REPORT
Identification of a basolateral Clminus /HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger specific to gastric parietal cells

Snezana Petrovic1,2, Xie Ju1, Sharon Barone1, Ursula Seidler3, Seth L. Alper4, Hannes Lohi5, Juha Kere6, and Manoocher Soleimani1,2

1 Department of Medicine, University of Cincinnati, Cincinnati 45267; 2 Veterans Affairs Medical Center at Cincinnati, Cincinati, Ohio 45220; 3 Department of Medicine, University of Tubingen, 72076 Tubingen, Germany; 4 Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215; 5 Department of Medical Genetics, University of Helsinki Finland, 00014 Helsinki, Finland; and 6 Department of Biosciences at Novum, Karolinska Institute, 14157 Huddinge, Sweden


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in parietal cells plays an essential role in gastric acid secretion mediated via the apical gastric H+-K+-ATPase. Here, we report the identification of a new Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger, which shows exclusive expression in mouse stomach and kidney, with expression in the stomach limited to the basolateral membrane of gastric parietal cells. Tissue distribution studies by RT-PCR and Northern hybridizations demonstrated the exclusive expression of this transporter, also known as SLC26A7, to stomach and kidney, with the stomach expression significantly more abundant. No expression was detected in the intestine. Cellular distribution studies by RT-PCR and Northern hybridizations demonstrated predominant localization of SLC26A7 in gastric parietal cells. Immunofluorescence labeling localized this exchanger exclusively to the basolateral membrane of gastric parietal cells, and functional studies in oocytes indicated that SLC26A7 is a DIDS-sensitive Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger that is active in both acidic and alkaline pHi. On the basis of its unique expression pattern and function, we propose that SLC26A7 is a basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in gastric parietal cells and plays a major role in gastric acid secretion.

SLC26A7; AE2


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

GASTRIC EPITHELIUM IS COMPRISED of several structurally and physiologically distinct cell populations that are essential to the digestive process. Parietal cells secrete an acidic juice, whereas mucous cells (or surface epithelial cells) secret a bicarbonate-rich fluid that is essential in protecting the gastric mucosa from the acid injury and ulcer (7, 9, 10, 12, 13, 37, 38). Acid secretion in parietal cells occurs via the apical gastric H+-K+-ATPase, a P-type ATPase that is present in tubulovesicular and canalicular membranes of the gastric parietal cells (1, 11, 18, 38). Extrusion of acid across the apical membrane results in the generation of intracellular hydroxyl ions (OH-), which are converted to HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> by intracellular carbonic anhydrase and then transported across the basolateral membrane of parietal cell via a Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger (10). By transporting the intracellular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in exchange for the extracellular Cl-, the basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger plays an essential role in acid secretion by preventing intracellular alkalinization and, at the same time, providing intracellular Cl-, thereby maintaining HCl secretion across the apical membrane during acid stimulation (10, 24, 26).

The anion exchanger AE2 is located on the basolateral membrane of gastric parietal cells (6, 20, 30, 47). On the basis of functional studies demonstrating mediation of Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange and based on its localization, it has been presumed that AE2 is the major HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-absorbing transporter in parietal cells (29, 30). However, certain functional properties of basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in parietal cells differ from the known functional properties of AE2 (26, 35, 36), raising the possibility that other basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger(s) may be present in parietal cells and play an important role in bicarbonate exit.

Recent studies (3, 8, 14, 15, 21-23, 25, 34, 39, 41, 46, 49) have identified a family of anion exchangers referred to as the SLC26A family, which include at least 10 distinct genes. Three well-known members of this family are SLC26A3 (or DRA), SLC26A4 (or pendrin), and SLC26A6 (PAT1 or CFEX) (8, 16, 21, 22, 46). Each of the above transporters is located apically in a limited and distinct number of epithelia and functions as a Cl-/OH-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger (25, 41, 46). DRA is predominantly expressed in the large intestine, with lower levels in the small intestine (25), whereas PAT1 is predominantly expressed in the small intestine with very low expression levels in the large intestine (46). Pendrin is located on the apical membrane of thyroid follicular cells (31) and kidney collecting ducts (32, 40).

A recently cloned member of the SLC26A family is SLC26A7, which has been shown to be expressed in kidneys and testes (23). In these studies, we investigated the gastrointestinal distribution and functional identity of SLC26A7, because little is known about this transporter. Our results indicate that SLC26A7 expression in the gastrointestinal tract is limited to the stomach, with predominant expression in parietal cells. SLC26A7 functions as a Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger and is located on the basolateral membrane of parietal cells. Due to its unique expression in the stomach and its functional mode, SLC26A7 may play an important role in acid secretion by exchanging the intracellular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> for the extracellular Cl- across the basolateral membrane of gastric parietal cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

RT-PCR of SLC26A7 in Mouse and Rabbit Tissues

A blast search of mouse expressed sequence tag (EST) database against the human SLC26A7 sequence (GenBank accession no. AF331521) identified a mouse EST (Genbank accession no. BB666404) with a high degree of sequence homology. On the basis of the cDNA sequence of the mouse EST, the following oligonucleotide primers: 5'-CTC ACC ACC GAA CCT ATT AC-3' (sense) and 5'-AAC TCG GAT AAG CCC AAC AC-3' (antisense), were designed and used for RT-PCR on RNA isolated from the mouse kidney and other tissues. A <50-bp PCR fragment was purified and sequenced that corresponded to the human nucleotides 8-550, 199.

For studies in rabbit stomach, the following mouse primers were designed and used for RT-PCR on RNA isolated from gastric parietal cells or mucous cells: 5'-TTG TTC TCG TTA AAG AGC TG-3' (sense) and 5'-ATA TTA GAC AAG CCA CCT GC-3' (antisense). These primers were designed based on the full-length mouse slc26a7 cDNA (GenBank accession no. BC026928), which was deposited in the GenBank after the original studies on mouse EST had been performed in our laboratory. The above primers encode nucleotides 831-1265. To amplify rabbit SLC26A7 by RT-PCR, a gradient-based PCR machine, which allows varying stringency conditions during the amplification process, was used with mouse specific primers. The primers were used for RT-PCR in various tissues and the purified PCR fragments were used as a probe for Northern blot hybridization in mouse tissues.

For PAT1 (SLC26A6), the following primers were designed based on a full-length mouse PAT-1 cDNA (GenBank accession no. AY 032863) and used for RT-PCR on mouse tissues: 5'-CGTCTGCACTGCTCCCTCCTCCATTG-3' (sense) and 5'-GAGTCCCAGG GCATCCATCCATG-3' (antisense). These primers encode nucleotides 45-2498 of the mouse PAT1 cDNA.

RNA Isolation and Northern Blot Hybridization

Total cellular RNA was extracted from various mouse tissues, including gastrointestinal tract segments, kidney, liver, heart, brain, and lung, using Tri Reagent (4). Hybridization was performed according to Church and Gilbert (5). The membranes were washed, blotted dry, and exposed to a PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA). 32P-labeled mouse PCR fragment for SLC26A7 (corresponding to nucleotides 8-550 of human SLC26A7 cDNA) was used as a probe for Northern hybridizations. For gastric H+-K+-ATPase, a ~380 PCR fragment corresponding to nucleotides 1681-2060 of the mouse alpha -subunit cDNA (GenBank accession no. NM_018731) was used for Northern hybridization. For studies in rabbit stomach, RT-PCR was performed on RNA isolated from gastric parietal and mucous cells.

Immunocytochemistry of SLC26A7 in Stomach

Antibodies. For SLC26A7, antibodies against human or mouse sequence were used. For mouse, a synthetic peptide corresponding to the amino acids residues CGAKRKKRSVLWGKMHTP of mouse slc26a7 (using the mouse EST with GenBank accession no. BB666404) was used for polyclonal antibody generation in two rabbits. For human, antibodies were raised against a synthetic peptide based on human SLC26A7 sequence (23). Gastric H+-K+-ATPase beta -subunit antibody was a generous gift from Dr. Forte (Univ. of California, Berkley). For AE2, an isoform-specific antibody was used (29, 30).

Immunoblotting of SLC26A7 in stomach. Mouse microsomal membranes from the stomach were isolated according to established methods (44). Immunoblotting experiments were carried out as previously described (46). Briefly, the solubilized membrane proteins were size fractionated on 8% SDS polyacrylamide minigels (Novex, San Diego, CA) under denaturing conditions, electrophoretically transferred to nitrocellulose membranes, blocked with 5% milk proteins, and then probed with the affinity-purified anti-SLC26A7 immune serum at an IgG concentration of 0.6 µg/ml. The secondary antibody was donkey anti-rabbit IgG conjugated to horseradish peroxidase (Pierce, Rockford, IL). The sites of antigen-antibody complex formation on the nitrocellulose membranes were visualized using a chemiluminescence method (SuperSignal Substrate; Pierce) and captured on light-sensitive imaging film (Kodak).

Immunofluorescense labeling studies. Mice were euthanized with an overdose of pentobarbital sodium and perfused through the left ventricle with 0.9% saline followed by cold 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). Stomachs were removed, cut in tissue blocks, and left in the fixative solution overnight at 4°C. For cryosections, tissue blocks were removed from the fixative solution and soaked in 30% sucrose overnight. The tissue was frozen on dry ice, and 5-µm sections were cut with a cryostat and stored at -80°C until used.

Single and double immunofluorescence labeling were performed as described recently (27). For double labeling with H+-K+-ATPase, gastric H+-K+-ATPase beta -subunit antibody was diluted at 1:250. Goat anti-rabbit IgG conjugated with Alexa Fluor 568 Dye (Molecular Probes, Eugene, OR) and goat anti-mouse IgG conjugated with Alexa Fluor 488 (Molecular Probes) were used at 1:200 and 1:100 dilutions for SLC26A7 and H+-K+-ATPase, respectively. Sections were examined, and images were acquired on a Nikon PCM 2000 laser confocal scanning microscope as 0.5-µm "optical sections" of the stained cells.

Cloning of Human and Mouse SLC26A7

Full-length human SLC26A7 cDNA was cloned from a human kidney cDNA library by PCR using the following primers: 5'-AAA TGA CAG GAG CAA AGA G-3' and 5'-TTA TTG TAG CAG AGG TCA TC-3' (GenBank accession no. AF331521). An ~2-kb PCR fragment was obtained that contained the full-length coding region of the exchanger (corresponding to nucleotides 208-2297). Full-length mouse SLC26A7 cDNA was cloned from a mouse stomach by RT-PCR using the following primers: 5'-AGA AGT TGA CTA CTA CAG GAG G-3' (sense) and 5'-AGT TGC CAA GTC ATA TCA TTC-3' (antisense). These primers encode nucleotides 68-2208 of a mouse SLC26A7 cDNA (GenBank accession no. BC026928). Amplification of the human or mouse SLC26A7 cDNA by PCR was performed according to Clontech Advantage 2 PCR kit (Clontech, Palo Alto, CA) protocol. Each PCR reaction contained 5 µl cDNA, 5 µl 10× PCR buffer, 1 µl 10 mM dNTPs, 10 pmol of each primer, and 1 µl Advantage 2 Polymerase mix in a final volume of 50 µl. Cycling parameters were 95°C, 1 min; 95°C, 30 s; and 68°C, 4 min. After PCR, the product was gel purified (revealing a single band of ~2 kb). Sequence analysis of the PCR products verified the sequences as SLC26A7. The PCR products were ligated into pGEM-T easy vector for expression studies.

Synthesis of SLC26A7 cRNA

The capped SLC26A7 was generated using mMESSAGE mMACHINE Kit (from Ambion, Austin, TX) according to the manufacturer's instructions. Briefly, the plasmids containing the full-length human or mouse cDNA were linearized, and the products were then in vitro transcribed to cRNAs, as described previously (45).

Expression of n SLC26A7 in Xenopus Oocytes

Xenopus oocytes were injected with the human or mouse SLC26A7 cRNA. Briefly, 50 nl cRNA (0.5-1.0 µg/µl) were injected with a Drummond 510 microdispenser via a sterile glass pipette.

pHi studies. pHi in oocytes was measured with the pH-sensitive fluorescent probe 2',7'-bis-(3-carboxypropyl)-5-(6)-carboxyfluorescein acetoxymethyl ester (BCPCF-AM; Molecular Probes), a close analog of BCECF-AM (Molecular Probes) as previously described (33, 45, 46). Oocytes were loaded with 10 µM BCPCF-AM for 5 min at room temperature, transferred on a nylon mesh in a 1-ml perfusion chamber, and perfused at a rate of 3 ml/min with the following solution (in mM): 63 NaCl, 33 NaHCO3, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES. Solutions were constantly gassed with 95% O2-5% CO2 yielding the pH of 7.5 at room temperature. Fluid was delivered to the chamber by a peristaltic pump via CO2-impermeable tubing (Cole Palmer, IL). The chamber was closed by a lid and constantly superfused with the gas mixture of 5% CO2-95% O2 to prevent CO2 loss and keep the constant pH. Ratiometric fluorescence measurements were performed on a Zeiss Axiovert S-100 inverted microscope equipped with Attofluor RatioVision digital imaging system (Attofluor, Rockville, MD). Excitation wavelengths were alternated between 440 and 488 nm, and fluorescence emission intensity was recorded at 520 nm. Data analyses were performed using Attograph and Attoview software packages provided with the imaging system. The ratios were obtained from the submembrane region of the oocytes that were visualized with an achroplan ×40/0.8 water objective with 3.6 mm working distance. Measured excitation ratios were converted to pHi by using a calibration curve that was constructed with high K+/nigericin method (33, 43, 45, 46).

To examine the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger activity, the chamber was first perfused with a Cl-containing solution that consisted of 63 mM NaCl and 33 mM NaHCO3 (see above). Oocytes were then switched to a Cl-free medium (63 mM Na-gluconate and 33 mM NaHCO<UP><SUB>3</SUB><SUP>−</SUP></UP>). All other Cl-containing chemicals (KCl, etc.) were replaced with gluconate salts. This maneuver results in cell alkalinization due to reversal of Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger (41, 46, 48). On pHi stabilization in Cl-free medium, oocytes were switched back to the Cl-containing solution. This should result in cell acidification back to baseline due to activation of Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger. The initial rate of cell pHi recovery was used as the rate of Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger activity (41, 46, 48).

Electrophysiology. Membrane potentials in oocytes injected with SLC26A7 cRNA were measured in response to sequential removal and addition of Cl- using conventional microelectrode technique and as described previously (45). Glass microelectrodes (resistance 3-5 mOmega ) were filled with 3 M KCl and connected to an Axoclamp 2A amplifier (Axon Instruments, Foster City, CA). The digitized signals were stored and analyzed on a personal computer using Axotape (Axon Instruments).

Rabbit Gastric Cell Purification

Cells were purified exactly as described before (30). Briefly, rabbit gastric cells were enzymatically dispersed and loaded into an elutriator (JM 6-C with JE-5.0 rotor; Beckman, Munich, Germany) using a 40-ml chamber and a constant rotor speed of 1,000 rpm. The cells were eluted in six fractions with increasing flow rates (of 15, 25, 50, 65, and 120 ml/min). The cells from fractions 3 and 5 were pelleted, reelutriated in a 5-ml chamber with a constant rotor velocity of 1,780 rpm and increasing flow rates (of 7, 14, 28, 35, 65, and 100 ml/min), loaded onto a Percoll density gradient, and centrifuged at 800 g for 20 min, as described. The upper band of the gradients from fraction 3 (mucous cells) and the upper band of the fraction 5 gradients (parietal cells) were collected, washed, homogenized in cold lysis buffer, and stored at 80°C. The mucous cell fraction consisted of 90% periodic acid-Schiff granule-positive cells, whereas the parietal cell fraction showed a purity of 80-90%.

Materials

[32P]dCTP was purchased from New England Nuclear (Boston, MA). Nitrocellulose filters and other chemicals were purchased from Sigma (St. Louis, MO). RadPrime DNA labeling kit was purchased from GIBCO-BRL. BCECF was from Molecular Probes. mMESSAGE mMACHINE Kit was purchased from Ambion. The human multiple tissue blots were purchased from Clontech.

Statistical Analyses

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


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SLC26A7 mRNA Expression

To examine the distribution of SLC26A7 mRNA in mouse tissues, RT-PCR was performed on RNA isolated from various mouse tissues using mouse specific primers (MATERIALS AND METHODS). Figure 1A is an ethidium bromide staining of an agarose gel and shows that an expected PCR fragment (~550 bp) was identified in RNA isolated from mouse stomach and kidney. Sequencing of the purified band verified its identity as SLC26A7. No PCR fragment was identified in heart, brain, or liver. Figure 1B shows the expression of PAT1 (or SLC26A6) in the same tissues as examined by RT-PCR. As indicated, PAT1 shows a wider distribution pattern and is expressed in brain, kidney, heart, liver, and stomach. To better examine SLC26A7 mRNA expression levels in mouse tissues, RNA isolated from various tissues and segments of gastrointestinal tract was hybridized against an SLC26A7 specific cDNA probe. As indicated, SLC26A7 mRNA was abundantly expressed in the stomach, with lower levels in the kidney (Fig. 1C). These results further demonstrate that SLC26A7 mRNA expression in the gastrointestinal tract is exclusively limited to the stomach and is absent in the small and large intestines (Fig. 1C). This pattern of expression is distinct from SLC26A6 (PAT1), which is abundant in small intestine (duodenum, jejunum, and ileum) and stomach but is very low in the colon (cecum, proximal, and distal colon; see Ref. 46 and Fig. 1B).


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Fig. 1.   A: mRNA expression of SLC26A7 in mouse tissues. Expression of SLC26A7 in mouse tissues by RT-PCR. An ethidium bromide staining of an agarose gel shows that an expected PCR fragment (~550 bp) is amplified from mouse stomach and kidney. B: expression of SLC26A6 (PAT1) in mouse tissues by RT-PCR. An ethidium bromide staining of an agarose gel shows that an expected PCR fragment (~2.4 kb) is amplified from several mouse tissues. C: Northern hybridization of SLC26A7 in mouse tissues. RNA (30 µg) was loaded on each lane. The equity in RNA loading is verified by the abundance of the 28S rRNA. -, Absence of RT; +, presence of RT.

Differential Expression of SLC26A7 mRNA in Parietal and Mucous Cells of Stomach

To determine the cellular distribution of SLC26A7 mRNA in the stomach, RT-PCR and Northern hybridizations were performed on RNA isolated from rabbit gastric parietal and mucous cells. Figure 2A is an ethidium bromide staining of an agarose gel and shows that a PCR fragment (~430 bp) is amplified from both gastric parietal and mucous cells. Sequencing verified the identity of this band as rabbit SLC26A7. The rabbit cDNA fragment shows 88% homology to the human SLC26A7. The sequence of the fragment has been deposited in the GenBank with the accession no. AY 166770. 


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Fig. 2.   A: expression of SLC26A7 in gastric epithelial cells. Expression of SLC26A7 in rabbit gastric epithelial cells by RT-PCR. An ethidium bromide staining of an agarose gel shows that an expected PCR fragment (~430 bp) is amplified from rabbit gastric parietal and mucous cells. B: Northern hybridization of SLC26A7 and gastric H+-K+-ATPase in rabbit gastric epithelial cells. RNA (5 µg) was loaded on each lane. The RNA loading is verified by the abundance of the 28S rRNA.

Figure 2B is a representative Northern hybridization and examines the expression of SLC26A7 in gastric epithelial cells. Figure 2B, bottom shows the 28S rRNA abundance in each lane and indicates equity in RNA loading. SLC26A7 mRNA shows more abundance in parietal cells than mucous cells (Fig. 2B). To determine the degree of cross contamination of mucous cells by parietal cells, the membrane was stripped and reprobed for gastric H+-K+-ATPase alpha -subunit expression. Comparison of the mRNA levels of gastric H+-K+-ATPase (which should only be expressed in parietal cells) and SLC26A7 shows an identical expression pattern in both cells, consistent with mild cross-contamination of mucous cells by parietal cells. Together, these results indicate that SLC26A7 mRNA is predominantly expressed in parietal cells.

Immunoblotting and Immunofluorescent Labeling of SLC26A7 in Mouse Stomach

We first examined the specificity of the antibody in microsomal membranes isolated from mouse stomach. Figure 3A demonstrates the labeling of a ~94-kDa band that was blocked with the preadsorbed immune serum.


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Fig. 3.   A: immunocytochemical staining and immunoblotting of SLC26A7 in mouse stomach. Immunoblot analysis of SLC26A7 in microsomal membranes of the stomach. Left: SLC26A7 appears as ~94 kDa. Right: preadsorbed immune serum. B: immunocytochemical staining of SLC26A7 in the stomach. The immunocytochemical staining in the stomach indicated that SLC26A7 is localized on the basolateral membrane of cells in the glandular portion of the stomach (a: low magnification; b and c: high magnification). The labeling with the preadsorbed immune serum failed to detect any labeling in the stomach (C).

To determine the cellular distribution and subcellular localization of SLC26A7, immunofluorescent staining with the purified immune serum was performed in mouse stomach. As shown in Fig. 3B (low and high magnifications), SLC26A7 is localized on the basolateral membrane of cells that are mostly located in the glandular portion of the stomach. There was also intracellular staining in a number of these cells. Preadsorbed serum did not detect any labeling (Fig. 3C). These results are consistent with the basolateral membrane and intracellular localization of SLC26A7 in certain gastric epithelial cells. To determine the identity of SLC26A7-expressing cells, double immunocytochemical staining with gastric H+-K+-ATPase antibody was performed. As shown in Fig. 4, SLC26A7 and gastric H+-K+-ATPase localized to the same cells. These results demonstrate that SLC26A7 is located on the basolateral membrane of gastric parietal cells.


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Fig. 4.   Immunocytochemical double labeling with SLC26A7 and gastric H+-K+-ATPase antibodies. The distribution of gastric H+-K+-ATPase beta -subunit (C) and SLC26A7 (A) correspond to the same cells when dual image was acquired (B).

Immunofluorescent Labeling of AE2 in Mouse Stomach

In this series of experiments, the cell distribution and membrane localization of AE2 in mouse stomach was examined. Figure 5 is a double immunocytochemical staining with AE2 and gastric H+-K+-ATPase antibodies. As shown in both lower magnification and higher magnification in Fig. 5, AE2 and gastric H+-K+-ATPase localized to the same cells in the middle portion of glandular cells. These results demonstrate that AE2 is located on the basolateral membrane of gastric parietal cells.


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Fig. 5.   Immunocytochemical double staining with AE2 and gastric H+-K+-ATPase in mouse stomach. The immunocytochemical staining in the stomach indicated that AE2 is localized on the basolateral membrane of cells in the glandular portion of the stomach (left). The distribution of gastric H+-K+-ATPase beta -subunit (right) and AE2 (left) corresponded to the same cells when dual image was acquired (middle), indicating that AE2-expressing cells are parietal cells. A: low magnification. B: high magnification.

Functional Identity of SLC26A7

In these series of experiments, the functional identity of human SLC26A7 was examined using the oocyte expression system. On the basis of structural similarity with DRA and pendrin, we speculated that SLC26A7 could function in Cl-/base exchange mode. In the first series of experiments, oocytes were injected with human SLC26A7 cRNA, loaded with BCPCF-AM in the presence of a Cl--and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-containing solution (MATERIALS AND METHODS), and monitored for pHi. The representative pHi tracings in Fig. 6A demonstrate that switching to a Cl--free solution resulted in a rapid intracellular alkalinization in oocytes expressing SLC26A7. Switching back to the Cl--containing solution caused a return of pHi to baseline. Control oocytes did not demonstrate any pHi alteration in response to exposure to the Cl--free medium (Fig. 6A). The presence of 0.5 mM DIDS completely inhibited the intracellular alkalinization and the subsequent return to baseline pHi in response to sequential removal and readdition of Cl-, respectively (Fig. 6A). In the absence of bicarbonate, the rate of cell alkalinization in response to Cl- removal was much lower than in the presence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, consistent with the low affinity of SLC26A7 for OH-. The summary of multiple experiments is shown in Fig. 6B and demonstrates that the rate of Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange activity was 0.19 ± 0.04 pH/min in oocytes expressing SLC26A7, whereas it was close to zero in control oocytes (Fig. 6B). These results are consistent with SLC26A7 functioning as a DIDS-sensitive Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger.


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Fig. 6.   Functional identity of SLC26A7. A: Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange activity. Representative tracings (a and b) demonstrating DIDS-sensitive Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger activity in oocytes expressing mouse SLC26A7 cRNA. Oocytes were loaded with 2',7'-bis-(3-carboxypropyl)-5- (6)-carboxyfluorescein (BCPCF) and perfused with solutions corresponding to the figure labels (see MATERIALS AND METHODS for details). Solutions were gassed with 95% O2-5% CO2. As indicated, switching to a Cl--free solution resulted in intracellular alkalinization in oocytes injected with SLC26A7 cRNA (a). The pHi returned to baseline on switching back to the Cl--containing solution. The pHi in oocytes that were injected with water (control group; a) remained unchanged in response to Cl- removal or addition. DIDS, at 0.5 mM, completely inhibited pHi alteration in response to Cl- removal or addition (b). B: summary of pHi experiments. SLC26A7 mediates Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange at the rate of 0.19 pH/min (P < 0.002 vs. control). The anion exchange activity was completely inhibited in the presence of 0.5 mM DIDS. C: sensitivity of the SLC26A7-mediated Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange to inhibition by DIDS. The rate of cell alkalinization in response to Cl- removal was examined in oocytes in the presence of 50, 150, and 450 µM DIDS. DIDS inhibited the SLC26A7 in a dose-dependent manner, with an IC50 of ~126 µM.

Next, we examined the sensitivity of SLC26A7 to inhibition by DIDS. Oocytes that were injected with SLC26A7 were examined for Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger activity in the presence of varying concentrations of DIDS. Accordingly, the rate of cell alkalinization in response to Cl- removal was examined in the presence of increasing concentrations of DIDS (50, 150, 450 µM). The summary of the results is depicted in Fig. 6C. The results indicate that DIDS inhibits the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger mediated by SLC26A7 in a dose-dependent manner, with an IC50 of ~126 µM.

Activation of SLC26A7 at Acidic pHi

The results of the above experiments indicate that SLC26A7 is a Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger located on the basolateral membrane of gastric parietal cells. The functional studies in Fig. 6 demonstrate that switching to a Cl-containing solution results in rapid return of the alkaline pHi to baseline, indicating that SLC26A7 can be activated at alkaline pHi. The purpose of the next series of experiments was to determine whether SLC26A7 can be activated at acidic pHi. Oocytes that were injected with SLC26A7 cRNA were initially perfused with an HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2-free, Na-free solution. Once baseline pHi was established, the perfusate was switched to an HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2-containing, Na-free solution. This maneuver results in intracellular acidification in both control and SLC26A7-injected oocytes. As shown in Fig. 7A, the pHi rapidly recovered to baseline levels in SLC26A7-injected oocytes but remained relatively acidic in control oocytes. The rate of pHi recovery from acidosis was 0.32 pH/min in SLC26A7-injected oocytes and 0.04 in control oocytes (P < 0.004; Fig. 7B). The experiments were performed in the absence of Na in perfusate, indicating the SLC26A7-mediated pHi recovery from acidosis was independent of Na. In oocytes depleted of Cl-, the pHi recovery was almost abolished (data not shown). Together, the results of experiments in Figs. 6 and 7 demonstrate that SLC26A7 is an Na-independent Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger and is functional at both acidic as well as alkaline pHi.


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Fig. 7.   pHi sensitivity of SLC26A7. A: the role of SLC26A7 in pHi recovery from acidosis. Representative pHi tracings (a) demonstrating that oocytes injected with SLC26A7 cRNA recover from acidic pHi induced by exposure to CO2. Control oocytes (b) did not demonstrate any recovery from acidosis. No Na was present in the perfusate (RESULTS). B: summary of pHi experiments. SLC26A7 mediates the recovery from acidic pHi in oocytes at the rate of 0.32 pH/min (P < 0.004 vs. control).

Examination of Electrogenecity of SLC26A7

To determine whether SLC26A7 is electrogenic, membrane potentials were recorded in oocytes injected with SLC26A7 cRNA using a conventional microelectrode technique (45) under conditions that favor Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger activation. Briefly, oocytes were first incubated in a bicarbonate/CO2-containing solution (pH 7.5) and then switched to a bicarbonate-free, CO2-containing solution (pH 6.0). This generates an outward HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> gradient and should result in exchange of intracellular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> for extracellular Cl- in SLC26-A7-injected oocytes. There was a comparable degree of depolarization of membrane potential in both groups (19.2 vs. 17.1 mV in control vs. SLC26A7-injected oocytes, n = 5, P > 0.05). The membrane depolarization that is observed in control oocytes in response to the generation of outward HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> gradient is likely due to the presence of an endogenous anion channel. Together, these results indicate that SLC26A7 is not electrogenic.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SLC26A7 displays a unique expression pattern in the gastrointestinal tract, with abundant mRNA levels in the stomach and no detectable levels in small and large intestines (Fig. 1). Lower levels of SLC26A7 mRNA were detected in the kidney (Fig. 1). This pattern of expression is distinct from the expression of PAT1 (SLC26A6), which shows a wider tissue distribution and is expressed in the small intestine, stomach, kidney, and heart (Fig. 1). SLC26A7 expression in the stomach is predominantly limited to the parietal cells (Fig. 2). Immunofluorescence labeling exclusively localized SLC26A7 to the basolateral membrane of gastric parietal cells (Figs. 3 and 4). Expression studies in oocytes demonstrated that SLC26A7 functions in Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange mode (Fig. 6) and is not electrogenic (RESULTS). SLC26A7 is active at both alkaline and acidic pHi and has an IC50 of ~126 µM for inhibition by DIDS (Figs. 6 and 7).

Acid secretion by gastric parietal cells is dependent on the activity of the basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger, because the extrusion of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and the entry of Cl- across the basolateral membrane via this exchanger are essential for maintaining the pHi and Cl- gradient within a physiological range. This, in turn, allows the secretion of HCl to proceed across the apical membrane during acid stimulation. Reductions in the rate of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> extrusion or Cl- entry will perturb intracellular ionic composition in gastric parietal cells and will result in decreased acid secretion. In support of this mechanism, it was found that inhibition of the basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger inhibited acid secretion in rabbit gastric parietal cells (35, 36).

Northern hybridizations and immunolabeling studies have localized the anion exchanger AE2 on the basolateral membrane of gastric parietal cells (20, 29, 30, 42). On the bases of functional studies in in vitro expression systems demonstrating mediation of Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange and on its localization, it has been presumed that AE2 is the major basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in gastric parietal cells (17, 42). However, a glance at certain functional properties of basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in parietal cells shows differences versus the known functional properties of AE2. For example, the basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in gastric parietal cells is active at acidic pHi and helps with pHi recovery from acidosis, a property not demonstrated by AE2 (26, 35, 36). Furthermore, the basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in parietal cells requires an inhibitory concentration of disulfonic stilbene DIDS that is more than ~100-fold higher (35) than what is needed to inhibit AE2. Together, these observations suggest that basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger(s) distinct from AE2 may play a major role in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> extrusion and the entry of Cl- in gastric parietal cells. Interestingly, SLC26A7 shows a pHi sensitivity profile that is distinct from AE2; whereas AE2 is only active at neutral and alkaline pHi and remains silent at acidic pHi (17), SLC26A7 is active at both alkaline and acidic pHi (Figs. 6 and 7). This latter property of SLC26A7 is in agreement with functional data on Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in basolateral membranes of gastric parietal cells (35, 36). The SLC26A7 is less sensitive to inhibition by DIDS than AE2, with an IC50 of 126 µM for SLC26A7 (vs. a much lower inhibitory concentration for AE2 based on published reports).

The most salient feature of these studies is demonstration of the unique expression pattern of SLC26A7. SLC26A7 expression in the gastrointestinal tract is exclusively limited to the stomach and is absent in the small and large intestines (Figs. 1-4). Furthermore, the expression of SLC26A7 in the stomach is predominantly limited to the basolateral membrane of parietal cells (Figs. 3-4). Coupled to functional studies (Figs. 6 and 7), our studies demonstrate that SLC26A7 is an electroneutral basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in gastric parietal cells. This is the first report on the identification of a basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger specific to gastric parietal cells. This contrasts with AE2, which is ubiquitous in its expression. The unique expression pattern of SLC26A7 in the stomach raises the possibility that this exchanger may play a major role in gastric acid secretion or parietal cell physiology. With respect to epithelial cells, the presence of two proteins with similar function on the same membrane domain usually suggests differential regulation or distinct roles related to the function or physiology of that cell or tissue. We, therefore, like to suggest that although both AE2 and SLC26A7 are basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchangers in gastric parietal cells, they may be essential to apical acid secretion or basolateral HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> transport under different conditions (i.e., distinct regulation by different stimulators such as cAMP, calcium, etc.).

cDNA analysis indicates that SLC26A7 is closely related to a family of anion transport proteins (SLC26A) that include the sulfate-anion transporter (Sat-1 or SLC26A1), the human diastrophic dysplasia sulfate transporter (DTDST or SLC26A2), the downregulated in adenoma (DRA or SLC26A3), pendrin (or SLC26A4), prestin, and PAT1 (SLC26A6) (2, 3, 8, 14, 16, 21, 22, 49). Sequence comparison revealed that SLC26A7 has ~31, 29, and 31% homology with DRA, Pendrin, and PAT1, respectively, at the amino acid level (3, 8, 16, 22). DRA, pendrin, and PAT1 mediate Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange (25, 41, 46). DRA can function in more than one anion exchange mode. Indeed, several studies have suggested that DRA can function as Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (25), Cl-/OH- (28), and sulfate/OH- exchanger (2). PAT1 can function in Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Cl-/OH- exchange modes (46) as well as in Cl-/oxalate and Cl-/formate exchange modes (19, 21). Whether SLC26A7 can function in other anion exchange modes is currently under investigation. A recent study indicates that SLC26A7 can function as a Cl-/oxalate and a Cl-/sulfate exchanger when expressed in oocytes (23).

In conclusion, SLC26A7 shows unique expression in the stomach (and kidney tubules), with no expression in the intestine or other tissues. Northern hybridizations and immunofluorescent staining studies localized SLC26A7 to the basolateral membrane of gastric parietal cells. Functional studies demonstrated that SLC26A7 functions as a Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger. We conclude that SLC26A7 is a basolateral Cl-/base exchanger specific to gastric parietal cells. On the basis of its unique expression and function, we propose that SLC26A7 plays a major role in acid secretion in gastric parietal cells.


    ACKNOWLEDGEMENTS

These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-52820 and DK-54430, a Merit review grant, a Cystic Fibrosis Foundation grant, and grants from the Dialysis Clinic (to M. Soleimani).


    FOOTNOTES

Address for reprint requests and other correspondence: M. Soleimani, Div. of Nephrology and Hypertension, Dept. of Medicine, Univ. of Cincinnati, 231 Albert Sabin Way, MSB G259, Cincinnati, OH 45267-0585 (E-mail: Manoocher.Soleimani{at}uc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpgi.00454.2002

Received 23 October 2002; accepted in final form 7 January 2003.


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