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Colocalization of the apical Clminus /HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger PAT1 and gastric H-K-ATPase in stomach parietal cells

Snezana Petrovic1,5, Zhaohui Wang1, Liyun Ma1, Ursula Seidler4, John G. Forte3, Gary E. Shull2, and Manoocher Soleimani1,5

Departments of 1 Medicine and 2 Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, Ohio 45267-0585; 3 Department of Molecular and Cell Biology, University of California, Berkley, California 94720; 4 University of Tubingen, 72076 Tubingen, Germany; and 5 Veterans Affairs Medical Center at Cincinnati, Cincinnati, Ohio 45220


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger called the putative anion transporter (PAT1; SLC26A6) is expressed on apical membranes of villus cells in the duodenum, but its location in the stomach remains unknown. Here we examined the cell distribution and membrane location of PAT1 in mouse stomach. Immunofluorescence labeling studies with anti-PAT1 antibodies and Dolichos biflorus agglutinin indicated the exclusive expression of PAT1 in gastric parietal cells. Double immunocytochemical staining revealed colocalization of PAT1 with the gastric H-K-ATPase, consistent with expression in tubulovesicles and/or the secretory canaliculus. Radiolabeled 36Cl flux studies demonstrated the functional presence of Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange in purified tubulovesicles of parietal cells. The expression of PAT1 was significantly decreased in parietal cells of gastric H-K-ATPase-null mice, which exhibit a sharp reduction in tubulovesicle membranes. These data indicate that the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger PAT1 is localized on tubulovesicular membranes, and they are consistent with the hypothesis that it functions in the maintenance of intravesicular ion concentrations in the resting state and dehydration of vesicles derived from the secretory membranes following the transition from the stimulated to the resting state.

Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange; putative anion transporter; acid secretion; bicarbonate transport; SLC26A6


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

THE GASTRIC EPITHELIUM COMPRISES several structurally and physiologically distinct cell populations that are essential for the digestive process and for mucosal protection. Secretion of acid into the lumen of the stomach is mediated by the apical H-K-ATPase of the gastric parietal cell (1, 27, 28). Acid is generated within the parietal cell by the conversion of H2O and CO2 to H+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> via the catalytic activity of carbonic anhydrase II (7). HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exits across the basolateral membrane in exchange for Cl-, which is required for sustained secretion of HCl across the apical membrane (7, 20). The available evidence suggests that basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange in the parietal cell, as well as in mucous cells, is mediated by multiple variants of anion exchanger 2 (4, 12, 21, 37), although the presence of other basolateral anion exchangers cannot be ruled out. The identities of apical anion transporters that might be involved in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion, which contributes to mucosal protection (1, 8, 13, 26), have not been determined.

Recent studies have identified a family of anion exchangers, referred to as the SLC26A family, that includes at least nine distinct genes (2, 5, 9, 10, 14, 16, 17, 19, 22-25, 29, 31, 33, 36, 38). Three well-known members of this family are SLC26A3 [downregulated in adenoma (DRA) or congenital Cl- diarrhea], SLC26A4 (pendrin), and SLC26A6 [putative anion transporter (PAT1) or Cl-/formate exchanger] (5, 10, 16, 24). Each of these transporters is located apically in a limited number of epithelial tissues and can function as a Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (OH-) exchanger (19, 33, 36). In addition, PAT1 has been shown to mediate Cl-/formate exchange (14), and pendrin is thought to mediate Cl-/I- exchange (25). DRA is expressed predominately in the colon, with lower levels in the small intestine (19), PAT1 is expressed at high levels in the small intestine (36) and renal proximal tubule (14), and pendrin is expressed in thyroid follicular cells (22) and in kidney collecting ducts (23, 31).

In addition to the kidney and small intestine, PAT1 is also expressed in the stomach (36). In the current studies, we investigated the cell distribution and membrane localization of PAT1 in the stomach. Given its apical location in other tissues, we anticipated that it might contribute to the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion observed in mucous cells (1, 7, 8, 13, 26). Contrary to this hypothesis, our experiments yielded the surprising observations that PAT1 is expressed exclusively in parietal cells, colocalizes with the gastric H-K-ATPase in a pattern consistent with expression in tubulovesicular and/or secretory canalicular membranes, and that Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange activity is present in tubulovesicles isolated from resting parietal cells. These findings raise the intriguing possibility that PAT1 might play important roles in countering acid influx into resting tubulovesicles via the gastric H-K-ATPase and in dehydrating membrane vesicles and vacuoles derived from the secretory canaliculus following transition from the stimulated to the resting state.


    EXPERIMENTAL PROCEDURES
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EXPERIMENTAL PROCEDURES
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Animals

Black Swiss mice (30-35 g) were used for PAT1 localization studies in stomach, duodenum, and colon. Wild-type and gastric H-K-ATPase-null mice (30) were used for colocalization studies of PAT1 and gastric H-K-ATPase in the stomach. Animals had free access to water and food during their stay. For acid stimulation, animals were fasted overnight, injected in the morning with a single dose of intraperitoneal histamine (2 µg/g body wt), and killed 30 min later. All animal protocols were approved by the institutional review committee.

Immunocytochemical Staining

Antibodies. A PAT1-specific antibody, raised against the amino-terminal sequence RRDYHMERPLLNQE (36), was used for these studies. For gastric H-K-ATPase, a monoclonal antibody to the beta -subunit, 2G11, was used for double-labeling studies (3). The alpha -subunit Na-K-ATPase antibody was a generous gift from Dr. J. Lingrel (University of Cincinnati).

Immunofluorescence labeling studies. Animals were euthanized with an overdose of sodium pentobarbital 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, duodena, and colons were removed, cut in tissue blocks, and left in the same 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.

For staining, sections were washed twice in 0.01 M PBS (pH 7.4) and blocked with 10% goat serum/0.3% Triton X-100-PBS solution for 45-60 min. PAT1 antibodies were diluted 1:40 in 1% BSA-0.3% Triton X-100-PBS solution and applied to tissue sections overnight at room temperature. Sections were rinsed and then incubated with a secondary antibody for 2 h at room temperature. Goat anti-rabbit IgGs conjugated with Oregon Green 488 (Molecular Probes, Eugene, OR) were used as a secondary antibody for PAT1 at 1:150 dilution. For double labeling with Dolichos biflorus TRITC-labeled agglutinin (Sigma-Aldrich, St. Louis, MO), sections were simultaneously incubated with the agglutinin (40 µg/ml) and goat anti-rabbit IgGs conjugated with Oregon Green 488. For double labeling with H-K-ATPase, gastric H-K-ATPase beta -subunit antibody was diluted at 1:250 in 1% BSA-0.3% Triton X-100-PBS solution and applied to tissue sections overnight at room temperature simultaneously with PAT1 antibody. Sections were rinsed and then incubated with secondary antibodies for 2 h at room temperature. Goat anti-rabbit IgG conjugated with Oregon Green 488 and goat anti-mouse IgG conjugated with Alexa Fluor 568 dye (Molecular Probes) were used at 1:150 dilution for PAT1 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. The 488-nm line of the argon laser, isolated with the standard argon laser exciter filter supplied with PCM 2000, was used for the green dye excitation. The PCM 2000 standard 515/30-nm emission filter was used for the green-emitting dye. Red dye was excited with the 543.5 nm single line output of the HeNe laser. The PCM 2000 standard red channel long-pass 565-nm filter was used as the emission filter for the red dye. Alexa Fluor 568 excites efficiently at 543 nm and has more red-shifted emission compared with TRITC, making separation from the green channel easier. Digital images of the green and red dyes were simultaneously acquired through a single illumination and detection pinhole. Subsequently, the image was discretely resolved into two separate image channels, so that red, green, and dual-channel, simultaneously-acquired fluorescence images (we refer to these as "dual images") could be separately analyzed.

Preparation of Purified Tubulovesicles From Rabbit Gastric Parietal Cells

Tubulovesicles were isolated from the gastric homogenates of New Zealand White rabbits and purified by differential centrifugation and density gradient flotation as described by Tyagarajan et al. (34).

Immunoblot Analysis of Gastric H-K-ATPase

Tubulovesicle membranes from rabbit parietal cells were prepared as above, resolved by SDS-PAGE (30 µg/lane), and transferred to nitrocellulose membranes. The membrane was blocked with 5% milk proteins and then incubated for 6 h with the H-K-ATPase beta -subunit monoclonal antibody diluted at 1:4,000. The secondary antibody was a goat anti-mouse IgG conjugated to horseradish peroxidase (Pierce, Rockford, IL). The site of antigen-antibody complexation on the nitrocellulose membranes was visualized by chemiluminescence (SuperSignal Substrate; Pierce) and captured on light-sensitive imaging film (Kodak).

Functional Cl-/HCO<UP><SUB><UP>3</UP></SUB><SUP><UP>−</UP></SUP></UP> Exchanger Assay in Tubulovesicles Isolated From Rabbit Parietal Cells

The timed uptake of 36Cl by tubulovesicle suspensions was assayed at room temperature in triplicate by a rapid filtration technique as described (32). All experiments were performed in the presence of valinomycin (0.5 mg/ml), and intracellular and extracellular K+ concentrations were equal during uptake measurement. The details of the experiments are included in the legend to Fig. 5.

RT-PCR of PAT1 in Rabbit Gastric Parietal Cells

Rabbit gastric parietal cells were isolated as described (21) and processed for total RNA isolation. The resulting RNA was poly(A+) selected using Oligotex latex beads (Qiagen) and then reverse transcribed at 47°C by using SuperScript II reverse transcriptase (Life Technologies) and oligo(dT) primers. The oligonucleotide primers (AAG GCC AGC CTG ACT GCA ATA C sense and GGG AGA TTG AAG TGG AAG TGT ACA TC antisense), which encode nucleotides 2162-2550, were designed based on mouse PAT1 cDNAs (GenBank accession no. AY032863). This area is conserved between mouse and human PAT1 (GenBank accession no. AF279265). The expected size of PCR product is 388 bp. These primers are specific for PAT1 and do not recognize other members of SLC26A family. Amplification of the PAT1 cDNA by PCR was performed according to established methods. Each PCR contained 10 µl cDNA, 5 µl 10× PCR buffer (with 20 mM MgCl2), 1 µl 10 mM dNTPs, 10 pmol each primer, and 2.5 units Taq DNA polymerase in a final volume of 50 µl. Cycling parameters were 95°C, 45 s; 47°C, 45 s; and 72°C, 2 min.

Materials

Nitrocellulose filters and other chemicals were purchased from Sigma. RadPrime DNA labeling kit was purchased from GIBCO-BRL. 36Cl was purchased from New England Nuclear (Boston, MA). Valinomycin was dissolved in 95% ethanol and added to the membranes in a 1/100 dilution.

Statistical Analyses

Values are expressed as means ± SE. The substantial difference between mean values was examined by using ANOVA. P < 0.05 was considered statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Immunocytochemical Staining of PAT1 in the Gastrointestinal Tract

In this series of experiments, the localization of PAT1 in mouse colon, duodenum, and stomach was examined by immunocytochemistry. PAT1 expression was virtually undetectable in the colon (Fig. 1A), consistent with Northern blot hybridization experiments (36). PAT1 is expressed on the apical membrane of the villi of the duodenum, with lower expression levels on the apical membrane of crypt cells (Fig. 1, B and C). These results are consistent with the localization of PAT1 on brush-border membranes of small intestine and confirm previous studies from our laboratory (36). Immunocytochemical staining in the stomach indicated that PAT1 is localized in the glandular portion of the stomach with no labeling in surface cells (Fig. 1, D and E). This reaction was specific, because the labeling was completely prevented by preadsorption of the immune serum (Fig. 1F).


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Fig. 1.   Immunocytochemical staining of putative anion transporter (PAT1) in colon (A), small intestine (B and C), and stomach (D-F). PAT1 expression in the colon was virtually undetectable. Staining indicates that PAT1 is expressed on the apical membrane of the villi of small intestine, with lower expression levels on the apical membrane of crypt cells (B). This reaction was specific, because the labeling was completely prevented by immune preadsorption (C). The immunocytochemical staining in the stomach indicated that PAT1 is localized in the glandular portion of the stomach with no labeling in the surface cells (D and E). Similar to duodenum, the labeling with the preadsorbed immune serum failed to detect any labeling in the stomach (F).

Colocalization of PAT1 With D. Biflorus Agglutinin or With the Gastric H-K-ATPase

Mouse stomach sections were stained with PAT1 antibody and with D. biflorus agglutinin, a parietal cell marker (18). As indicated in Fig. 2, when the dual images of cells that were stained with PAT1 (Fig. 2A) and D. biflorus agglutinin (Fig. 2C) were acquired, it became evident that cells that were stained with D. biflorus agglutinin were the same cells that expressed PAT1 (Fig. 2B). These results indicate the exclusive expression of PAT1 in parietal cells.


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Fig. 2.   Immunocytochemical double labeling with PAT1 and Dolichos biflorus agglutinin. Cells were stained with PAT1 (A) and D. biflorus agglutinin (C), and images were obtained by confocal microscopy. The dual image (B) indicates that cells that were stained with D. biflorus agglutinin were the same cells that expressed PAT1.

To determine the localization of PAT1 with respect to gastric H-K-ATPase in parietal cells, stomach sections were double labeled with antibodies directed against PAT1 and the beta -subunit of the gastric H-K-ATPase. Figure 3 [lower (A) and higher (B) magnifications] shows that the patterns of distribution of gastric H-K-ATPase and PAT1 are identical when a dual image was acquired. These results indicate that gastric H-K-ATPase and PAT1 are present in the same intracellular pool in parietal cells, namely tubulovesicular and/or secretory canalicular membranes.


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Fig. 3.   Immunocytochemical double labeling with PAT1 and gastric H-K- ATPase. The distribution of gastric H-K-ATPase beta -subunit and PAT1 is completely identical when dual image was acquired. Magnification: lower (A) and higher (B).

Expression of PAT1 is Reduced in Gastric H-K-ATPase Knockout Mice

The colocalization of PAT1 and the gastric H-K-ATPase suggests that PAT1 expression should be reduced in parietal cells in which the tubulovesicle and canalicular membranes are reduced. Parietal cells from gastric H-K-ATPase alpha - and beta -subunit knockout mice exhibit sharp reductions in both types of membranes.1 Accordingly, we examined the expression and abundance of PAT1 in the stomach of gastric H-K-ATPase alpha -subunit knockout mice. The staining with PAT1 antibody was significantly diminished in H-K-ATPase-deficient parietal cells when compared with those of wild-type mice (Fig. 4A). Staining with the gastric H-K-ATPase beta -subunit antibody was also less intense in H-K-ATPase-deficient cells (Fig. 4B), consistent with published reports (30). These results demonstrate a correlation between a reduction in H-K-ATPase-containing membranes and a reduction in PAT1 expression.


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Fig. 4.   PAT1 expression in gastric H-K-ATPase knockout (KO) mice. The staining with PAT1 antibody was significantly diminished in gastric H-K-ATPase KO mice vs. wild-type (WT) animals. Staining with the gastric H-K-ATPase beta -subunit antibody is less intense in gastric H-K-ATPase KO vs. WT animals. See text for description.

Functional Presence of Cl-/HCO<UP><SUB><UP>3</UP></SUB><SUP><UP>−</UP></SUP></UP> Exchange in Tubulovesicle Membranes

The results of the above studies indicated that parietal cell membranes that contain the gastric H-K-ATPase also contain PAT1, thereby raising the question of whether PAT1 Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange activity might be required under certain circumstances to counter the activity of the gastric H-K-ATPase. Such would be the case if, for example, the presence of PAT1 were essential to counteract low-level activity of the H-K-ATPase in tubulovesicles during the resting state. The purpose of the following series of experiments was to test for the presence of Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange in tubulovesicle preparations containing the H-K-ATPase. Because protocols for the isolation of tubulovesicular membranes from the mouse stomach have not been established (due to high contamination and low yield), we used rabbit tubulovesicle membrane for functional studies.

Tubulovesicles were isolated from rabbit parietal cells, subjected to several purification steps as described before (34), and examined for the expression of H-K-ATPase. Figure 5A shows an immunoblot analysis of tubulovesicle membranes using the gastric H-K-ATPase beta -subunit antibody. As indicated, the fraction containing pure tubulovesicle membranes (lane 3) showed the highest gastric H-K-ATPase abundance when compared with the initial homogenate (lane 1) and semipurified membranes (lane 2). To examine the presence of basolateral membrane contamination in tubulovesicle proteins, immunoblot analysis of alpha -subunit Na-K-ATPase was performed on the initial homogenate (Fig. 5B, corresponding to lane 1 in A) and purified tubulovesicles (Fig. 5B, corresponding to lane 3 in A). As shown in Fig. 5B, Na-K-ATPase labeling was very faint in tubulovesicle membranes vs. the initial homogenate, indicating the absence of a significant amount of basolateral contamination in tubulovesicle membranes. Because our PAT1 polyclonal antibody that was raised in rabbit does not recognize the rabbit PAT1 in stomach, duodenum, or kidney (data not shown), we examined the mRNA expression of PAT1 in rabbit stomach parietal cells. Parietal cells were isolated as before (21) and subjected to RT-PCR as described in EXPERIMENTAL PROCEDURES. The ethidium bromide staining of the agarose gel demonstrated the amplification of a ~390-bp band (Fig. 5C). The product was gel purified and subjected to sequence analysis, which verified the sequence as rabbit PAT1.


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Fig. 5.   Functional presence of Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange in tubulovesicle membranes. A: immunoblot analysis of the tubulovesicles with the gastric H-K-ATPase antibody. Three fractions of crude (lane 1), semipure (lane 2), and pure (lane 3) tubulovesicle membranes were isolated and examined for expression of the H-K-ATPase beta -subunit. As indicated, the fraction that contained the pure tubulovesicular membranes (lane 3) had the highest gastric H-K-ATPase abundance. Protein loaded was 10 µg/lane. B: immunoblot analysis with the alpha -subunit Na-K-ATPase antibody. Na-K-ATPase labeling was minimal in tubulovesicle membranes (corresponding to lane 3 of A) vs. the initial homogenate (corresponding to lane 1 of A). Protein loaded was 10 µg/lane. C: detection of PAT1 transcript by RT-PCR in rabbit parietal cells. Ethidium bromide staining of an agarose gel indicating the presence of a transcript of expected size in rabbit parietal cells using PAT1-specific primers (see EXPERIMENTAL PROCEDURES). Lane 1, +RT; lane 2, no RT; lane 3, DNA ladder. D: HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent 36Cl influx in tubulovesicles. intracellular to extracellular pH (pHi/pHo) = 7.5/7.5 or 7.5/6.0 ± HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2. Vesicles were preequilibrated for 90 min at 20°C in a medium that consisted of 100 mM mannitol, 50 mM K-gluconate, 20 mM HEPES, 10 mM tetramethylammonium hydroxide, and either 25 mM KHCO3 or 25 mM K-gluconate, pHi 7.5, and gassed with either 5% CO2 or no CO2. The uptake of 4 mM 36Cl into the vesicles was measured at 30 s after 1:10 dilution and incubation of the vesicles in a medium that consisted of 1) 100 mM mannitol, 50 mM K-gluconate, and 20 mM HEPES with either 25 mM KHCO3 (and gassed with 5% CO2, pHo 7.5) or 25 mM K-gluconate (and no CO2, pHo 7.5) or 2) 75 mM mannitol, 25 mM MES, 20 mM HEPES, and 75 mM K-gluconate, pHo 6.0. DIDS was used at 0.5 mM concentration. The experiments were performed in triplicate.

Figure 5D shows a functional assay examining the presence of Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange in tubulovesicle membranes. As indicated, imposing an outward pH and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> gradient {intracellular pH (pHi) 7.5/extracellular (pHo) pH 6.0, intracellular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration ([HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]i) 25 mM/extracellular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration ([HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]o) 0 mM, 5% CO2} increased the 30-s influx of 36Cl by ~5.8-fold when compared with that observed in the absence of pH and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> gradients (pHi 7.5/pHo 7.5, [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]i 25 mM/[HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>]o 25 mM, 5% CO2). The 30-s influx of 36Cl was increased by ~2.7-fold in the presence of a pH gradient alone (pHi 7.5/pHo 6.0, no HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> or CO2). The pH and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-stimulated 36Cl influx was inhibited by >74% in the presence of 0.5 mM DIDS (Fig. 5). The 36Cl influx initial rate was 6.31 ± 0.51 and decreased to 1.55 ± 0.20 nmol · mg protein-1 · min-1 in the presence of DIDS. Whether there is more than one anion exchanger or both Cl-/OH- and Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange modes (Fig. 5D) are mediated via PAT1 remains to be determined.

In the last series of experiments, we examined the distribution of PAT1, vis-à-vis H-K-ATPase, in mouse parietal cells in response to stimulation of acid secretion. Mice were treated with intraperitoneal histamine (2 µg/g body wt) after an overnight fast and euthanized 30 min later. Stomach sections were double labeled with PAT1 and H-K-ATPase antibodies and analyzed by high-resolution confocal microscopy. The results demonstrate remarkably similar labeling pattern for PAT1 and H-K-ATPase during the resting and the stimulated states (Fig. 6), with both transporters showing a diffuse pattern in a majority of cells during the resting state (Fig. 6A) but a reticular pattern during the stimulated state (Fig. 6B).


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Fig. 6.   Immunocytochemical double labeling with PAT1 and gastric H-K-ATPase during acid stimulation. A: immunofluorescent labeling with PAT1 and H-K-ATPase shows diffuse and superimposable staining pattern in a majority of parietal cells during the resting state. B: staining pattern of PAT1 is identical to H-K-ATPase and is reticular in appearance in the majority of parietal cells during the stimulation of acid secretion by histamine (see RESULTS).


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The results of the above experiments indicate that PAT1 is expressed on apical membranes in the small intestine, as shown previously (34), and in the glandular portion of the stomach (Fig. 1). The expression of PAT1 in the stomach is limited to the parietal cells (Fig. 2) and mirrors the expression pattern of the H-K-ATPase, indicating that PAT1 is present in secretory canalicular and/or tubulovesicular membranes (Fig. 3). PAT1 expression was significantly decreased in parietal cells of gastric H-K-ATPase knockout mice (Fig. 4), in which H-K-ATPase-containing membranes are sharply reduced, and functional studies using purified tubulovesicles demonstrated the presence of a DIDS-sensitive Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger (Fig. 5).

It is difficult to conceive of a physiological function for Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange in the secretory canaliculus during acid secretion, because it would directly counter HCl secretion. Indeed, there is no evidence for active Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange in the secretory canaliculus of stimulated parietal cells. However, such an activity could play an important role in H-K-ATPase-containing membranes immediately following the transition from the stimulated to the resting state and also in resting tubulovesicles. Ito et al. (11) presented striking electron micrographs showing that transition of the parietal cell to the resting state can result in closing of the apical opening to the lumen of the gland while extended canaliculi remain within the cell. As far as we are aware, no mechanism has been proposed by which the voluminous acid contents of these entirely intracellular canaliculi could be dissipated. If PAT1 were present and active in these structures, then its Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange activity might provide a mechanism for removing HCl and ultimately dehydrating the canaliculus. Similarly, because the H-K-ATPase is retrieved from the canalicular membranes by endocytosis, the lumen of the tubulovesicles would remain acidic if there were no transport mechanism, such as Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange, for removal of HCl.

In the studies described here, we showed that purified tubulovesicles contain robust Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange activity. This is the first report on the presence and identification of a Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in tubulovesicular membranes of parietal cells. A critical role of PAT1 in pH and ion homeostasis of tubulovesicles is suggested by its downregulation in parietal cells of achlorhydric gastric H-K-ATPase knockout mice (Fig. 5), which exhibit a sharp reduction in the number of tubulovesicles.1 Because no apical HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion has been observed in parietal cells during active acid secretion, these results strongly suggest that the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger PAT1 is functional only during the resting state. The absence of any apparent cAMP/PKA consensus binding site in the PAT1 amino acid sequence (16) supports the notion that cAMP, which activates acid secretion in response to a number of secretogogs (7), does not stimulate PAT1. The transformation from a diffuse staining pattern for both PAT1 and H-K-ATPase during the resting state (Fig. 6A) to a predominantly reticular pattern during acid secretion (Fig. 6B) strongly suggests that both transporters are located on tubulovesicular membranes during the resting state and move to the secretory canaliculi during the stimulated state. Whether PAT1 is inactive during acid stimulation or is active but has insufficient activity to counter acid secretion via the H-K-ATPase remains speculative at the present time. It is worth mentioning that the presence of an apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in secretory tubulovesicle membranes does not conflict with published studies showing low CO2 permeability of apical membranes of parietal cells (35). The base species transported via PAT1 into tubulovesicle membranes are preformed HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and not CO2 and water. It should be noted that a previous report failed to detect Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange activity in gastric tubulovesicle membranes (15). Although those results may seem to be at variance with the experiments depicted in Fig. 5D, it is possible that differences in experimental conditions, such as incomplete voltage clamping or differences in the values of imposed intra/extravesicular pH, may account for the apparent absence of Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange activity in tubulovesicle membranes in the previous study. Although markers for tubulovesicle membranes (such as the gastric H-K-ATPase) are enriched and markers of basolateral membranes (such as the Na-K-ATPase) are depleted in tubulovesicle membrane preparations in the current studies, the possibility that contamination by basolateral membrane anion exchanger(s) contributes to the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange activity observed in isolated tubulovesicle membranes in Fig. 5 cannot be excluded.

Although further studies will be needed to determine the physiological function of Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange in tubulovesicles, it seems likely that it is involved in maintaining an intravesicular ionic milieu that is appropriate for resting tubulovesicles. Unless there are mechanisms capable of maintaining the H-K-ATPase in a completely inactive state when it resides in tubulovesicles, some low level of acid transport would be expected to occur. PAT1-mediated Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange would appear to be an ideal mechanism to prevent the accumulation of acid in tubulovesicles by exchanging intravesicular Cl- for cytoplasmic HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Testing of this hypothesis will require the development of PAT1 knockout mice or specific inhibitors of the exchanger.

In conclusion, PAT1 or SLC26A6 expression in the stomach occurs exclusively in the parietal cells. PAT1 colocalizes with the gastric H-K-ATPase and mediates Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange in tubulovesicle membranes of parietal cells. These data suggest that PAT1 is essential for neutralization of H+ and removal of Cl- from secretory membranes during the passage of parietal cells from the stimulated state to the resting state and for maintaining the normal resting state of tubulovesicles.


    ACKNOWLEDGEMENTS

These studies were supported by a Merit Review Grant from the Department of Veterans Affairs, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant DK-54430, a Cystic Fibrosis Foundation Grant, grants from Dialysis Clinic Incorporated (to M. Soleimani), and by NIDDK Grants DK-50594 (to G. E. Shull) and DK-38972 (to J. G. Forte).


    FOOTNOTES

Address for reprint requests and other correspondence: M. Soleimani, Division 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).

1 The volume density of total secretory membranes and tubulovesicle membranes were 3.5-fold and 22-fold greater, respectively, in wild-type parietal cells than in H-K-ATPase-deficient parietal cells; M. Miller, L. M. Judd, I. R. van Driel, A. Andringa, M. Flagella, S. M. Bell, P. J. Schultheis, and G. E. Shull (unpublished observations).

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.

July 17, 2002;10.1152/ajpgi.00137.2002

Received 9 April 2002; accepted in final form 12 July 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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