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Originally published In Press as doi:10.1074/jbc.M403779200 on April 30, 2004

J. Biol. Chem., Vol. 279, Issue 29, 30531-30539, July 16, 2004
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Mice with a Targeted Disruption of the AE2 Exchanger Are Achlorhydric*

Lara R. Gawenis{ddagger}, Clara Ledoussal{ddagger}, Louise M. Judd{ddagger}§, Vikram Prasad{ddagger}, Seth L. Alper¶, Alan Stuart-Tilley¶, Alison L. Woo{ddagger}, Christina Grisham{ddagger}, L. Philip Sanford{ddagger}, Thomas Doetschman{ddagger}, Marian L. Miller||, and Gary E. Shull{ddagger}**

From the {ddagger}Department of Molecular Genetics, Biochemistry and Microbiology and the ||Department of Environmental Health, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267 and the Molecular Medicine and Renal Units, Harvard Medical School, Boston, Massachusetts 02215

Received for publication, April 5, 2004 , and in revised form, April 23, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The AE2 exchanger is expressed in numerous cell types, including epithelial cells of the kidney, respiratory tract, and alimentary tract. In gastric epithelia, AE2 is particularly abundant in parietal cells, where it may be the predominant mechanism for efflux and Cl- influx across the basolateral membrane that is needed for acid secretion. To investigate the hypothesis that AE2 is critical for parietal cell function and to assess its importance in other tissues, homozygous null mutant (AE2-/-) mice were prepared by targeted disruption of the AE2 (Slc4a2) gene. AE2-/- mice were emaciated, edentulous (toothless), and exhibited severe growth retardation, and most of them died around the time of weaning. AE2-/- mice exhibited achlorhydria, and histological studies revealed abnormalities of the gastric epithelium, including moderate dilation of the gastric gland lumens and a reduction in the number of parietal cells. There was little evidence, however, that parietal cell viability was impaired. Ultrastructural analysis of AE2-/- gastric mucosa revealed abnormal parietal cell structure, with severely impaired development of secretory canaliculi and few tubulovesicles but normal apical microvilli. These results demonstrate that AE2 is essential for gastric acid secretion and for normal development of secretory canalicular and tubulovesicular membranes in mouse parietal cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Anion exchanger 2 (AE2)1 is a member of the Slc4a gene family, which includes both exchangers and Na-HCO3 cotransporters (1–4). For the AE2 exchanger, alternative promoters in the Slc4a2 gene give rise to mRNAs encoding four NH2-terminal protein variants (1, 2). AE2a is expressed in all tissues, AE2b1 and AE2b2 are largely restricted to epithelial tissues (2, 5), and AE2c is expressed at significant levels only in stomach (2). The ubiquitous distribution of AE2a and the tissue- and cell type-specific expression of the other NH2-terminal variants suggest that AE2 serves both housekeeping and tissue-specific functions.

One of the most striking examples of a tissue-specific physiological process that requires high levels of exchange is gastric acid secretion. During acid secretion, the combined activities of the gastric H+,K+-ATPase, a Cl- channel, and a K+ channel at the apical and canalicular membranes of parietal cells mediate the secretion of HCl and KCl (6). Along with the generation of H+ for secretion, intracellular carbonic anhydrase activity produces ions, which are extruded across the basolateral membrane by the activity of one or more exchangers (7, 8). exchange also serves as a Cl- uptake pathway across the basolateral membrane to support Cl- secretion through the apical membrane Cl- channels (8, 9).

exchange in the basolateral membrane of parietal cells has generally been attributed to the activity of AE2 (2, 10–12) because: 1) it is expressed at higher levels in stomach than in any other tissue and at much higher levels in parietal cells than in any other gastric epithelial cell type (2, 12); 2) at least three NH2-terminal variants of AE2 are expressed in the parietal cell (12); 3) it has been localized to the basolateral membrane of the parietal cell using multiple antibodies that identify all three variants (13); and 4) isolated parietal cells exhibit 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-inhibitable Cl- and transport consistent with AE2 activity (14, 15). Although it has been suggested that the NKCC1 Na+-K+-2Cl- cotransporter may provide a Cl- uptake pathway that is essential for acid secretion (16), studies using NKCC1 null mutant mice showed that the loss of NKCC1 did not reduce gastric acid secretion (17).

Together, these observations support the hypothesis that basolateral exchange mediated by AE2 is critical for HCl secretion. However, recent studies have shown that two exchangers of the Slc26a family, Slc26a6 (also termed PAT-1 or CFEX (Ref. 18)) and Slc26a7, are expressed in parietal cells (19, 20). Although the Slc26a6 exchanger is present in tubulovesicles (19) and therefore cannot mediate basolateral exchange, Slc26a7 has been localized to the basolateral membrane (20). Thus, it is unclear whether AE2 alone mediates the basolateral exchange that is thought to be essential for acid secretion, or whether the activity of Slc26a7 can support a significant level of acid secretion. To understand the role of AE2 in gastric acid secretion, we developed a mouse model carrying a targeted disruption of the Slc4a2 gene. Analyses of AE2 null mutant mice demonstrated that they were achlorhydric and that their gastric parietal cells failed to develop an extensive secretory canalicular membrane system.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Targeting Construct and Generation of Mutant Mice— The targeting construct was prepared using the pMJKO vector (21) and PCR-generated fragments of the mouse AE2 gene. A 2.8-kb genomic fragment spanning exons 8–14 (beginning at codon 352 of the AE2a transcription unit and ending at codon 702) was inserted into a cloning site 5' to the promoter of the neo gene, and a 2.0-kb fragment spanning exons 17–23 (beginning at codon 859 and ending 13 nucleotides beyond codon 1237) was inserted between the 3' end of the neo gene and the herpes simplex virus thymidine kinase gene. Targeting of ES cells, derived from 129S6/SvEv Tac (Taconic, Germantown, NY) mice, was performed as previously described (21). ES cells in which homologous recombination had occurred were identified by Southern blot analysis of BamHI-digested DNA using a 5' probe spanning genomic sequences from exon 7 to exon 8 and then confirmed using a 3' probe spanning genomic sequences from exon 17 to exon 21. Chimeric mice carrying the targeted ES cells were bred with wild-type Black Swiss females (Taconic), and studies were performed using mice of the mixed 129S6/SvEv and Black Swiss background.

Measurement of Gastric pH and Acid/Base Equivalents—Gastric pH and the acid-base content of the gastric secretions were measured as previously described (21). Briefly, 15 min following subcutaneous injection of histamine (2 µg/g of body weight), mice were euthanized and the intact stomach was exposed via a midline laparotomy and clamped at the pyloric and gastroesophageal junctions prior to removal. The stomachs were immersed in 5 ml of oxygen-saturated normal saline solution at room temperature, opened along the lesser curvature, everted, and rinsed of their contents. The gastric contents were pelleted by centrifugation, and the pH and acid-base equivalents of the gastric secretions were determined. Data for the acid-base content of the gastric secretions were reported as microequivalents (µmol of H+ or ions)/g wet stomach weight.

Immunofluorescence—Stomachs from 18-day-old mice were fixed in 10% neutral buffered formalin, dehydrated, and embedded in paraffin. Sections were cut and then deparaffinized and rehydrated in a graded series of ethanols for use with the anti-gastric H+,K+-ATPase antibody. For the anti-AE2 antibody, cryosections were prepared from stomach tissues following perfusion fixation in 2% paraformaldehyde. Slides were stained overnight at 4 °C with either rabbit anti-{alpha}-subunit of the porcine gastric H+,K+-ATPase (Calbiochem, San Diego, CA) or affinity-purified rabbit anti-mouse AE2 antibody (raised against amino acids 1224–1237, which are found in all variants). Tissues stained with the anti-H+,K+-ATPase antibody were incubated with secondary donkey anti-rabbit IgG antibodies conjugated to Texas red fluorophore and analyzed by fluorescence microscopy as described previously (22). Tissues stained with the anti-AE2 antibody were incubated with secondary donkey anti-rabbit IgG antibodies conjugated to Cy3 fluorophore (Jackson Immunoresearch, West Grove, PA) and analyzed by confocal laser immunofluorescence microscopy.

Northern Blot Analysis—Total RNA was isolated from tissues of 18-day-old mice using TRI Reagent according to the directions from the manufacturer (Molecular Research Center, Inc., Cincinnati, OH). RNA (10 µg) was denatured with glyoxal, fractionated by electrophoresis in 1% agarose, and transferred to a nylon membrane. Blots were analyzed using 32P-labeled cDNA probes specific for AE2 5' of the neo insertion site (codons 461–765), AE2 3' of the neo insertion site (codons 861–1237), gastric H+,K+-ATPase {alpha} and {beta} subunits, Slc26A7 exchanger, intrinsic factor, pepsinogen, the neo gene product, and the L32 ribosomal subunit protein (as a loading control) (22). Following autoradiography, bands were quantified using a PhosphorImager and densitometric analysis carried out using ImageQuant software version 5.1 (Molecular Dynamics, Sunnyvale, CA). Band densities were normalized to the band density of L32 for each sample to control for differences in loading and the -fold change in expression relative to wild type determined.

Western Blot Analysis of Gastric H+,K+-ATPase—Because of the small size of the stomach samples, whole tissue homogenates were used for Western blot analysis. Samples were powdered in liquid nitrogen and homogenized in 20 mM HEPES, 10 mM KCl, 1 mM EDTA,1 mM dithiothreitol, 0.2% Nonidet P-40, and 10% glycerol. Protease and phosphatase inhibitors (NaF, 50 mM; cantharidin, 20 ng/ml; Na3VO4, 0.1 mM) were added to the buffer just prior to use. Protein samples (20 µg/lane) were resolved by electrophoresis on an 8% reducing sodium dodecyl sulfate-polyacrylamide gel and transferred to nitrocellulose. The blot was incubated overnight at 4 °C with both a mouse anti-gastric H+,K+-ATPase {alpha} subunit antibody (23) that recognizes Asp682–Leu688 (1:4000 dilution) and an anti-actin antibody (1:4000 dilution; Sigma), and then probed with an horseradish peroxidase-conjugated secondary anti-mouse antibody. Chemiluminescence was developed using the SuperSignal West Pico reagent (Pierce). Autoradiography was performed and densitometric analysis was carried out using an HP scanner and ImageQuant software (version 5.1).

Histology, Electron Microscopy, and Morphometry—Stomachs were fixed in 10% formalin, dehydrated, and embedded in paraffin, and hematoxylin- and eosin-stained sections were examined by light microscopy. For electron microscopy, stomachs were fixed in 4% paraformaldehyde, post-fixed in 1% osmium tetroxide, dehydrated in ethanol and propylene oxide, embedded in Spurr's resin, and sectioned. Morphometric analysis of the gastric glandular epithelium was performed as previously described (21, 22). The criteria for enteroendocrine cells were cells with a visible nucleus and a small amount of very pale cytoplasm. Chief cells were identified as having a profile of nucleus, basophilic cytoplasm (rough endoplasmic reticulum), and a minimum of three birefringent granules, which differed from previous studies that used a minimum of six birefringent granules (22). The criteria for parietal cells were cells containing a nuclear profile and less dense cytoplasm that were predominantly large and exhibited well organized mitochondria distributed around canaliculi and tubulovesicles. Mucous cells were defined as cells with a nuclear profile, and either a cap of mucous granules or apical granules. Cells not fulfilling the criteria of any of these categories were classified as "other."

Statistics—Data from two treatment groups were compared using a two-tailed unpaired Student's t test assuming equal variances. Data from more than two treatment groups were compared using a one-way ANOVA with a post hoc Tukey's t test. Morphometric data were analyzed using the General Linear Model of SAS (version 8; SAS, Cary, NC). A probability value of p < 0.05 was considered statistically significant. All values are reported as the mean ± S.E.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of AE2 Null Mutant Mice—The targeting procedure replaced sequences from exons 14 to 17, which are present in the transcripts of all known AE2 variants, with the neomycin resistance (neo) gene (Fig. 1A). Chimeric male mice generated using the targeted ES cells were bred to wild-type females to produce AE2 heterozygous mutant mice, and breeding of AE2+/- mice resulted in live offspring of all three genotypes (Fig. 1B). Because the neo gene was inserted in the same orientation as the AE2 transcription units, we anticipated that any transcripts from the mutant allele would utilize the neo gene polyadenylation site, thereby eliminating codons 703–1237, which encode the transmembrane domains. Northern blot analysis using an AE2 cDNA probe consisting of sequences 3' to the neo gene insertion site demonstrated that the wild-type AE2 mRNAs were ablated in AE2-/- mice (Fig. 1C, top panel). However, an AE2 probe derived from sequences 5' to the insertion site of the neo gene identified an mRNA in AE2-/- tissues that was consistent in size with that of AE2a and AE2b mRNAs (Fig. 1C, second panel); also, a transcript similar in size to that of the stomach-specific AE2c mRNA was observed at trace levels. This suggested that mutant transcripts consisting of the 5' end of the AE2 mRNA and the neo gene were being expressed. Hybridization analysis confirmed that the mutant AE2 mRNAs do contain the neo gene (Fig. 1C, third panel).



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  		FIG. 1.
Generation of AE2 null mutant mice. A, gene targeting strategy showing a map of the wild-type gene, a diagram of targeting construct containing the neomycin resistance gene (NEO) and the thymidine kinase gene (TK), and the predicted structure of the targeted allele and location of diagnostic probes. B, Southern blot analysis of tail DNA from offspring of a heterozygous mating. DNA was digested with BamHI and hybridized with 5' outside or 3' inside probes, which identify 8.6- and 1.9-kb fragments, respectively, in the wild-type allele and 6.1- and 3.0-kb fragments, respectively, in the mutant allele. C, Northern blot analysis of total RNA (5 µg/lane) from stomach, small intestine, and colon of 18-day-old wild-type (+/+), heterozygous (+/-), and homozygous mutant (-/-) mice hybridized with cDNA probes corresponding to AE2 coding sequences located either 3' or 5' of the NEO insertion site, NEO coding sequences, and a cDNA for the L32 ribosomal protein (as a loading control).

 
Northern blot analysis demonstrated that the mutant mRNAs lacked sequences encoding the transmembrane-spanning regions of the protein, which are required for anion transport activity. To confirm the absence of AE2 protein in the AE2-/- gastric mucosa, immunofluorescence studies were performed using an antibody that recognizes the AE2 carboxyl terminus, which is present in all AE2 protein variants. In contrast to the robust staining of basolateral membranes in AE2+/+ parietal cells, AE2 expression was not detected in the knockout (Fig. 2).



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  		FIG. 2.
Immunostaining of AE2 in gastric mucosa of AE2+/+ and AE2-/- mice. Confocal images showing immunostaining of gastric glands from AE2+/+ (A) and AE2-/- (B) mice using an anti-AE2 antibody that recognizes all known AE2 protein variants. The arrows indicate the basolateral membranes of parietal cells. Arrowheads indicate staining of an erythrocyte (because of the presence of the AE1 exchanger), and asterisks indicate the lumen of gastric glands. Bar = 20 µm.

 
Gross Phenotype—Genotype frequencies of pups obtained from heterozygous breeding pairs did not exhibit a normal 1:2:1 Mendelian ratio. Among 697 pups, there were 213 wild-type (30.5%), 409 heterozygous (58.5%), and 75 knockout (11%) mice. Among the null mutant mice observed at birth, there was no substantial alteration in the percentage of males (45%) or females (55%). On the day of birth, ~20% of the AE2-/- pups were found dead and ~25% of the live AE2-/- mice were notably smaller than their AE2+/+ and AE2+/- littermates. During the first 10 days of life, the remaining AE2-/- mice exhibited little evidence of a disease phenotype other than mild growth retardation, and fed well, as indicated by milk in the stomach. Between 10 and 15 days of age, AE2-/- mice exhibited a progressive decline relative to AE2+/+ and AE2+/- littermates. During this period, AE2-/- mice stopped feeding well, exhibited severe growth retardation (Fig. 3A), became emaciated, and showed evidence of dehydration (skin tenting). AE2-/- mice also became mildly ataxic and exhibited a failure of both molar and incisor tooth eruption (Fig. 3B). After 15 days of age, AE2-/- mice exhibited high morbidity and mortality, with the oldest AE2-/- mouse surviving to only 40 days of age (Fig. 3C). In addition to these defects, the mutants had a severe hearing loss and defective development of bone.2 Providing powdered rodent chow to the toothless AE2-/- mice did not improve their growth or survival.



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  		FIG. 3.
Gross phenotype of AE2 null mutant mice. A, compared with gender-matched AE2+/+ littermates, 18-day-old AE2-/- mice exhibited growth retardation and emaciation. B, in contrast to gender-matched AE2+/+ littermates, AE2-/- mice lack incisor tooth eruption. C, survival curve for AE2+/+ (n = 126) and AE2-/- (n = 33) mice. The percentage of surviving mice was plotted against age in days, with day 0 being the day of birth.

 

Gastric Achlorhydria in AE2 Null Mutant Mice—To test the hypothesis that AE2 is necessary for gastric acid secretion, the pH and acid-base content of gastric secretions were analyzed in AE2+/+, AE2+/-, and AE2-/- mice following treatment with histamine to stimulate acid secretion. Because of the poor survival, these studies and the histological studies described below were performed using 18–19-day-old mice. The pH of gastric secretions was 7.83 ± 0.11 in AE2-/- mice, 3.73 ± 0.38 in AE2+/- mice, and 3.73 ± 0.14 in AE2+/+ mice (Fig. 4A). The amount of net acid or base in the gastric secretions of the same animals was quantified by titrating the pH of the gastric contents using HCl or NaOH (Fig. 4B). The amount of base was 8.8 ± 1.6 µeq/g wet weight in AE2-/- stomachs; the amount of acid was 68.9 ± 4.5 µeq/g wet weight in AE2+/- stomachs and 62.1 ± 5.0 µeq/g wet weight in AE2+/+ stomachs.



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  		FIG. 4.
Gastric pH and acid-base microequivalents in AE2+/+, AE2+/-, and AE2-/- stomachs. Samples were collected from the stomachs of 18-day-old mice (n = 6 animals for each genotype) 15 min after subcutaneous injection of histamine (2 µg/g body weight). A, pH of gastric secretions; B, acid-base content (expressed as µeq/g stomach wet weight). The mean of each measurement is indicated by a horizontal bar. *, p < 0.01 for the difference between AE2-/- and either AE2+/+ or AE2+/- mice.

 
Northern Blot Analysis of Gastrin mRNA and Both Parietal and Chief Cell Markers—Achlorhydria is associated with hypergastrinemia and an increase in gastrin mRNA expression in stomach (21, 22, 24, 25). Consistent with achlorhydria, gastrin mRNA expression was elevated in AE2-/- stomach (Fig. 5). H+,K+-ATPase {alpha} subunit and H+,K+-ATPase {beta} subunit mRNAs were reduced to ~70% of the levels observed in wild-type stomach, suggesting that the loss of AE2 does not lead to a major reduction in the number of parietal cells, as occurred in mice lacking the NHE2 Na+/H+ exchanger (21). Slc26a7 mRNA, which encodes a exchanger that is expressed on the basolateral membrane of parietal cells (20), was reduced to ~40% of wild-type levels. Expression of pepsinogen and intrinsic factor mRNA, both chief cell markers in rodents (26, 27), was reduced to ~30 and ~50% of wild-type levels, respectively, suggesting that there was a reduction in the number of chief cells, which has also been observed in other models of achlorhydria (21, 22, 28, 29).



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  		FIG. 5.
Northern blot analysis of gastrin mRNA and parietal and chief cell markers in AE2+/+ and AE2-/- stomachs. Total RNA (10 µg) was isolated from stomachs of 18-day-old animal AE2+/+ and AE2-/- littermate pairs. Blots were hybridized with probes to gastrin, gastric H+,K+-ATPase {alpha} and {beta} subunits, Slc26A7, pepsinogen, intrinsic factor, and the L32 ribosomal protein subunit (as a loading control). Numbers (right side) indicate -fold change in gene expression in AE2-/- stomach as compared with control.

 
Immunohistochemical and Immunoblot Analysis of Gastric H+,K+-ATPase—To exclude the possibility that the loss of gastric acid secretion in AE2 null mice might be because of the absence of viable parietal cells expressing the gastric H+,K+-ATPase, immunofluorescence studies were performed using primary antibodies to the gastric H+,K+-ATPase {alpha} subunit. Gastric glands from AE2-/- mice exhibited significant staining with the anti-H+,K+-ATPase {alpha} subunit antibody (Fig. 6A), indicating that H+,K+-ATPase {alpha} subunit-expressing cells were abundant in AE2-/- gastric glands. However, the staining was more diffuse in AE2-/- parietal cells than in AE2+/+ cells, and the fluorescence appeared to be of less intensity than in AE2+/+ parietal cells (Fig. 6B).



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  		FIG. 6.
Immunostaining of gastric H+,K+-ATPase {alpha} subunit in AE2+/+ and AE2-/- stomachs. Immunostaining of gastric glands from AE2+/+ (left panels) and AE2-/- (right panels) mice using an anti-gastric H+,K+-ATPase {alpha} subunit antibody at magnification x100 (A) and x400 (B). The dark panels to the far left in panel A are controls with no primary antibody (bars = 50 µm). The glandular stomach of AE2-/- mice exhibited more diffuse staining of the gastric H+,K+-ATPase, including a reduction in the number of very brightly fluorescing cells.

 
Both the immunofluorescence and Northern blot data suggested that, despite the lack of measurable gastric acid secretion, there was significant expression of the gastric H+,K+-ATPase in AE2-/- parietal cells. Immunoblot analysis was performed to quantify the level of gastric H+,K+-ATPase {alpha} subunit. Expression of the gastric H+,K+-ATPase {alpha} subunit protein in AE2-/- stomachs was ~42% of wild-type levels (Fig. 7). This degree of reduction in H+,K+-ATPase expression is not sufficient to account for the observed achlorhydria.



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  		FIG. 7.
Immunoblot analysis of gastric H+,K+-ATPase {alpha} subunit protein in AE2+/+ and AE2-/- stomachs. Whole stomach homogenates from AE2+/+ and AE2-/- mice were probed with an anti-gastric H+,K+-ATPase {alpha} subunit antibody. Blots were also probed with an anti-actin antibody as a loading control. A, representative Western blot showing the results from two pairs of AE2+/+ and AE2-/- mice. The lower band of the doublet is a breakdown product of the gastric H+,K+-ATPase {alpha} subunit. B, quantification of gastric H+,K+-ATPase {alpha} subunit expression relative to actin expression for AE2+/+ (n = 4) and AE2-/- (n = 3) mice. Expression of the gastric H+,K+-ATPase {alpha} subunit was reduced by ~ 58% in AE2-/- stomachs. *, significantly different from AE2+/+, p < 0.007.

 
Histopathologic and Ultrastructural Changes in the Gastric Mucosa of AE2 Null Mice—Moderate hyperkeratosis was observed in the epithelium lining the nonglandular mucosa of AE2-/- stomachs (data not shown). This was also observed in the esophagus, however, and may have been secondary to poor feeding ability and the consequent absence of the abrasive action of ingested food. As compared with AE2+/+ stomach (Fig. 8A), the glandular stomach of AE2-/- mice exhibited dilation of the gastric gland lumens with the accumulation of basophilic material (Fig. 8, B and C). Despite abundant cellularity and no apparent increase in the number of dead or dying cells, there were fewer readily discernable parietal cells in the AE2-/- gastric glands than in wild-type glands. Morphometric analysis revealed a decreased number of total and mature parietal cells and a decrease in the mean number of chief cells in AE2-/- gastric glands (Table I), although the difference was not statistically significant. However, the thickness of the glandular epithelium was not reduced significantly in the knockout and many cells in the AE2-/- gastric glands were difficult to identify because of their small size, abnormal appearance, and low number of visible granules within the cells (Table I). The deficit in easily identifiable parietal cells suggested that there were either a reduced number of parietal cells or that the loss of canalicular structures and mitochondrial organization made the parietal cells difficult to recognize.



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  		FIG. 8.
Hematoxylin- and eosin-stained sections of gastric epithelium from AE2+/+ and AE2-/- mice. As compared with 18-day-old AE2+/+ mice (A), the glandular stomach of 18-day-old AE2-/- mice (B and C) was characterized by dilation of the gland lumens with accumulation of debris and a reduction in the number of readily identifiable parietal cells despite the abundant cellularity (bars = 50 µm).

 


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  		TABLE I
Epithelial cell populations of gastric glands All values are mean ± S.E. and represent the percentage of the total epithelial population. n = 7 for AE2+/+; n = 8 for AE2-/-. NS, not significant.

 
Based on the light microscopy and immunofluorescence studies, parietal cell morphology appeared to be altered in AE2-/- gastric glands, and ultrastructural analysis confirmed that there were significant changes. In contrast to wild-type parietal cells, in which well developed secretory canaliculi extended deep within the cell (Fig. 9A), canalicular structures in the cytoplasm of AE2-/- parietal cells were greatly reduced in number. Although there was little, if any, intracellular secretory canaliculus in most AE2-/- parietal cells, the apical membrane had abundant secretory-type microvilli (long and flexible) that were not different in length or width than AE2+/+ apical microvilli (Fig. 9B). In areas where gastric gland lumena were dilated and contained electron-dense material that appeared similar to that of zymogen granules in chief cells, the AE2-/- parietal cells were somewhat flattened (Fig. 9C). The best developed AE2-/- parietal cell observed to date is shown in Fig. 9D. In this cell, there is a small secretory canaliculus extending into the cell from the apical surface, but it is not nearly as well developed as the canaliculi observed in wild-type cells. The lack of foci of canalicular formation in the deep cytoplasm of AE2-/- parietal cells was supported by the apparent lack of tubulovesicular structures, which normally occur at the interface between secretory microvilli and the cytoplasm.



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  		FIG. 9.
Electron micrographs of AE2+/+ and AE2-/- parietal cells. A, AE2+/+ parietal cell showing well developed secretory canaliculi (bar = 2 µm). B, AE2-/- parietal cell with the secretory microvilli confined to the apical surface of the cell. Note that membranes with secretory microvilli do not extend into the cytoplasm to form secretory canaliculi (bar = 2 µm). C, flattened AE2-/- parietal cell with the secretory microvilli confined to the apical surface of the cell. Note that electron-dense material, visible in the gland lumen, is embedded in the microvilli (bar = 5 µm). D, the most highly developed parietal cell observed in AE2-/- stomachs showed membranes with secretory microvilli extending from the apical surface into the cytoplasm to form a short canaliculus (bar = 5 µm). C, canaliculi; N, nucleus; MV, microvilli; L, lumen; BM, basal membrane; JC, junctional complex.

 
Additional changes in AE2-/- parietal cells involved the basolateral membrane and mitochondria. Because AE2 is expressed in the basolateral membrane, it is interesting that invaginations or interdigitations of the basolateral membrane in AE2-/- parietal cells were less compact and less regular than in wild-type cells, sometimes appearing as large blebs rather than the typical finger-like structures (Fig. 10). The volume density of mitochondria was decreased in AE2-/- parietal cells, consistent with the apparent quiescence of these cells. Mitochondrial profiles often appeared as cup or donut shapes, and large intramitochondrial densities were observed frequently (Fig. 10).



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  		FIG. 10.
Electron micrographs of the basal aspect of AE2+/+ and AE2-/- parietal cells. A, AE2+/+ parietal cell with normal canalicular (C) and basolateral membranes (arrows). B, AE2-/- parietal cells exhibited altered basolateral membrane (arrows) morphology characterized by an increase in the number and extent of infolding into the cell cytoplasm. Also note mitochondria (M) with altered ring-shaped or "donut" morphology. Bar = 2 µm.

 
Ultrastructural analysis also revealed abnormalities in AE2-/- chief cells. Compared with those of wild-type mice, chief cells in most AE2-/- mice exhibited a reduction in the number of zymogen granules as well as a deficiency in the amount of rough endoplasmic reticulum. These changes, which likely contributed to the difficulty in identifying chief cells by light microscopy, could reflect impaired maturation resulting from either the achlorhydric state or from the primary loss of AE2 expression. However, evidence of some chief cell maturation was present in the AE2-/- gastric glands, including the presence of granules within the cytoplasm. A gastric gland of an AE2-/- mouse bordered by one parietal cell and three chief cells (with electron-dense intracellular granules) is shown in Fig. 11. The lumen of the gastric gland is filled with material of an electron density similar to that of pepsinogen-containing zymogen granules. This finding suggests that pepsinogen may be a significant component of the dense material observed in the gastric gland lumens of AE2-/- mice.



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  		FIG. 11.
Electron micrograph of a gastric gland lumen from an 18-day-old AE2-/- mouse bordered by a parietal cell and three chief cells. AE2-/- chief cells had intracellular granules. Note that the material in the lumen is of a similar electron density as the granules contained within the chief cells (bar = 2 µm). MV, microvilli; L, lumen; JC, junctional complex; G, zymogen granule.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The reduced number of AE2 null mutants observed at birth and the high incidence of neonatal mortality suggest that the loss of AE2 causes some embryolethality or fetal death. Nevertheless, many of the AE2-/- mice appeared to be normal at birth and could not be readily distinguished from their wild-type or heterozygous littermates. Those that survived beyond day 1 began to exhibit a general failure to thrive at around day 10, deteriorated quickly after that point, and few survived beyond 25 days of age. Given the relatively normal appearance of many mutants at birth, it seems unlikely that the morbidity and mortality is because of serious impairments of major housekeeping functions such as regulation of intracellular pH or cell volume, although these cannot be ruled out as contributing factors. In addition to the acid secretory defect, which was the focus of this study, several other abnormalities were observed. For example, the mutants have a defect in bone development, which may contribute directly to their growth retardation, and they are ataxic and virtually deaf. Their smaller size, lack of tooth eruption, ataxia, and hearing loss may put them at a competitive disadvantage with their rapidly developing wild-type siblings. Other factors, such as impaired function of not only the stomach, but of other components of the digestive system, might also play a role in the progression of the disease state. These factors are currently under investigation

A mouse model in which a Cre/loxP strategy was used to disrupt expression of 3 of the 4 protein variants of AE2 was recently reported (30), but the phenotype was very mild compared with that of our model. Although male infertility and some perinatal lethality were observed, AE2 homozygous mutants had no other apparent abnormalities. The targeting strategy used in that study (30) deleted exons 2, 1b1, and 1b2, which encode the first 17, 3, or 8 amino acids for the AE2a, AE2b1, and AE2b2 variants, respectively. However, because exons 3–23, which encode 1220 amino acids that include the anion transport domains, were left intact, it is possible that mutant AE2 mRNAs might be transcribed using the AE2a promoter and that production of the AE2c transcript would be unaffected. RT-PCR analysis of testis RNA was performed; however, the PCR primers were based on sequences from the exons that were deleted and therefore only confirmed the targeting procedure rather than testing whether transcripts were produced. No information was presented regarding expression in any tissue of AE2 protein or AE2 mRNAs that might have been transcribed from the AE2a or AE2c promoters. Thus, it is possible that the failure of spermiogenesis (30) was because of AE2 deficiency in testis, but that there was sufficient expression of AE2 protein and activity in other tissues to avoid the severe phenotype observed in our own study.

There is strong evidence that exchange across the basolateral membrane of the parietal cell is essential for acid secretion (2, 10–12) and, until recently, AE2 was the only exchanger known to be expressed on this membrane (11, 13, 31). However, localization of the Slc26a7 exchanger to the basolateral membrane of the parietal cell (19, 20) raised the possibility that some of the activity attributed to AE2 might be because of this transporter. However, analysis of gastric secretions after stimulation with histamine revealed that AE2-/- mice were achlorhydric. In fact, the gastric secretions were alkaline. This finding is similar to that observed in NHE2 knockout mice (21) and likely indicates that the mechanisms for secretion by surface cells are intact in the AE2-/- mice. Although the finding of achlorhydria does not rule out a subsidiary role for Slc26a7 in acid secretion, these results demonstrate that, in the absence of AE2, the activity of Slc26a7 is unable to support even minimal acidification of the luminal contents of the stomach and that AE2 is, therefore, essential for net acid secretion.

The achlorhydria phenotype of AE2-/- mice differed from that of mice lacking the NHE2 Na+/H+ exchanger (21), which is expressed in the parietal cell along with NHE1 and NHE4 (32, 33), and might therefore work in concert with AE2. AE2-deficient mice were achlorhydric at 18–19 days of age, whereas NHE2-deficient mice were only mildly hypochlorhydric at this age, ranged from hypochlorhydric to achlorhydric at 45 days, and were achlorhydric to alkaline by 12 weeks of age (21). Although the number of viable parietal cells was sharply reduced in achlorhydric adult NHE2 knockouts and the number of dead and dying parietal cells was increased, occasional mature parietal cells were observed that appeared to be secreting acid at a high rate. It should be noted that with high levels of base secretion in the adult NHE2-deficient stomach (21), most likely as a result of secretion by surface cells, acid secretion by a small number of parietal cells would not necessarily result in net acid secretion. Thus, achlorhydria in the NHE2 knockout appeared to be because of severely impaired viability of parietal cells rather than a direct perturbation of acid secretion by individual parietal cells (21). This may involve a failure of intracellular pH and volume homeostasis during acid secretion, as there is evidence that NHE2 contributes to both processes (32).

In light of the NHE2 phenotype, we anticipated that the loss of AE2 might also cause a severe impairment of parietal cell viability via perturbations of both pH and volume homeostasis. However, the data provided little support for this hypothesis. Although a reduction in the number of parietal cells was observed in AE2-/- stomachs, there was no increase in the number of cells undergoing mitosis or in the number of necrotic parietal cells, as observed in other models in which the production of parietal cells was substantially increased to replace cells being lost (21, 28). Although the apparent reduction in the number of parietal cells in AE2-/- stomachs could be indicative of a reduction in parietal cell life span, which has been estimated at 54 days in mice (34), it is also possible that the small size and morphological changes in these cells led to their classification as undifferentiated or other cells. The immunocytochemical studies revealed a substantial number of parietal cells; Northern blot analysis showed only a 30% reduction in the amount of gastric H+,K+-ATPase {alpha} and {beta} subunit mRNAs; and Western blot analysis showed a 58% reduction in gastric H+,K+-ATPase {alpha} subunit protein. These findings suggested that the loss of AE2 did not have a major effect on parietal cell viability, and indicated that any reduction in the number of parietal cells was modest at most, and not sufficient in itself to account for the achlorhydria phenotype.

The ultrastructural changes in AE2-/- parietal cells differed from those of parietal cells from achlorhydric mice in which NHE2 (21) or the gastric H,K-ATPase {alpha} (22) or {beta} (25) subunits were ablated. In wild-type cells, a secretory canaliculus with well developed microvilli extends from the plasma membrane to deep within the cell. However, this architecture, which is necessary for high rates of acid secretion, was almost entirely absent in AE2-deficient cells. Although the number of mature parietal cells was sharply reduced in NHE2-deficient mice, there were some cells in which the secretory canaliculus was massively expanded (21), indicating that the loss of NHE2 did not, in all cells, perturb either development of secretory membranes or the acid secretory mechanism itself. Parietal cells lacking either the H+,K+-ATPase {alpha} or {beta} subunits had abnormal secretory membrane microvilli because of the loss of interactions between the H+,K+-ATPase {alpha} subunit (which is degraded in {beta} subunit-deficient mice) and the actin cytoskeleton (25, 35), and contained dilated canaliculi (22, 25). The latter observation may have reflected the continued presence of mechanisms for both fluid and electrolyte uptake across basolateral membranes and secretion across canalicular membranes of H+,K+-ATPase-deficient parietal cells.

AE2-/- parietal cells had secretory microvilli on the apical membrane, but only in rare instances was there any penetration of a canaliculus into the cytoplasm, and even in those cases the penetration was very shallow. Impaired secretion was indicated not only by the achlorhydria but also by the observation that the apical microvilli were frequently surrounded with luminal material that appeared to contain pepsinogen (Figs. 9 and 11). In addition to the absence of a well developed secretory canaliculus, there was a virtual absence of tubulovesicles in AE2-/- parietal cells. Under normal conditions, the secretory canaliculus is formed by exocytic insertion of tubulovesicles into the basal aspect of the secretory microvilli-containing apical plasma membrane as the cell undergoes the transition from the resting to the stimulated state. Tubulovesicles are reformed by endocytic retrieval from canalicular membranes as the cell returns to the resting state (36, 37). The ultrastructural data presented here demonstrate that the absence of AE2 decreases the abundance of the membranes of the parietal cell secretory apparatus. This, in turn, would be expected to reduce the overall secretory capacity of the cell, which should lessen the imbalance between apical and basolateral transport mechanisms that might otherwise lead to parietal cell death.

It is conceivable that the loss of AE2 affects development of secretory membranes by blocking terminal differentiation of the parietal cell; however, the relatively high levels of H+,K+-ATPase {alpha} and {beta} subunit mRNAs and H+,K+-ATPase {alpha} subunit protein, which are expressed during terminal differentiation of parietal cells, argue against this hypothesis. Another possibility is that development of secretory membranes is impaired as a direct result of the absence of AE2 activity in intracellular membranes. The demonstration that AE2 overexpressed in HEK-293 cells can be active in a pre-Golgi compartment led to the suggestion that it might normally contribute to ionic homeostasis of intracellular organelles (38). An argument against this hypothesis is that other anion exchangers such as the tubulovesicular and apical SLC26a6 exchanger (19) and the basolateral SLC26a7 (20) might also contribute to this function in early compartments of the secretory pathway. A third, yet more speculative possibility is that the development of a mature secretory membrane system requires at least some level of ion and water transport across the parietal cell and that this capacity is absolutely dependent on AE2.

How might such an absolute dependence of secretion on AE2 be explained? Given the achlorhydria phenotype and the fact that AE2 is expressed at higher levels in parietal cells than in any other cell type (2, 12), it is reasonable to conclude that AE2 is responsible for the very high rates of exchange-mediated Cl- influx and efflux across the basolateral membrane. Because the parietal cell uses carbonic anhydrase to catalyze the formation of H+ and from H2O and CO2, basolateral exchange can completely balance HCl secretion across the canalicular membrane; in effect, it provides both the major cation (H+, via extrusion of ) and anion (Cl-) in the secreted fluid. However, previous studies indicate that it might play an even broader role (39–41). exchange coupled with Na+/H+ exchange could serve as a mechanism for loading the parietal cell with both Cl- and Na+. Intracellular Cl- can exceed 60 mM in the resting state (41) and is required for stimulated HCl and KCl secretion. Intracellular Na+ drives the Na+,K+-ATPase, which mediates uptake of K+ and generates the membrane potential. K+ is a major component of gastric secretions and is an essential counter-ion for the H+,K+-ATPase, and an appropriate membrane potential is necessary for secretion of Cl- that must accompany the secreted K+ and H+. Although it could be argued that the NKCC1 Na+-K+-2Cl- cotransporter might provide an alternative basolateral uptake mechanism for Na+,K+, and Cl- (16), NKCC1 is not necessary for acid secretion (17). Furthermore, it may not contribute to volume homeostasis in the parietal cell during acid secretion (42), and it is expressed at very low levels in the more vigorously acid-secreting parietal cells in the upper third of the gastric gland (43). Thus, exchange mediated by AE2, operating both by itself and coupled with Na+/H+ exchange with secondary coupling to the Na+,K+-ATPase, may be required to supply virtually all of the ions (H+, K+, and Cl-) secreted by the parietal cell and to contribute critically to the electrochemical driving force for Cl- secretion.

AE2-/- mice also had chief cell abnormalities, including a reduced number of secretory granules (22), and there was a reduction in the expression of pepsinogen and intrinsic factor mRNA in AE2-/- stomachs, consistent with a decrease in the number or maturation status of chief cells (26, 27). Reduction in pepsinogen mRNA expression, despite abundant pepsinogen protein stores, has been observed both in rats treated with inhibitors of acid secretion and in mouse models of achlorhydria (21, 22, 28, 29). These previous studies, as well as data from the current study, suggest that the loss of acid secretion may have a negative regulatory effect on pepsinogen mRNA synthesis and release of pepsinogen. In a study of the effects of inhibition of acid secretion on pepsinogen expression and secretion in the guinea pig stomach, inhibition of pepsinogen secretion following omeprazole treatment led to inhibition of pepsinogen mRNA synthesis (44). Although AE2-/- chief cells had fewer granules than those in wild-type glands, electron microscopy indicated that pepsinogen was a major component of the basophilic material observed in AE2-/- gastric gland lumens. The apparent accumulation of pepsinogen in the gastric gland lumens may result from inefficient flushing of the gastric glands because of the loss of HCl and fluid secretion.

In summary, the loss of AE2 caused a profound impairment of gastric acid secretion, as anticipated, but did not have a major impact on parietal cell viability. Although we hypothesized that, in the absence of the major exchanger on the basolateral membrane, the activity of intact secretory mechanisms on the apical/canalicular membranes would lead to cell death via severe perturbations of pH and volume homeostasis, parietal cell viability appeared to be minimally affected or unchanged. However, the observed achlorhydria, the impaction of dense luminal material on the apical surface of some parietal cells, and the ultrastructural evidence that a mature secretory membrane system fails to develop all indicate that secretion is virtually absent in the AE2-/- parietal cell. These results suggest that the exchanger is a central component of the basolateral ion transport system that supports gastric HCl and KCl secretion across the apical membrane. The absence of AE2 leads to loss of critical electrochemical driving forces required for both gastric acid secretion and, perhaps, also for secretory membrane development, resulting in viable, but nonfunctional parietal cells.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants DK50594 and HL61974 (to G. E. S.), DK43495 and DK34854 (to S. L. A.), and T32-HL7571 and DK67749 (to L. R. G.) and by National Institutes of Health NIEHS Center Grant P30 ES06096 (to M. L. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Current address: Dept. of Medicine, Melbourne University, Western Hospital, Gordon St., Footscray, Victoria 3011, Australia. Back

** To whom all correspondence should be addressed: Dept. of Molecular Genetics, Biochemistry, and Microbiology, 231 Albert Sabin Way, ML524, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0524. Tel.: 513-558-0056; Fax: 513-558-1885; E-mail: shullge{at}ucmail.uc.edu.

1 The abbreviations used are: AE2, anion exchanger isoform 2; ES, embryonic stem; AE2-/-, AE2+/-, and AE2+/+, AE2 homozygous mutant, heterozygous mutant, and wild-type, respectively; neo gene, neomycin resistance gene. Back

2 L. R. Gawenis, M. L. Miller, and G. E. Shull, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Adam Smolka (Medical University of South Carolina) for providing the anti-gastric H+,K+-ATPase antibody; Moying Yin, Sharon Pawlowski, and Ilona Ormsby for expert technical assistance in performing blastocyst-mediated transgenesis; and Angel Whittaker for expert animal husbandry.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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