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MUCOSAL BIOLOGY

PAT-1 (Slc26a6) is the predominant apical membrane Cl^–/HCO₃^– exchanger in the upper villous epithelium of the murine duodenum

Departments of ¹Biomedical Sciences and ²Veterinary Pathobiology and ³Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri; ⁴Department of Medicine, Medical University of South Carolina, Charleston, South Carolina; and Departments of ⁵Molecular Genetics, Biochemistry, and Microbiology and ⁶Medicine, University of Cincinnati, Cincinnati, Ohio

Submitted 1 August 2006 ; accepted in final form 12 December 2006

	ABSTRACT

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Basal HCO₃^– secretion across the duodenum has been shown in several species to principally involve the activity of apical membrane Cl^–/HCO₃^– exchanger(s). To investigate the identity of relevant anion exchanger(s), experiments were performed using wild-type (WT) mice and mice with gene-targeted deletion of the following Cl^–/HCO₃^– exchangers localized to the apical membrane of murine duodenal villi: Slc26a3 [down-regulated in adenoma (DRA)], Slc26a6 [putative anion transporter 1 (PAT-1)], and Slc4a9 [anion exchanger 4 (AE4)]. RT-PCR of the isolated villous epithelium demonstrated PAT-1, DRA, and AE4 mRNA expression. Using the pH-sensitive dye BCECF, anion exchange rates were measured across the apical membrane of epithelial cells in the upper villus of the intact duodenal mucosa. Under basal conditions, Cl^–/HCO₃^– exchange activity was reduced by 65–80% in the PAT-1(–) duodenum, 30–40% in the DRA(–) duodenum, and <5% in the AE4(–) duodenum compared with the WT duodenum. SO₄^2–/HCO₃^– exchange was eliminated in the PAT-1(–) duodenum but was not affected in the DRA(–) and AE4(–) duodenum relative to the WT duodenum. Intracellular pH (pH_i) was reduced in the PAT-1(–) villous epithelium but increased to WT levels in the absence of CO₂/HCO₃^– or during methazolamide treatment. Further experiments under physiological conditions indicated active pH_i compensation in the PAT-1(–) villous epithelium by combined activities of Na⁺/H⁺ exchanger 1 and Cl^–-dependent transport processes at the basolateral membrane. We conclude that 1) PAT-1 is the major contributor to basal Cl^–/HCO₃^– and SO₄^2–/HCO₃^– exchange across the apical membrane and 2) PAT-1 plays a role in pH_i regulation in the upper villous epithelium of the murine duodenum.

bicarbonate secretion; intestine; cystic fibrosis transmembrane conductance regulator; Slc4; putative anion transporter 1; downregulated in adenoma; anion exhanger isoform 4

The principal candidates for this process are two members of the multifunctional anion exchanger (Slc26a) family, i.e., Slc26a3 [known as downregulated in adenoma (DRA)] and Slc26a6 [known as putative anion transporter 1 (PAT-1) or Cl^–/formate exchanger] and one member of the HCO₃^– transporter family (Slc4a), i.e., Slc4a9 [known as anion exchanger isoform 4 (AE4)]. All three anion exchangers have been immunolocalized to the apical membrane of the intestinal epithelium, especially along the villous axis (18, 22, 45, 48). Studies utilizing recombinant proteins have shown that DRA transports HCO₃^– in exchange for luminal Cl^–, SO₄^2–, or oxalate (29). PAT-1 is more versatile, demonstrating Cl^–, oxalate, SO₄^2–, formate, HCO₃^–, and hydroxyl transport in Xenopus oocytes (29). Although controversial (7), recent studies have suggested that both DRA and PAT-1 exhibit electrogenic properties, leading to the proposal that that DRA and PAT-1 have apparent stoichiometries of 2 Cl^–:1 HCO₃^– and 1 Cl^–:2 HCO₃^–, respectively (33). Less well characterized is the AE4 protein, but a functional expression study (48) has shown that AE4 exhibits Cl^–/HCO₃^– exchange (48).

Disease-causing loss-of-function mutations have not been reported for human PAT-1 (SLC26A6) or AE4 (SLC4A9). However, loss-of-function mutations in DRA (SLC26A3) are the causal factor in the inherited disease congenital chloride-losing diarrhea (CLD). These patients have defective intestinal Cl^–/HCO₃^– exchange resulting in diarrhea with high Cl^– content and systemic alkalinization (20, 28). Likewise, initial reports of clinical disease in the DRA knockout mouse model (i.e., diarrhea, elevated chloride stool concentration, and growth retardation) closely resemble that of human CLD (6). The severe intestinal manifestations of CLD may reflect an interaction between DRA and the major apical membrane Na⁺/H⁺ exchangers (NHE2 and NHE3) responsible for electroneutral NaCl absorption, as based on evidence of functional coupling of these exchangers in recombinant cell systems and changes in DRA expression in the NHE3 knockout intestine (26, 30).

Duodenal villi are directly exposed to gastric acid chyme from the stomach, but little is known regarding the physiology of HCO₃^– transport in the villous epithelium where DRA, PAT-1, and AE4 are reportedly expressed. Recently, we (35) have demonstrated the capability to directly measure Cl^–/HCO₃^– exchange activity across the apical membrane of epithelial cells in the upper half of villi in the intact murine duodenal mucosa using BCECF-AM microfluorometry of intracellular pH (pH_i). This investigation found robust Cl^–/HCO₃^– exchange activity in the upper villus with exchange rates similar to or greater than those reported for exchanger isoforms expressed in heterologous cell systems (7, 38, 45); however, the in situ exchange process varied from recombinant protein studies by exhibiting relative insensitivity to distilbenes but not niflumic acid. It is important to examine the activity of specific anion exchangers in the native intestinal epithelium, but, unfortunately, there are no known inhibitors of anion exchangers that effectively discriminate between exchanger isoforms or lack additional effects on other anion transport proteins. Therefore, in the present study, we used the intestinal mucosa from mice with gene-targeted deletions of PAT-1, DRA, and AE4 and their wild-type (WT) littermates to assess the relative contributions of each exchanger to the process of apical Cl^–/HCO₃^– exchange across the duodenal upper villous epithelium.

	MATERIALS AND METHODS

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Animals. The experiments in this study were performed on mice with gene-targeted disruptions of the murine homologs of PAT-1 (44), DRA (6), or AE4 on a mixed genetic background. AE4-deficient mice were prepared by deleting a portion of exon 12 and all of exon 13, thereby eliminating codons 558–626 of mouse AE4 (Accession No. AK142545) and terminating the open reading frame at the beginning of the transmembrane domains (L. R. Gawenis and G. E. Shull, unpublished data). All comparisons of homozygous knockout (–/–) mice were made with gender- and age-matched (+/+; WT) siblings. Mutant mice were identified using a PCR-based analysis of tail-snip DNA, as previously described (9). All mice were maintained ad libitum on standard laboratory chow (Formulab 5008 Rodent Chow, Ralston Purina) and tap water. Mice were housed singly in a temperature (22–26°C)- and light (12:12-h light-dark cycle)-controlled room in the American Association for Accreditation of Laboratory Animal Care-accredited animal facility at Dalton Cardiovascular Research Center. Intestinal tissues for experiments were obtained from mice of 2–4 mo of age. These mice were fasted overnight prior to 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 measurements of pH_i and image analysis. The method used for imaging villous epithelial cells in the intact murine intestine has been previously described (16, 35). Briefly, after the mouse had been euthanasized, the proximal duodenum was isolated, stripped of the outer muscle layers by blunt dissection, and mounted luminal side up in a horizontal Ussing-type perfusion chamber where luminal and serosal surfaces of the tissue were independently bathed. The luminal superfusate was an isethionate-bicarbonate-Ringer (IBR) solution containing (in mmol/l) 140.0 Na⁺, 55.0 Cl^–, 55.0 isethionate^–, 25.0 HCO₃^–, 5.2 K⁺, 5.0 TES, 4.8 gluconate^–, 2.8 PO₄^3–, 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). The serosal superfusate was a Cl^–-free IBR solution (with Cl^– replaced with isethionate) gassed with 95% O₂-5% CO₂ at 37°C (pH 7.4). All duodena were treated with indomethacin (1 µM, bilateral) and TTX (0.1 µM, serosal) to minimize the effect of endogenous prostaglandins and neural tone, respectively (5, 34). During superfusion, the duodenum was treated on the serosal side with 1 µM EIPA to block the activity of NHE1. Villi immobilized by a fine nylon mesh overlay were incubated for 5 min with a luminal solution containing 100 µM DL-DTT to remove mucus and then incubated on the luminal side with 16 µM BCECF-AM for 10 min before superfusion of the luminal surface. Using a x40 water-immersion objective (Olympus, Melville, NY), 10 epithelial cells from the mid to upper region of a single villus were selected for ratiometric analysis. Changes in pH_i were measured by dual-excitation wavelength techniques (440 and 495 nm), and villi were imaged at 535-nm emission. Ratiometric images were obtained at 20-s intervals with a Sensi-Cam digital camera (Cooke, Auburn Heights, MI) and processed using Axon Imaging Workbench 2.2 (Axon Instruments, Union City, CA). The 495-to-440-nm ratios were converted to pH_i using a standard curve generated by the K⁺/nigericin technique (4, 40). The intrinsic buffering capacity (_i) of duodenal villous cells was estimated by the ammonium prepulse technique, and the total buffering capacity (_total) was calculated from the equation _total = _i + _HCO3^– = _i + 2.3 x [HCO₃^–]_i, where _HCO3^– is the buffering capacity of the HCO₃^–/CO₂ system and [HCO₃^–]_i is the intracellular concentration of HCO₃^–. The rate of pH_i change during the initial 90-s period of linear pH/t change was converted to the transmembrane flux (J) of HCO₃^– or H⁺ (J_HCO3^– and J_H⁺; measured in mM/min) using the following equation: J = pH/t x _total (46).

Measurement of apical Cl^–/HCO₃^– and Na⁺/H⁺ exchange. For measurements of Cl^–/HCO₃^– exchange, duodenal preparations were superfused with IBR solution on the luminal side, and pH_i alkalinization was induced by replacement of Cl^– with isethionate^– on an equimolar basis. After a stable pH_i (2 min) had been attained, pH_i recovery was initiated by the readdition of Cl^– to the luminal superfusate. In some experiments, SO₄^2– was used to replace Cl^– on an equimolar basis during pH_i recovery. For methazolamide experiments, 100 µM methazolamide was initially added to the superfusate from a 10 mM stock in Cl^–-free IBR solution. For bilateral Krebs-bicarbonate-Ringer (KBR) experiments, the luminal superfusate was KBR solution, which contained (in mM) 140.0 Na⁺, 5.2 K⁺, 25 HCO₃^–, 5 TES, 2.8 PO₄^3–, 110 Cl^–, 1.2 Ca²⁺, 1.2 Mg²⁺, 4.8 gluconate^–, and 16.8 mannitol and was gassed with 95% O₂-5% CO₂ at 37°C (pH 7.4). The serosal perfusate was similar in composition to the luminal solution except that 10 mM mannitol was replaced (equimolar) with glucose. Rates of anion exchange during alkalinization and recovery (pH_i/min) were calculated from a linear regression of the values from the first 90 s of the initial pH_i changes during Cl^– removal and replacement, respectively, and these rates were converted to J_HCO3–.

For measurements of apical membrane Na⁺/H⁺ exchange activity, the proximal duodenum was superfused with nominally CO₂/HCO₃^–-free solutions where NaHCO₃ was replaced equimolar with NaTES and gassed with 100% O₂. Experiments to measure Na⁺/H⁺ exchange consisted of pH_i acidification induced by replacement of luminal Na⁺ with NMDG⁺ on an equimolar basis. After a stable pH_i had been obtained (2 min), pH_i recovery was initiated by replacing NMDG⁺ with Na⁺. Rates of anion exchange during Na⁺/H⁺ exchange were calculated from a linear regression of the values from the first 90 s of the initial pH_i changes during Na⁺ replacement, and these rates were converted to J_H⁺.

Experiments of recombinant murine DRA. Murine (m)DRA (a gift of J. Melvin, University of Rochester) was inserted into the multiple cloning site of a bicistronic pIRES plasmid (Invitrogen, Carlsbad, CA) containing DSRed2, a red fluorescent protein (RFP). The resulting plasmid was transformed into DH5-competent cells (Invitrogen) by heat shock, and single colonies were picked for plasmid isolation (after overnight growth) by the alkaline lysis method. The plasmid was digested with HincII to confirm the proper orientation of the DRA insert. Plasmid containing mDRA or empty vector (control) was transfected using Superfect transfection reagent (Qiagen, Valencia, CA) according to the manufacturer's directions into CHO cells grown on coverslips (50% confluent). Cells expressing RFP were selected for pH_i experiments 24 h after transfection.

Isolation of villous epithelium and RT-PCR. Duodenal segments were resected, opened longitudinally along the mesenteric border, and washed in oxygenated physiological Ringer solution containing 100 µM DTT. Under a dissecting microscope, the segment was placed mucosal side up over a parallel ridge (1-mm height) on a plastic surface and drawn beneath a no. 11 scalpel blade (perpendicular to the villi), thereby scraping the villous epithelium from the villous cores. Isolated villi were dispersed in a small volume (0.5 ml) of oxygenated Ringer solution containing 800 U/ml RNAse inhibitor (Qiagen) using a large-bore transfer pipette and immediately frozen in liquid nitrogen. Total RNA was isolated using TRI-Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's instructions and dissolved in 100 µl RNAse-free water. The Superscript preamplification system (Invitrogen) was used for first-strand cDNA synthesis according to the manufacturer's instructions. Each PCR of 25 µl contained 1 µl cDNA, 1x PCR Mg²⁺-free buffer, 2 mM MgCl₂, 0.2 mM dNTP, 0.4 mM primer, and 0.5 U/reaction Taq DNA polymerase (Promega, Madison, WI). A master mix was made and added to 0.2-ml reaction tubes containing either cDNA (±reverse transcriptase) or water (as the negative control). Primers for DRA, PAT-1, and AE4 were designed based on murine sequences from the National Center for Biotechnology Information database. The primers for DRA were 5'-GGTTTAGCATTTGCTCTGCTGG-3' and 5'-TTACAGTCATGATGAGTTCGATG-3'. The primers for PAT-1 were 5'-GCGACTCTCTGAAAGAGAAGTG-3' and 5'-TCAGAGTTTGGTGGCCAAAACA-3'. The primers for AE4 were 5'-CATGCCTGGTCAAGAAAGCTAG-3' and 5'-CACTCATGTTACTGGGCCTGGTGG-3'. RT-PCR was performed on samples using RT-PCR cycles consisting of a 5.0-min denaturation at 94°C followed by 60 cycles of 94°C for 30 s, 60°C for 1.0 min, and 72°C for 1.0 min and ended with 7.0 min at 72°C using a MWG Primus 96 plus thermocycler (MWG-Biotech, Highpoint, NC). RT-PCR products were electrophoresed on a 2% agarose gel for analysis. To evaluate genomic DNA contamination of the RNA samples, RT-PCR was performed on samples using the following primers designed to amplify genomic DNA and cDNA of -actin: 5'-TGTTACCAACTGGGACGACA-3' and 5'-TCTCAGCTGTGGTGGTGAAG-3'. The absence of an amplicon in the no reverse transcriptase sample indicated minimal genomic DNA contamination. mRNA expression for each exchanger was compared against a similarly treated tissue control reported to express high levels of the specific transcript.

Western blot analysis of NHE3. For Western blot analysis, duodenal microsomal membranes were prepared from age-matched PAT-1(–) and WT adult mice as previously described (3, 41, 45). Briefly, membrane preparations were separated by SDS-PAGE and transferred to nitrocellulose membranes, blocked with 5% milk proteins, and probed with affinity-purified primary antibodies against NHE3 or -actin. The monoclonal anti-NHE3 antibody was directed against the COOH-terminal 131 amino acids of rabbit NHE3 (41), and -actin monoclonal antibodies were purchased from Alpha Diagnostics (San Antonio, TX). The secondary antibody was an anti-rabbit IgG conjugated to horseradish peroxidase (GIBCO-BRL, Gaithersburg, MD), which was visualized using chemiluminescence (SuperSignal Substrate, Pierce) and captured on light-sensitive imaging film (Kodak). Bands corresponding to NHE3 and -actin proteins were quantified by densitometric analysis (UN-SCAN-IT gel software, Silk Scientific, Orem, UT) and were expressed as percentages of control. The equity in protein loading in all blots was verified by gel staining using Coomassie brilliant blue R-250 (Bio-Rad, Hercules, CA).

Materials. The fluorescent dye BCECF-AM was obtained from Invitrogen. TTX was obtained from BioMol. 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 using a two-tailed unpaired Student t-test assuming equal variances between groups. A probability value of P < 0.05 was considered statistically significant.

	RESULTS

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Expression of DRA, PAT-1, and AE4 in the isolated duodenal villous epithelium. DRA, PAT-1, and AE4 have previously been localized to the apical membrane of the intestinal epithelia (18, 22, 45, 48). To verify their expression in murine duodenal villi, mRNA expression in isolated murine duodenal villi was determined by RT-PCR and compared against a tissue control with high expression levels of the particular transcript (the cecum for DRA and the kidney for PAT-1 and AE4) (21, 22, 26, 45, 47). As shown in Fig. 1, RT-PCR of the microdissected villous epithelium from the murine duodenum showed prominent expression of DRA and PAT-1 and lesser expression of AE4 compared with high-expressing tissue controls.

View larger version (20K): [in this window] [in a new window]   Fig. 1. mRNA expression of downregulated by adenoma (DRA), putative anion transporter 1 (PAT-1), and anion exchanger isoform 4 (AE4) in microdissected murine duodenal villi. RT-PCR showed mRNA expression of DRA, PAT-1, and AE4 in the isolated villous epithelium (v) from wild-type (WT) duodena (representative of 3 experiments). Positive tissue controls were cDNA from the cecum (Ce) for DRA and the kidney (k) for PAT-1 and AE4. Water (w) and no reverse transcriptase RT(–) samples were negative controls. The absence of a band for -actin in the RT(–) control lanes indicates minimal contamination of the sample with genomic DNA.

View larger version (13K): [in this window] [in a new window]   Fig. 2. Anion exchange activity in the duodenal villous epithelium of WT and AE4(–) mice. A and B: summary of HCO3– influx (A) and HCO3– efflux (B) rates during luminal Cl– removal and replacement, respectively, in duodenal villous epithelial cells from WT and AE4(–) mice (n = 5). Cumulative data showed no significant differences between the rates of Cl–/HCO3– exchange in AE4(–) compared with WT mice. C: summary of baseline intracellular pH (pHi) in duodenal villous epithelial cells of WT and AE4(–) mice (n = 5). Cumulative data showed no significant differences between the baseline pHi in AE4(–) compared with WT mice. D: summary of SO42–-dependent HCO3– efflux rates in duodenal villous epithelial cells of WT and AE4(–) mice (n = 3). Cumulative data showed no significant differences between the rates of SO42–/HCO3– exchange in AE4(–) compared with WT mice.

View larger version (20K): [in this window] [in a new window]   Fig. 3. Anion exchange activity in the duodenal villous epithelium of WT and DRA(–) mice. A. and B: summary of HCO3– influx (A) and HCO3– efflux (B) rates in duodenal villous epithelial cells of WT (n = 10) and DRA(–) (n = 7) mice. Cumulative data showed a moderate but significant reduction in the rates of Cl–/HCO3– exchange following luminal Cl– removal (HCO3– influx) and replacement (HCO3– efflux) in DRA(–) compared with WT mice. C: summary of baseline pHi in duodenal villous epithelial cells of WT (n = 10) and DRA(–) (n = 7) mice. Cumulative data showed significantly increased baseline pHi in DRA(–) compared with WT mice. D: summary of SO42–-dependent HCO3– efflux rates in duodenal villous epithelial cells of WT and DRA(–) mice (n = 3). Cumulative data showed no significant differences between the rates of SO42–/HCO3– exchange in DRA(–) compared with WT mice. E: representative pHi trace from murine (m)DRA-transfected CHO cells during extracellular Cl– removal and replacement followed by a second Cl– removal period and extracellular SO42– addition (representative of 4 experiments). Removal of extracellular Cl– resulted in intracellular alkalinization, and the readdition of Cl– led to pHi recovery, demonstrating Cl–/HCO3– exchange. However, SO42– replacement following alkalinization did not lead to appreciable pHi recovery. *Significantly different from WT.

Cl^–/HCO₃^– exchange in the PAT-1(–) upper villous epithelium of the murine duodenum. Previous studies conducted in upper murine duodenal villous epithelia have revealed characteristics of basal anion exchange (e.g., high rates of SO₄^2– transport and enhanced Cl^–/HCO₃^– exchange activity during epithelial cell depolarization) that were most consistent with PAT-1 activity based on recombinant protein expression studies (19, 23, 35, 45, 47). To evaluate the contribution of PAT-1, we compared the rates of apical membrane Cl^–/HCO₃^– exchange activity between PAT-1(–) and WT duodenal villi. As shown by the experiment in Fig. 4A, the rates of alkalinization and recovery during Cl^– substitution/replacement were significantly reduced in the PAT-1(–) duodenum compared with the WT duodenum. Cumulative data from several pH_i experiments on PAT-1(–) and WT epithelia demonstrated that the rates of HCO₃^– influx during Cl^– removal and HCO₃^– efflux during Cl^– replacement were significantly reduced by 65% and 80%, respectively, in PAT-1(–) villi relative to WT villi (Fig. 4, B and C). Paradoxically, the baseline pH_i was significantly more acidic in PAT-1(–) villi relative to WT villi under the conditions of our study (Fig. 4D).

View larger version (20K): [in this window] [in a new window]   Fig. 4. Comparison of Cl–/HCO3– exchange activity and baseline pHi in the duodenal villous epithelium of WT and PAT-1(–) mice. A: representative pHi traces of WT and PAT-1(–) duodenal villous epithelial cells during luminal Cl– removal and replacement. B and C: summary of HCO3– influx (B) and HCO3– efflux (C) rates in duodenal villous epithelial cells from WT and PAT-1(–) mice (n = 5). Cumulative data showed significantly reduced rates of Cl–/HCO3– exchange following luminal Cl– removal (HCO3– influx) and replacement (HCO3– efflux) in PAT-1(–) compared with WT mice. D: summary of baseline pHi in duodenal villous epithelial cells of WT and PAT-1(–) mice (n = 5). Cumulative data showed significantly reduced baseline pHi in PAT-1(–) compared with WT mice. *Significantly different from WT.

View larger version (14K): [in this window] [in a new window]   Fig. 5. Comparison of SO42–/HCO3– exchange in the duodenal villous epithelium of WT and PAT-1(–) mice. A: representative pHi traces of WT and PAT-1(–) duodenal villous epithelial cells. Following alkalinization secondary to luminal Cl– removal, SO42– was introduced in the luminal superfusate for SO42–in/HCO3–out exchange. B: summary of HCO3– efflux rates induced by luminal SO42– application in duodenal epithelial cells from WT (n = 3) and PAT-1(–) (n = 5) mice. Cumulative data showed significantly reduced rates of SO42–/HCO3– exchange in PAT-1(–) compared with WT mice. *Significantly different from WT.

View larger version (20K): [in this window] [in a new window]   Fig. 6. Comparison of Na+/H+ exchanger isoform 3 (NHE3) expression and apical membrane Na+/H+ exchange activity in WT and PAT-1(–) small intestines. A, top: representative Western blot of NHE3 and -actin protein (as a loading control) in duodenal microsomes of gender-matched WT and PAT-1(–) littermate pairs. Bottom, densiometric analysis of protein expression for NHE3 normalized to expression of -actin (n = 4 mouse pairs). These data showed no significant differences of NHE3 protein expression in PAT-1(–) compared with WT brush-border membrane vesicles. B: summary of Na+-dependent H+ efflux rates in duodenal villous cells of WT and PAT-1(–) mice (n = 3). Cumulative data showed no significant differences between the rates of Na+/H+ exchange in PAT-1(–) compared with WT duodenal villi.

View larger version (14K): [in this window] [in a new window]   Fig. 7. Baseline pHi experiments in the duodenal villous epithelium of WT and PAT-1(–) mice. A: summary of baseline pHi in duodenal villous epithelial cells of WT and PAT-1(–) (n = 3) mice in the absence of CO2/HCO3–. Cumulative data showed no significant differences in baseline pHi of PAT-1(–) compared with WT mice. B: summary of baseline pHi in duodenal villous epithelial cells of WT and PAT-1(–) (n = 3) mice in the presence of bilateral methazolamide (100 µM) to inhibit carbonic anhydrase activity. Cumulative data showed no significant differences in baseline pHi of PAT-1(–) compared with WT mice. C: summary of baseline pHi in duodenal villous epithelial cells of WT and PAT-1(–) (n = 5) mice in the absence of serosal EIPA. Cumulative data showed significantly reduced baseline pHi in PAT-1(–) compared with WT mice. D: summary of baseline pHi in duodenal villous epithelial cells of WT and PAT-1(–) (n = 3) mice with bilateral Krebs-bicarbonate-Ringer (KBR) solution. Cumulative data showed no significant differences in baseline pHi of PAT-1(–) compared with WT mice under physiological conditions. *Significantly different from WT.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

In the present investigation, BCECF microfluorometry experiments of the intact PAT-1(–) duodenum revealed that apical Cl^–/HCO₃^– exchange was significantly reduced by 65–80% compared with the WT duodenum. This finding confirms the results of our previous study (35), which revealed characteristics of anion exchange in the upper villous epithelium that were most consistent with PAT-1 activity. In addition to PAT-1, a finite rate of Cl^–/HCO₃^– exchange was still present in the PAT-1(–) duodenum, indicating the contribution of other anion exchangers such as DRA and AE4. The examination of AE4(–) mice compared with WT mice revealed minimal involvement of AE4 in basal Cl^–/HCO₃^– exchange in the upper villous epithelium. In contrast, the examination of Cl^–/HCO₃^– exchange activity in DRA(–) mice compared with WT mice demonstrated a 30–40% reduction in exchange rates. Thus, these data indicate that PAT-1 is the principal Cl^–/HCO₃^– exchanger in the apical membrane of the upper villous epithelium with a smaller but significant contribution by DRA and minimal contribution from AE4.

The results of the functional experiments suggested that there was limited alteration in the apical surface activity of the remaining anion exchangers in each knockout mouse. In this regard, we noted a minor discrepancy in the estimates for the relative contribution of DRA to the total Cl^–/HCO₃^– exchange activity in the murine upper villous epithelium. Taking the residual Cl^–/HCO₃^– exchange activity in the PAT-1(–) duodenum as a measure of DRA activity, we estimate a minimum contribution of 20% to total Cl^–/HCO₃^– exchange activity (Fig. 4C). On the other hand, the reduction of Cl^–/HCO₃^– exchange activity in the DRA(–) villous epithelium yields a maximal estimate of 40% (Fig. 3B). This discrepancy suggests decreased expression/activity of the remaining anion exchanger in each knockout, e.g., DRA in the PAT-1(–) duodenum and PAT-1 in the DRA(–) duodenum. Thus, compensatory increases in anion exchanger activity are not apparent in the villous epithelium. Precise estimation of the contribution of DRA to the total Cl^–/HCO₃^– exchanger activity will require measurements of anion exchanger expression specifically in the upper villous epithelium, e.g., by using laser capture microdissection or differential cell isolation techniques, which are beyond the scope of the present investigation.

The present investigation found that a distinguishing feature of PAT-1 is its ability to transport SO₄^2–, because SO₄^2–/HCO₃^– exchange was essentially eliminated in the PAT-1(–) villous epithelium compared with the WT villous epithelium. Both PAT-1 and DRA have been reported to be SO₄^2– transporters (29); however, recent studies using recombinant protein expression systems have shown that human DRA does not efficiently transport SO₄^2–, i.e., the rate of SO₄^2– transport is two to three orders of magnitude less than the rate of Cl^–/HCO₃^– exchange (8, 29). In the present study, SO₄^2–/HCO₃^– exchange in DRA(–) duodenal villi was not significantly different from that observed in WT villi, indicating that mDRA also does not appreciably transport SO₄^2–. To verify that mDRA, like human DRA, does not effectively transport SO₄^2–_, we measured SO₄^2–/HCO₃^– exchange activity of recombinant mDRA expressed in CHO fibroblasts. These experiments revealed robust Cl^–/HCO₃^– exchange but little evidence of SO₄^2–/HCO₃^– exchange in mDRA-transfected cells. In addition, the examination of SO₄^2– transport in the surface epithelium of the cecum, where DRA is the dominant apical membrane exchanger, also revealed no detectable SO₄^2– transport (data not shown). Rates of SO₄^2–/HCO₃^– exchange were also measured in the AE4(–) duodenal villous epithelium because studies examining AE4 exchange properties are limited (48). Like the experiments with the DRA(–) epithelium, these experiments revealed no significant differences in SO₄^2–-dependent HCO₃^– efflux in the AE4(–) villous epithelium compared with the WT villous epithelium. Thus, these data show that a distinguishing characteristic of PAT-1 is the capability for SO₄^2–/HCO₃^– exchange in the upper villous epithelium of the murine duodenum.

Measurements of pH_i in the DRA(–) and PAT-1(–) villous epithelium revealed opposite effects of these transporters on pH_i regulation. In the DRA(–) villous epithelium, baseline pH_i was significantly increased compared with the WT epithelium. This change is consistent with the loss of a Cl^–/HCO₃^– exchanger that is normally coupled to NHE3 for electroneutral NaCl absorption, i.e., continued proton efflux via NHE3 without concurrent HCO₃^– efflux would increase pH_i. Although NHE3 may not normally contribute to pH_i regulation in the intestine (14), the genetic ablation of a partnering anion exchanger possibly removes an element of normal regulation for NHE3. Additional studies of DRA(–) mice using the jejunum, where electroneutral NaCl absorption predominates, will be necessary to test this hypothesis. In contrast to the deletion of DRA, measurements of pH_i in the PAT-1(–) villous epithelium revealed a significantly reduced baseline pH_i compared with the WT epithelium. This phenomenon was investigated further because cell acidification resulting from deletion of a base exporter is counterintuitive. One potential explanation is that loss of a coupled interaction between PAT-1 and NHE3 reduces the activity or expression of the NHE. However, apical membrane Na⁺/H⁺ exchange activity and NHE3 protein expression were not different between PAT-1(–) and WT epithelia. An alternative hypothesis is that PAT-1 acts as a base importer (i.e., Cl^–_out/HCO₃^–_in exchange) under the conditions of our study. Consistent with this hypothesis was the demonstration that removal of CO₂/HCO₃^– or treatment with the CA inhibitor methazolamide normalized pH_i between PAT-1(–) and WT epithelia. Based on evidence that PAT-1 may functionally and physically interact with CA II (1), the latter finding suggests a novel interaction between PAT-1 and intracellular CA, i.e., a "reversed" HCO₃^– transport metabolon (25, 39) that facilitates HCO₃^– influx to protect the epithelial cell against intracellular acidification. This may be of physiological importance as the upper villous epithelium of the duodenum is exposed to acid challenge not only from gastric effluent but also from H⁺ influx during nutrient absorption (e.g., apical H⁺/peptide cotransporter 1) (12). Given the high PCO₂ in gastric effluent (17), the source of HCO₃^– for PAT-1 Cl^–_out/HCO₃^–_in exchange may be dependent on extracellular CA activity (27). Although rapid diffusion of CO₂ through cell membranes induces pH_i acidification in the duodenal villous epithelium during luminal exposure to a high-PCO₂/low-HCO₃^– solution (pH 6.4) (15), we found that a greater extent of pH_i acidification occurs during exposure to high-PCO₂/low-HCO₃^– solution (pH 5.4) in the PAT-1(–) duodenum compared with the WT duodenum (WT pH_i = –0.9 ± 0.1 and PAT-1(–) pH_i = –1.3 ± 0.2, n = 3; J. Simpson and L. Clarke, unpublished observations). The recent proposal that PAT-1 is electrogenic with a stoichiometry of 1 Cl^–:2 HCO₃^– (33) would also favor the HCO₃^– influx activity of PAT-1 during depolarization of the villous epithelium, e.g., during Na⁺- or H⁺-coupled nutrient absorption. The resting apical membrane potential and intracellular Cl^– concentrations of the upper villous epithelium under the conditions of our study are presently unknown, so whether the electrochemical gradient favors Cl^–_out/HCO₃^–_in activity by PAT-1 cannot be determined at this time. However, as we (35) have shown previously, depolarization of the upper villous epithelium using high extracellular K⁺ concentration specifically increases the rate of Cl^–_out/HCO₃^–_in exchange. Together, these considerations raise the possibility that PAT-1 plays a role in pH_i regulation, especially during nutrient absorption, by providing a HCO₃^– influx pathway across the apical membrane of the villous enterocyte.

In our study, isolation of apical membrane acid-base transporters required inhibition of basolateral membrane NHE1 and Cl^–/HCO₃^– exchange by EIPA treatment and removal of basolateral Cl^–, respectively. We first investigated whether the activity of NHE1 could normalize basal pH_i in the PAT-1(–) epithelium by removal of basolateral EIPA treatment. Although a trend toward increased pH_i was noted, the PAT-1(–) villous epithelium remained acidic compared with the WT epithelium, indicating that activity of NHE1 alone is apparently unable to compensate for the loss of PAT-1. Next, a physiological Cl^– concentration was included in the basolateral bath in addition to removal of basolateral EIPA treatment. This change corrected the pH_i difference between PAT-1(–) and WT villous epithelia. Thus, under physiological conditions, pH_i of the upper villous epithelium can be normalized in the absence of PAT-1 by a complex process apparently requiring the activity of basolateral transporters.

The activity of PAT-1 in the upper villous epithelium is likely affected by the presence of low levels of CFTR. In our previous study (35) of the CFTR(–) mouse intestine, it was shown that both Cl^–_in/HCO₃^–_out and Cl^–_out/HCO₃^–_in exchange rates were reduced in the upper villous epithelium compared with the WT mouse. The mechanism of decrease was traced to the loss of a CFTR Cl^– leak pathway that facilitated apical membrane Cl^–/HCO₃^– exchange activity. The role of CFTR was considered passive and not due to a direct intermolecular interaction with the anion exchanger(s) (24) because CFTR is expressed at low levels in the upper villous epithelium (thereby minimizing the opportunity for 1:1 stoichiometry between CFTR and a Slc26a exchanger) and the effect could be reproduced by acute CFTR channel blockade. Given the dominance of PAT-1 activity (Fig. 4), it is reasonable to conclude that the CFTR Cl^– leak pathway primarily facilitates PAT-1 and perhaps lesser amounts of DRA activity in the upper villous epithelium. Since CFTR demonstrates ohmic conductance of Cl^– (2), it is reasonable that the Cl^– leak pathway may operate to the advantage of PAT-1 Cl^–/HCO₃^– exchange activity independent of the direction of transport, i.e., providing Cl^– uptake during Cl^–_out/HCO₃^–_in exchange and Cl^– exit during Cl^–_in/HCO₃^–_out exchange.

Although we (31) have previously suggested that the reduced Cl^–/HCO₃^– exchange activity of the upper villous epithelium contributes to deficient basal HCO₃^– secretion across the CFTR(–) duodenum, a subsequent pH stat study (42) of PAT-1(–) mice showed that PAT-1 only contributes 20% to basal HCO₃^– secretion in the WT duodenum. Thus, a 30% reduction in PAT-1 activity in the upper villous epithelium of the CFTR(–) duodenum would have a negligible effect on basal HCO₃^– secretion. Apparently, most transepithelial HCO₃^– secretion under basal conditions comes from the lower villous and crypt epithelium where the activities of DRA or AE4 may provide a major contribution. Given evidence that PAT-1 plays a minor role in transepithelial HCO₃^– secretion under basal conditions, it is possible that the abundant PAT-1 activity in the upper villous epithelium serves other functions, such as pH_i regulation, as suggested above.

In conclusion, our study of PAT-1(–), DRA(–), and AE4(–) mice indicated that PAT-1 is the major contributor to basal Cl^–/HCO₃^– and SO₄^2–/HCO₃^– exchange across the apical membrane of the upper villous epithelium in the murine duodenum. Based on past studies of CFTR activity in this region of the villus, it is likely that PAT-1 activity is facilitated indirectly by a CFTR Cl^– leak pathway that sustains the gradient for Cl^–/HCO₃^– exchange. However, additional experiments in the PAT-1(–) duodenum revealed a propensity for PAT-1 to operate in Cl^–_out/HCO₃^–_in mode for HCO₃^– influx, thereby providing a potentially important mechanism for protecting the upper villous epithelial cell from intracellular acidification. DRA was also found to contribute a smaller but significant portion of Cl^–/HCO₃^– exchange in the upper villous epithelium. DRA deletion resulted in intracellular alkalinization, which may indicate unregulated activity of Na⁺/H⁺ exchange and evidence of DRA's involvement in coupled NaCl absorption in this locale. Therefore, DRA may not significantly contribute to net HCO₃^– secretion under basal conditions from the upper villous epithelium, whereas a recent report (36) from our laboratory suggested that DRA may have a different role in the lower villus epithelium. Thus, it will be necessary to examine anion transport in the lower villus/crypt epithelium where CFTR is predominantly expressed to fully evaluate the contributions of AE4, DRA, and PAT-1 to transepithelial HCO₃^– secretion.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by National Institutes of Health Grants T32-RR-07004 (to J. E. Simpson), DK-48816 (to L. L. Clarke), DK-50594 (to G. E. Shull), CA-95172 (to C.W. Schweinfest), and DK-62809 (to M. Soleimani) and by Cystic Fibrosis Foundation Grant CLARKE05G0 (to L. L. Clarke).

	FOOTNOTES

 

Address for reprint requests and other correspondence: L. L. Clarke, 324D Dalton Cardiovascular Research Center, 134 Research Park Dr., Univ. of Missouri, 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.

¹ No endogenous Cl^–/HCO₃^– exchange was observed in mock-transfected CHO cells, whereas inconsistent levels of activity were found when mock-transfected NIH 3T3 and HEK-293 cells were examined.

² Pharmacological inhibition of NHE1 by 1 µM EIPA added to the basolateral superfusate during a similar time period, i.e., 20 min, has been previously demonstrated in this preparation by a study (43) showing significant acidification of the pH_i in the upper villous epithelium of the NHE3(–) duodenum during 1 µM EIPA treatment.

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