, 2003.[CrossRef][ISI][Medline]
  20. Gawenis LR, Stien X, Shull GE, Schultheis PJ, Woo AL, Walker NM, and Clarke LL. Intestinal NaCl transport in NHE2 and NHE3 knockout mice. Am J Physiol Gastrointest Liver Physiol 282: G776–G784, 2002.[Abstract/Free Full Text]
  21. Gray MA, Plant S, and Argent BE. cAMP-regulated whole cell chloride currents in pancreatic duct cells. Am J Physiol Cell Physiol 264: C591–C602, 1993.[Abstract/Free Full Text]
  22. Greeley T, Shumaker H, Wang Z, Schweinfest CW, and Soleimani M. Downregulated in adenoma and putative anion transporter are regulated by CFTR in cultured pancreatic duct cells. Am J Physiol Gastrointest Liver Physiol 281: G1301–G1308, 2001.[Abstract/Free Full Text]
  23. Hirokawa M, Takeuchi T, Chu S, Akiba Y, Wu V, Guth PH, Engel E, Montrose MH, and Kaunitz JD. Cystic fibrosis gene mutation reduces epithelial cell acidification and injury in acid-perfused mouse duodenum. Gastroenterology 127: 1162–1173, 2004.[CrossRef][ISI][Medline]
  24. Illek B, Tam AWK, Fischer H, and Machen TE. Anion selectivity of apical membrane conductance of Calu 3 human airway epithelium. Pflügers Arch 437: 812–822, 1999.[CrossRef][ISI][Medline]
  25. Jacob P, Rossmann H, Lamprecht G, Kretz A, Neff C, Lin-Wu E, Gregor M, Groneberg DA, Kere J, and Seidler U. Down-regulated in adenoma mediates apical Cl/HCO3 exchange in rabbit, rat, and human duodenum. Gastroenterology 122: 709–724, 2002.[ISI][Medline]
  26. Jiang Z, Grichtchenko II, Boron WF, and Atonson PS. Specificity of anion exchange mediated by mouse Slc26a6. J Biol Chem 277: 33963–33967, 2002.[Abstract/Free Full Text]
  27. Ko SBH, Luo X, Hager H, Rojek A, Choi JY, Licht C, Suzuki M, Muallem S, Nielsen S, and Ishibashi K. AE4 is a DIDS-sensitive Cl/HCO3 exchanger in the basolateral membrane of the renal CCD and the SMG duct. Am J Physiol Cell Physiol 283: C1206–C1218, 2002.[Abstract/Free Full Text]
  28. Ko SBH, Shcheynikov N, Choi JY, Luo X, Oshibashi K, Thomas PJ, Kim JY, Kim KH, Lee MG, Naruse S, and Muallem S. A molecular mechanism for aberrant CFTR-dependent HCO3 transport in cystic fibrosis. EMBO J 21: 5662–5672, 2002.[Abstract/Free Full Text]
  29. Ko SBH, Zeng W, Dorwart MR, Luo X, Kim KH, Millen L, Goto H, Naruse S, Soyombo A, Thomas PJ, and Muallem S. Gating of CFTR by the STAS domain of SLC26 transporters. Nat Cell Biol 6: 343–350, 2004.[CrossRef][ISI][Medline]
  30. Lamprecht G, Heil A, Baisch S, Lin-Wu E, Yun CC, Kalbacher H, Gregor M, and Seidler U. The down regulated in adenoma (dra) gene product binds to the second PDZ domain of the NHE3 kinase A regulatory protein (E3KARP), potentially linking intestinal Cl/HCO3 exchange to Na+/H+ exchange. Biochemistry 41: 12336–12342, 2002.[CrossRef][ISI][Medline]
  31. 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]
  32. 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]
  33. Linsdell P, Tabcharani JA, Rommens JM, Hou YX, Chang XB, Tsui LC, Riordan JR, and Hanrahan JW. Permeability of wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride channels to polyatomic anions. J Gen Physiol 110: 355–364, 1997.[Abstract/Free Full Text]
  34. Lohi H, Lamprecht G, Markovich D, Heil A, Kujala M, Seidler U, and Kere J. Isoforms of SLC26A6 mediate anion transport and have functional PDZ interaction domains. Am J Physiol Cell Physiol 284: C769–C779, 2003.[Abstract/Free Full Text]
  35. Luo X, Choi JY, KOSBH, Pushkin A, Kurtz I, Ahn W, Lee MG, and Muallem S. HCO3 salvage mechanisms in the submandibular gland acinar and duct cells. J Biol Chem 276: 9808–9816, 2001.[Abstract/Free Full Text]
  36. Melvin JE, Park K, Richardson L, Schultheis P, and Shull GE. Mouse down-regulated in adenoma (DRA) is an intestinal Cl/HCO3 exchanger and is up-regulated in colon of mice lacking the NHE3 Na+/H+ exchanger. J Biol Chem 274: 22855–22861, 1999.[Abstract/Free Full Text]
  37. Moczydlowski EG. Electrophysiology of the cell membrane. In: Medical Physiology, edited by Boron WF and Boulpaep EL. New York: Elsevier Science, 2002, chapt. 6, p. 266–302.
  38. Moseley RH, Hoglund P, Wu GD, Silberg DG, Haila S, de la Chapelle A, Holmberg C, and Kere J. Downregulated in adenoma gene encodes a chloride transporter defective in congenital chloride diarrhea. Am J Physiol Gastrointest Liver Physiol 276: G185–G192, 1999.[Abstract/Free Full Text]
  39. Mount DB and Romero MF. The SLC26 gene family of multifunctional anion exchangers. Pflügers Arch 447: 710–721, 2004.[CrossRef][ISI][Medline]
  40. Novak I and Greger R. Properties of the luminal membrane of isolated perfused rat pancreatic ducts. Pflügers Arch 411: 546–553, 1988.[CrossRef][ISI][Medline]
  41. O'Loughlin EV, Hunt DM, Bostrom TE, Hunter D, Gaskin KJ, Gyory A, and Cockayne DJH. X-ray microanalysis of cell elements in normal and cystic fibrosis jejunum: evidence for chloride secretion in villi. Gastroenterology 110: 411–418, 1996.[ISI][Medline]
  42. Ohrui T, Skach W, Thompson M, Matsumoto-Pon J, Calayag C, and Widdicombe JH. Radiotracer studeis of cystic fibrosis transmembrane conductance regulator. Am J Physiol Cell Physiol 266: C1586–C1593, 1994.[Abstract/Free Full Text]
  43. Petrovic S, Wang Z, Ma L, Seidler U, Forte JG, Shull GE, and Soleimani M. Colocalization of the apical Cl/HCO3 exchanger PAT1 and gastric H-K-ATPase in stomach parietal cells. Am J Physiol Gastrointest Liver Physiol 283: G1207–G1216, 2002.[Abstract/Free Full Text]
  44. 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/Free Full Text]
  45. Powell DW. Intestinal water and electrolyte transport. In: Physiology of the Gastrointestinal Tract, edited by Johnson LR. New York: Raven, 1987, p. 1267–1306.
  46. Praetorius J, Friis UG, Ainsworth MA, De Muckadell OBS, and Johansen T. The cystic fibrosis transmembrane conductance regulator is not a base transporter in isolated duodenal epithelial cells. Acta Physiol Scand 174: 327–336, 2002.[CrossRef][ISI][Medline]
  47. Pratha VS, Hogan DL, Martensson BA, Bernard J, Zhou R, and Isenberg JI. Identification of transport abnormalities in duodenal mucosa and duodenal enterocytes from patients with cystic fibrosis. Gastroenterology 118: 1051–1060, 2000.[ISI][Medline]
  48. Reddy MM and Quinton PM. Control of dynamic CFTR selectivity by glutamate and ATP in epithelial cells. Nature 423: 756–760, 2003.[CrossRef][ISI][Medline]
  49. 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 505: 411–423, 1997.[Abstract]
  50. Shcheynikov N, Kim KH, Kim K, Dorwart MR, Ko SBH, Goto H, Naruse S, Thomas PJ, and Muallem S. Dynamic control of cystic fibrosis transmembrane conductance regulator Cl/HCO3 selectivity by external Cl. J Biol Chem 279: 21857–21865, 2004.[Abstract/Free Full Text]
  51. Sheldon RJ, Malarchik ME, Fox DA, Burks TF, and Porreca F. Pharmacological characterization of neural mechanisms regulating mucosal ion transport in mouse jejunum. J Pharmacol Exp Ther 249: 572–582, 1988.[ISI]
  52. Short DB, Trotter KW, Reczek D, Kreda SM, Bretscher A, Boucher RC, Stutts MJ, and Milgram SL. An apical PDZ protein anchors the cystic fibrosis transmembrane conductance regulator to the cytoskeleton. J Biol Chem 273: 19797–19801, 1998.[Abstract/Free Full Text]
  53. 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, 1999.
  54. Snouwaert JN, Brigman KK, Latour AM, Malouf NN, Boucher RC, Smithies O, and Koller BH. An animal model for cystic fibrosis made by gene targeting. Science 257: 1083–1088, 1992.[ISI][Medline]
  55. Spiegel S, Phillipper M, Rossmann H, Riederer B, Gregor M, and Seidler U. Independence of apical Cl/HCO3 exchange and anion conductance in duodenal HCO3 secretion. Am J Physiol Gastrointest Liver Physiol 285: G887–G897, 2003.[Abstract/Free Full Text]
  56. Strong TV, Boehm K, and Collins FS. Localization of cystic fibrosis transmembrane conductance regulator mRNA in the human gastrointestinal tract by in situ hybridization. J Clin Invest 93: 347–354, 1994.[ISI][Medline]
  57. Tabcharani JA, Rommens JM, Hou YX, Chang XB, Tsui LC, Riordan JR, and Hanrahan JW. Multi-ion pore behavior in the CFTR chloride channel. Nature 366: 79–82, 1993.[CrossRef][ISI][Medline]
  58. Thomas JA, Buchsbaum RN, Zimniak A, and Racker E. Intracellular pH measurements in ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 18: 2210–2218, 1979.[CrossRef][ISI][Medline]
  59. Walker NM, Flagella M, Gawenis LR, Shull GE, and Clarke LL. An alternate pathway of cAMP-stimulated Cl secretion across the NKCC1-null murine duodenum. Gastroenterology 123: 531–541, 2002.[CrossRef][ISI][Medline]
  60. Weintraub WH and Machen TE. pH regulation in hepatoma cells: roles for Na-H exchange, Cl-HCO3 exchange, and Na-HCO3 cotransport. Am J Physiol Gastrointest Liver Physiol 257: G317–G327, 1989.[Abstract/Free Full Text]
  61. Willumsen NJ and Boucher RC. Intracellular pH and its relationship to regulation of ion transport in normal and cystic fibrosis human nasal epithelia. J Physiol 455: 247–269, 1992.[Abstract]
  62. Xie Q, Welch R, Mercado A, Romero MF, and Mount DB. Molecular characterization of the murine Slc26a6 anion exchanger: functional comparison with Slc26a1. Am J Physiol Renal Physiol 283: F826–F838, 2002.[Abstract/Free Full Text]
  63. Xu J, Barone S, Petrovic S, Wang Z, Seidler U, Riederer B, Ramaswamy K, Dudeja PK, Shull GE, and Soleimani M. Identification of an apical Cl/HCO3 exchanger in gastric surface mucous and duodenal villus cells. Am J Physiol Gastrointest Liver Physiol 285: G1225–G1234, 2003.[Abstract/Free Full Text]
  64. Zeiher BG, Eichwald E, Zabner J, Smith AP, Puga PB, McCray PB, Capecchi MR, Welsh MJ, and Thomas KR. A mouse model for the {Delta}F508 allele of cystic fibrosis. J Clin Invest 96: 2051–2064, 1995.[ISI][Medline]




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
288/6/G1241    most recent
00493.2004v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (3)
Google Scholar
Articles by Simpson, J. E.
Articles by Clarke, L. L.
Articles citing this Article
PubMed
PubMed Citation
Articles by Simpson, J. E.
Articles by Clarke, L. L.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.