Stomachs of Mice Lacking the Gastric H,K-ATPase α-Subunit Have Achlorhydria, Abnormal Parietal Cells, and Ciliated Metaplasia

The H,K-ATPase of the gastric parietal cell is the most critical component of the ion transport system mediating acid secretion in the stomach. To study the requirement of this enzyme in the development, maintenance, and function of the gastric mucosa, we used gene targeting to prepare mice lacking the α-subunit. Homozygous mutant (Atp4a −/−) mice appeared healthy and exhibited normal systemic electrolyte and acid-base status but were achlorhydric and hypergastrinemic. Immunocytochemical, histological, and ultrastructural analyses of Atp4a −/−stomachs revealed the presence of chief cells, demonstrating that the lack of acid secretion does not interfere with their differentiation. Parietal cells were also present in normal numbers, and despite the absence of α-subunit mRNA and protein, the β-subunit was expressed. However, Atp4a −/− parietal cells had dilated canaliculi and lacked typical canalicular microvilli and tubulovesicles, and subsets of these cells contained abnormal mitochondria and/or massive glycogen stores. Stomachs of adultAtp4a −/− mice exhibited metaplasia, which included the presence of ciliated cells. We conclude that ablation of the H,K-ATPase α-subunit causes achlorhydria and hypergastrinemia, severe perturbations in the secretory membranes of the parietal cell, and metaplasia of the gastric mucosa; however, the absence of the pump appears not to perturb parietal cell viability or chief cell differentiation. The H,K-ATPase of the gastric parietal cell is the most critical component of the ion transport system mediating acid secretion in the stomach. To study the requirement of this enzyme in the development, maintenance, and function of the gastric mucosa, we used gene targeting to prepare mice lacking the α-subunit. Homozygous mutant (Atp4a −/−) mice appeared healthy and exhibited normal systemic electrolyte and acid-base status but were achlorhydric and hypergastrinemic. Immunocytochemical, histological, and ultrastructural analyses of Atp4a −/−stomachs revealed the presence of chief cells, demonstrating that the lack of acid secretion does not interfere with their differentiation. Parietal cells were also present in normal numbers, and despite the absence of α-subunit mRNA and protein, the β-subunit was expressed. However, Atp4a −/− parietal cells had dilated canaliculi and lacked typical canalicular microvilli and tubulovesicles, and subsets of these cells contained abnormal mitochondria and/or massive glycogen stores. Stomachs of adultAtp4a −/− mice exhibited metaplasia, which included the presence of ciliated cells. We conclude that ablation of the H,K-ATPase α-subunit causes achlorhydria and hypergastrinemia, severe perturbations in the secretory membranes of the parietal cell, and metaplasia of the gastric mucosa; however, the absence of the pump appears not to perturb parietal cell viability or chief cell differentiation. kilobase pair(s) embryonic stem periodic acid-Schiff Atp4a +/−, andAtp4a +/+, gastric H,K-ATPase α-subunit homozygous mutant, heterozygous mutant, and wild-type, respectively Secretion of hydrochloric acid in the stomach is dependent on the gastric H,K-ATPase, a P-type ATPase that is present in tubulovesicular and canalicular membranes of the gastric parietal cell. The enzyme consists of two subunits (1.Chow D.C. Forte J.G. J. Exp. Biol. 1995; 198: 1-17Crossref PubMed Google Scholar, 2.Shull G.E. Lingrel J.B. J. Biol. Chem. 1986; 261: 16788-16791Abstract Full Text PDF PubMed Google Scholar, 3.Shull G.E. J. Biol. Chem. 1990; 265: 12123-12126Abstract Full Text PDF PubMed Google Scholar), a 114-kDa α-subunit (gene locusAtp4a) and a 35-kDa (protein moiety) β-subunit (gene locusAtp4b). The α-subunit contains ATP and cation binding sites and carries out the catalytic and transport functions of the enzyme (1.Chow D.C. Forte J.G. J. Exp. Biol. 1995; 198: 1-17Crossref PubMed Google Scholar), and it also contains sequences responsible for apical membrane localization (4.Dunbar L.A. Courtois-Coutry N. Roush D.L. Muth T.R. Gottardi C.J. Rajendran V. Geibel J. Kashgarian M. Caplan M.J. Acta Physiol. Scand. 1998; 163 Suppl. 643: 289-295Google Scholar). The heavily glycosylated β-subunit is required for endocytic retrieval of the H,K-ATPase from the canilicular membranes as the cell passes from the stimulated to the resting state (5.Courtois-Coutry N. Roush D. Rajendran V. McCarthy J.B. Geibel J. Kashgarian M. Caplan M.J. Cell. 1997; 90: 501-510Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) and may also contribute to proper folding and membrane localization of the enzyme (1.Chow D.C. Forte J.G. J. Exp. Biol. 1995; 198: 1-17Crossref PubMed Google Scholar). When the resting parietal cell is stimulated by acid secretogogues, the tubulovesicles are transformed into the secretory canaliculus (6.Forte J.G. Yao X. Trends Cell Biol. 1996; 6: 45-48Abstract Full Text PDF PubMed Scopus (100) Google Scholar). HCl (∼160 mm) and KCl (∼17 mm) are then secreted via the combined activities of the H,K-ATPase, which mediates the electroneutral exchange of intracellular H+ and extracellular K+, and both K+ and Cl− channels, which allow the passage of these ions down their electrochemical gradients. Because it is a major component of the tubulovesicular and canalicular membranes, it is possible that the H,K-ATPase is necessary for the biosynthesis and/or integrity of these membranes, and it might also play a role in the reversible transformations from one membrane state to the other via interactions with cytoskeletal components and other proteins. The recently described phenotype of a mouse lacking the gastric H,K-ATPase β-subunit (7.Scarff K.L. Judd L.M. Toh B.-H. Gleeson P.A. van Driel I.R. Gastroenterology. 1999; 117: 605-618Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), in which there were severe perturbations of the tubulovesicular and canalicular membrane systems, is consistent with this hypothesis. There are indications that the acid secretory activity of the H,K-ATPase might be necessary for the viability and normal development of parietal cells (8.Karam S.M. Forte J.G. Am. J. Physiol. 1994; 266: G745-G758PubMed Google Scholar) and possibly for the differentiation of chief cells (9.Kakei N. Ichinose M. Tatematsu M. Shimizu M. Oka M. Yahagi N. Matsushima M. Kurokawa K. Yonezawa S. Furihata C. Shiokawa K. Kageyama T. Miki K. Fukamachi H. Biochem. Biophys. Res. Commun. 1995; 214: 861-868Crossref PubMed Scopus (18) Google Scholar). A frequent observation in gastric glands of animals treated with inhibitors of acid secretion is the presence of parietal cells with dilated canaliculi or vacuoles (8.Karam S.M. Forte J.G. Am. J. Physiol. 1994; 266: G745-G758PubMed Google Scholar, 10.Lehy T. Dubrasquet M. Am. J. Dig. Dis. 1972; 17: 887-901Crossref PubMed Scopus (8) Google Scholar, 11.Karasawa H. Tani N. Miwa T. Gastroenterol. Jap. 1988; 23: 1-8Crossref PubMed Scopus (30) Google Scholar, 12.Helander H.F. Mattsson H. Elm G. Ottosson S. Scand. J. Gastroenterol. 1990; 25: 799-809Crossref PubMed Scopus (23) Google Scholar). Treatment with omeprazole, an inhibitor of the H,K-ATPase, caused degeneration of parietal cells and an expansion of the number of preparietal cells, and it also caused a reduction in the number of mature chief cells (8.Karam S.M. Forte J.G. Am. J. Physiol. 1994; 266: G745-G758PubMed Google Scholar, 9.Kakei N. Ichinose M. Tatematsu M. Shimizu M. Oka M. Yahagi N. Matsushima M. Kurokawa K. Yonezawa S. Furihata C. Shiokawa K. Kageyama T. Miki K. Fukamachi H. Biochem. Biophys. Res. Commun. 1995; 214: 861-868Crossref PubMed Scopus (18) Google Scholar). Expression of diphtheria toxin (13.Li Q. Karam S.M. Gordon J.I. J. Biol. Chem. 1996; 271: 3671-3676Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar) or simian virus 40 large T antigen (14.Li Q. Karam S.M. Gordon J.I. J. Biol. Chem. 1995; 270: 15777-15788Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) in parietal cells of transgenic mice caused the loss of mature parietal cells, an expansion of the preparietal cell population, and an apparent block in the maturation of chief cells. Li et al.(14.Li Q. Karam S.M. Gordon J.I. J. Biol. Chem. 1995; 270: 15777-15788Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) suggested that the inhibition of chief cell maturation might be secondary to the accompanying defect in acid secretion or, alternatively, that the parietal cell might play a direct role in controlling the differentiation and maturation of gastric epithelial cell types. Although the function of the gastric H,K-ATPase in acid secretion is well established, the importance of its acid secretory activity for the viability of the parietal cell and for the normal development of the gastric mucosa is not well understood. On the basis of studies discussed above, it seems likely that gastric H,K-ATPase activity might be a critical factor in the development and maintenance of parietal cells and other cells of the gastric mucosa. To address this issue, we have developed and analyzed a mouse model in which expression of the H,K-ATPase α-subunit mRNA and protein was eliminated. Our studies show that there are significant differences between the histopathology that occurs in the gastric mucosa of mice lacking the H,K-ATPase α-subunit and that observed after treatment with H,K-ATPase inhibitors or elimination of the H,K-ATPase β-subunit (7.Scarff K.L. Judd L.M. Toh B.-H. Gleeson P.A. van Driel I.R. Gastroenterology. 1999; 117: 605-618Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). A portion of the gastric H,K-ATPase gene was isolated from a mouse strain 129/SvJ phage genomic library using a rat gastric H,K-ATPase cDNA probe. The clone was partially characterized by restriction mapping, Southern blot analysis, polymerase chain reaction, and DNA sequencing. Exons 4, 7, 16, 19, and 20 were amplified by polymerase chain reaction, and DNA sequence analysis showed that they matched the published mouse cDNA sequence (15.Mathews P.M. Claeys D. Jaisser F. Geering K. Horisberger J.-D. Kraehenbuhl J.-P. Rossier B.C. Am. J. Physiol. 1995; 268: C1207-C1214Crossref PubMed Google Scholar). A polymerase chain reaction strategy was used to obtain fragments for insertion into the MJK+KO (16.Schultheis P.J. Clarke L.L. Meneton P. Harline M. Boivin G.P. Stemmermann G. Duffy J.J. Doetschman T. Miller M.L. Shull G.E. J. Clin. Invest. 1998; 101: 1243-1253Crossref PubMed Scopus (212) Google Scholar) targeting vector. The 3.4-kb1 5′ arm extended from codon 71 in exon 4 to codon 359 in exon 8. The 3.4-kb 3′ arm extended from codon 390 in exon 8 to codon 600 in exon 13. Initial subcloning of the arms into the targeting vector resulted in rearrangements of the plasmid, most likely due to the presence of poison sequences in the genomic fragments, with subsequent selection of rearrangements during growth of the bacteria harboring the plasmid. Therefore, the targeting vector was modified by replacing the portion of the vector containing pBluescript plasmid sequences with the pBR322 plasmid, which converted the targeting vector to a low copy plasmid. Also, NotI,PacI, HindIII, and AscI cloning sites were added to the vector immediately 5′ of the neomycin resistance gene. The 3′ arm was blunt end-ligated into the NotI site, and the 5′ arm was blunt end-ligated into the XhoI site between the neomycin resistance and herpes simplex virus thymidine kinase genes. This strategy resulted in the replacement of 31 codons with the neomycin resistance gene. Electroporation of the targeting construct, after linearizing with PacI, into ES cells and selection of G418- and gancyclovir-resistant ES cell lines were carried out as described previously (16.Schultheis P.J. Clarke L.L. Meneton P. Harline M. Boivin G.P. Stemmermann G. Duffy J.J. Doetschman T. Miller M.L. Shull G.E. J. Clin. Invest. 1998; 101: 1243-1253Crossref PubMed Scopus (212) Google Scholar). ES cell lines that underwent homologous recombination were identified by Southern blot analysis using a 0.6-kb probe that consisted of a genomic fragment extending from codon 26 in exon 2 to codon 70 in exon 4. Chimeric mice were generated by blastocyst-mediated transgenesis and mated to Black Swiss mice. Offspring with ES cell-derived genetic material were identified by their agouti coat color, and those carrying the targeted allele were determined by Southern blot analysis of tail DNAs using a genomic probe extending from codon 418 in exon 9 to codon 559 in exon 11. Awake mice were gently warmed for 10–15 min on a heating pad to increase peripheral blood circulation. Blood (50 μl) from the tail vein was collected in heparin-treated capillary tubes and analyzed immediately for gases, electrolytes, and pH using a Chiron diagnostics model 348 pH/blood gas analyzer (Chiron, Norwood, MA). pH and acid-base equivalents of the gastric contents were measured as described previously with slight modifications (16.Schultheis P.J. Clarke L.L. Meneton P. Harline M. Boivin G.P. Stemmermann G. Duffy J.J. Doetschman T. Miller M.L. Shull G.E. J. Clin. Invest. 1998; 101: 1243-1253Crossref PubMed Scopus (212) Google Scholar, 17.Stechschulte Jr., D.J. Morris D.C. Jilka R.L. Dileepan K.N. Am. J. Physiol. 1990; 259: G41-G47Crossref PubMed Google Scholar). Sex-matched mice of all three genotypes (8–10 weeks old) were fasted for 2 h prior to the experiment. Histamine HCl in phosphate-buffered saline was injected subcutaneously (2 μg/g body weight; Sigma Chemical Co., St. Louis, MO). After 45 min, the mice were killed, and their stomachs were removed. Stomach contents were emptied into 2 ml of N2-saturated normal saline, insoluble material was pelleted by centrifugation, and the pH of the supernatant was measured. The supernatant was then titrated to pH 6.5 (pH of normal saline) with either 0.01 n NaOH or 0.01 n HCl, and the data were expressed as microequivalents per gram of wet stomach weight. Mice of all three genotypes, 8–12 weeks old, were fasted overnight and anesthetized with avertin, and blood was collected by cardiocentesis. Serum was prepared, and gastrin concentrations were determined using an 125I radioimmunoassay kit (Diagnostic Products Corp.) as described previously (16.Schultheis P.J. Clarke L.L. Meneton P. Harline M. Boivin G.P. Stemmermann G. Duffy J.J. Doetschman T. Miller M.L. Shull G.E. J. Clin. Invest. 1998; 101: 1243-1253Crossref PubMed Scopus (212) Google Scholar). Total RNA was isolated from the stomachs of Atp4a +/+,Atp4a +/−, and Atp4a −/−mice using Tri-Reagant (Molecular Research Center, Inc., Cincinnati, OH). RNA was denatured with glyoxal, separated by electrophoresis in a 1% agarose gel, and transferred to a nylon membrane. The following probes were sequentially hybridized with the blot using the method of Church and Gilbert (18.Church G.M. Gilbert W. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1991-1995Crossref PubMed Scopus (7251) Google Scholar): rat gastric H,K-ATPase α-subunit, rat gastric H,K-ATPase β-subunit, rat pepsinogen C, rat intrinsic factor, rat gastrin, and mouse L32 ribosomal subunit as a loading control. Blots were analyzed by autoradiography and PhosphorImager analysis (Molecular Dynamics, Inc., Sunnyvale, CA). Antibodies and lectin and the dilutions for each used in immunohistochemical analysis of stomach sections were as follows: rabbit anti-α-subunit of porcine gastric H,K-ATPase (1:100; Calbiochem-Novabiochem); rabbit anti-β-subunit of porcine gastric H,K-ATPase (1:25; Calbiochem-Novabiochem); sheep anti-human pepsinogen II (1:25; Biodesign International, Kennebunk, ME); donkey anti-rabbit IgG (1:100; Cortex Biochem, Inc., San Leandro, CA); donkey anti-sheep IgG (1:100; Cortex Biochem, Inc., San Leandro, CA); and fluorescein isothiocyanate-conjugated Dolichos biflorus agglutinin (40 μg/ml; Sigma). The secondary antibodies were conjugated to Texas Red fluorophore. For immunohistochemical staining, stomachs were removed from 10-week-old mice, fixed in 10% neutral buffered formalin, and embedded in paraffin. Sections (5 μm) were cut, deparaffinized with xylene, and rehydrated with graded concentrations of ethanol. Slides that were stained with antibodies were first blocked with normal donkey serum. The primary antibodies, diluted in normal donkey serum, were incubated overnight at 4 °C, while the secondary antibodies were incubated in the dark for 1 h at room temperature. The lectin, D. biflorus agglutinin, was diluted in phosphate-buffered saline with 1% bovine serum albumin and 0.3% Triton X-100. Stomach sections treated with D. biflorus agglutinin were first blocked with phosphate-buffered saline containing 1% bovine serum albumin and 0.3% Triton X-100, incubated overnight with the lectin at 4 °C, and washed three times in phosphate-buffered saline. Stomachs were removed from juvenile (17-day-old, n = 2 wild-type, 2 heterozygous, and 2 homozygous mutant) and adult (10–12-week-old, n= 4 wild-type, 2 heterozygous, and 4 homozygous mutant) mice, fixed in 10% neutral buffered formalin, and embedded in paraffin. Blocks were cut into 5-μm-thick sections and stained with either hematoxylin and eosin or periodic acid-Schiff (PAS) and Alcian blue for examination by light microscopy. Stomachs from another set of juvenile (n = 4 wild-type, 1 heterozygous, and 4 homozygous mutant) and adult (n = 4 wild-type, 3 heterozygous, and 4 homozygous mutant) mice were fixed in 4% paraformaldehyde in phosphate buffer (pH 7.3), embedded in Spurr's resin, sectioned 1 μm thick, and stained with toluidine blue for detailed light microscopy. Sections from the same wild-type and homozygous mutants (n = 4 of each genotype), 0.9 μm thick, were stained with uranyl acetate and lead citrate and examined by electron microscopy. Morphometry was performed as described previously (16.Schultheis P.J. Clarke L.L. Meneton P. Harline M. Boivin G.P. Stemmermann G. Duffy J.J. Doetschman T. Miller M.L. Shull G.E. J. Clin. Invest. 1998; 101: 1243-1253Crossref PubMed Scopus (212) Google Scholar) using 17-day-old (two of each genotype) and 10–12-week-old (four of each genotype) wild-type and null mutant mice. Only cells in which the nucleus was present in the plane of section were counted. Using phase contrast, all cells in the normal position of parietal cells in the gastric gland and having features typical of parietal cells (e.g. canaliculi or numerous and large mitochondria) were counted as parietal cells, regardless of whether their morphology was normal. Cells located in the base or neck of the gland and having at least six large birefringent granules were counted as chief cells. Cells with small apical mucous granules in the neck of the gastric gland and cells at the gastric pit and surface were counted as mucous cells. Cells without distinguishing features, such as secretory granules or large mitochondria or canaliculi, were counted as “other” cells. The mutant allele of Atp4a was generated in ES cells by replacing codons 360–390 in exon 8 with the neomycin resistance gene (Fig.1, A and B). The region that was deleted encodes sequences extending from the fourth transmembrane domain to just beyond the conserved phosphorylation site (Asp385), which is required for enzyme activity. This region was eliminated to ensure that the mutant allele would be functionally null. Chimeric mice were generated using one of the targeted ES cell lines, and the null allele was successfully transmitted through the germ line. Southern blot analysis of tail DNA samples from litters of heterozygous matings (Fig. 1 C) demonstrated that mice of all three genotypes were born in the expected Mendelian ratios (102 +/+, 172 +/−, and 99 −/−).Atp4a −/− mice thrived, were indistinguishable from their wild-type and heterozygous littermates in both behavior and outward appearance, and were fertile. The α-subunit of the gastric H,K-ATPase is expressed in mouse kidney (19.Nakamura S. Amlal H. Schultheis P.J. Galla J.H. Shull G.E. Soleimani M. Am. J. Physiol. 1999; 276: F914-F921PubMed Google Scholar), and it has been suggested that it might function in renal control of acid-base or potassium homeostasis (20.Silver R.B. Soleimani M. Am. J. Physiol. 1999; 276: F799-F811PubMed Google Scholar). As an initial test of this hypothesis, blood samples were taken from adult animals of all three genotypes, and plasma electrolytes, blood pH, and blood gasses were analyzed. As shown in Table I, no significant differences were observed among the three genotypes.Table IPlasma electrolytes and acid-base statuspHpCO2pO2HCO −3Na+K+Cl−mm Hgmm HgmmmmmmmmAtp4a +/+7.45 ± 0.0135.71 ± 1.3579.53 ± 1.0624.55 ± 0.93148.0 ± 0.76.10 ± 0.20115.3 ± 1.7Atp4a +/−7.45 ± 0.0136.66 ± 1.2673.13 ± 3.3224.91 ± 0.87148.5 ± 0.56.02 ± 0.19116.0 ± 0.8Atp4a −/−7.48 ± 0.0435.44 ± 1.374.89 ± 3.2724.06 ± 1.20148.0 ± 0.66.60 ± 0.31115.4 ± 0.5All values are means ± S.E. n = 8 for each genotype. Open table in a new tab All values are means ± S.E. n = 8 for each genotype. To confirm that the gastric H,K-ATPase is solely responsible for gastric acid secretion and to determine whether the loss of one copy of the gastric H,K-ATPase α-subunit gene leads to a reduction in net acid secretion, we measured the pH and acid-base content of gastric secretions from histamine-treatedAtp4a +/+, Atp4a +/−, andAtp4a −/− mice. The contents ofAtp4a +/+ and Atp4a +/−stomachs were similar with respect to both pH (3.17 ± 0.17 and 3.13 ± 0.21, respectively; Fig.2 A) and acid equivalents (35.4 ± 9.0 μeq/g and 32.5 ± 5.2 μeq/g, respectively; Fig. 2 B). In contrast, the pH of the gastric contents ofAtp4a −/− mice was close to neutrality (6.9 ± 0.1; Fig. 2 A) and contained net base (4.5 ± 2.3 μeq base/g; Fig. 2 B). The peptide hormone gastrin is known to play an important role in the regulation of acid secretion (21.Brand S.J. Schmidt W.E. Yamada T. Gastroenterology. 2nd Ed. J.B. Lippincott Co., Philadelphia1995: 25-71Google Scholar) and is induced in several mouse models of achlorhydria (7.Scarff K.L. Judd L.M. Toh B.-H. Gleeson P.A. van Driel I.R. Gastroenterology. 1999; 117: 605-618Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 16.Schultheis P.J. Clarke L.L. Meneton P. Harline M. Boivin G.P. Stemmermann G. Duffy J.J. Doetschman T. Miller M.L. Shull G.E. J. Clin. Invest. 1998; 101: 1243-1253Crossref PubMed Scopus (212) Google Scholar) or hypochlorhydria (22.Langhans N. Rindi G. Chiu M. Rehfeld J.F. Ardman B. Beinborn M. Kopin A.S. Gastroenterology. 1997; 112: 280-286Abstract Full Text PDF PubMed Scopus (183) Google Scholar). Northern blot analysis showed that gastrin mRNA was increased ∼4-fold inAtp4a −/− stomachs compared withAtp4a +/+ or Atp4a +/−stomachs (Fig. 3 A). In addition, serum gastrin concentrations (Fig. 3 B) were significantly higher in Atp4a −/− mice than in either heterozygous or wild-type mice (Atp4a −/−, 499 ± 120 pg/ml;Atp4a +/−, 131 ± 45 pg/ml;Atp4a +/+, 81 ± 21 pg/ml). High levels of the gastric H,K-ATPase α-subunit mRNA were present in Atp4a +/+stomachs, and there was little, if any, decrease inAtp4a +/− stomachs (Fig.4, top panel). Although trace levels of an ∼1-kb mRNA were detected in samples fromAtp4a −/− stomachs after long autoradiographic exposures (data not shown), the wild-type mRNA was absent in the knockout (Fig. 4, top panel). In contrast, mRNA for the H,K-ATPase β-subunit was ∼1.8-fold more abundant in theAtp4a −/− samples than in theAtp4a +/+ or Atp4a +/−samples (Fig. 4, second panel). In the rodent stomach, pepsinogen and intrinsic factor are expressed almost exclusively in chief cells (23.Lorenz R.G. Gordon J.I. J. Biol. Chem. 1993; 268: 26559-26570Abstract Full Text PDF PubMed Google Scholar, 24.Maeda M. Asahara S. Nishi T. Mushiake S. Oka T. Shimada S. Chiba T. Tohyama M. Futai M. J. Biochem. (Tokyo). 1995; 117: 1305-1311Crossref PubMed Scopus (15) Google Scholar), making them excellent molecular markers for this cell type. In theAtp4a −/− stomach, intrinsic factor mRNA was detected at levels comparable with those observed inAtp4a +/+ and Atp4a +/−stomachs (Fig. 4, third panel), suggesting that mature chief cells were present. Pepsinogen mRNA, however, was sharply reduced in the Atp4a −/− stomachs (Fig. 4, fourth panel). As noted above, the α-subunit probe, which corresponded to the N-terminal coding sequence, detected trace amounts of a 1-kb mRNA in Atp4a −/−stomachs. To address the possibility that a stable protein containing N-terminal sequences might be translated from the aberrant transcript and to further document the null mutation, immunocytochemistry of stomach sections was performed using an antibody directed against a peptide sequence from the N terminus of the protein. Parietal cells of adult Atp4a +/+ mice were heavily stained (Fig.5 A), but no specific staining was detected in Atp4a −/− parietal cells (Fig.5 B). To determine whether β-subunit protein was present inAtp4a −/− stomachs, as suggested by the abundance of its mRNA, and to assess the relative numbers of parietal cells in stomachs of wild-type and mutant mice, stomach sections were stained with an antibody directed against the gastric H,K-ATPase β-subunit and with D. biflorus agglutinin, both of which have been used as parietal cell markers (23.Lorenz R.G. Gordon J.I. J. Biol. Chem. 1993; 268: 26559-26570Abstract Full Text PDF PubMed Google Scholar). In bothAtp4a +/+ and Atp4a −/−stomachs, a comparable number of cells were stained with the β-subunit antibody (Fig. 6,B and F) and with D. biflorusagglutinin (Fig. 6, D and H). Staining with the β-subunit antibody seemed less intense inAtp4a −/− sections compared withAtp4a +/+ sections (Fig. 6, B andF), suggesting that β-subunit protein is present at lower levels in Atp4a −/− parietal cells than in wild-type cells, despite the higher mRNA levels. Positively staining cells in the wild-type stomach had the normal appearance of parietal cells (Fig. 6, B and D). In contrast, positively staining cells in Atp4a −/− stomachs had numerous vacuole-like structures in the cytoplasm (Fig. 6,F and H) reminiscent of the dilated canaliculi observed after treatment with omeprazole (8.Karam S.M. Forte J.G. Am. J. Physiol. 1994; 266: G745-G758PubMed Google Scholar, 11.Karasawa H. Tani N. Miwa T. Gastroenterol. Jap. 1988; 23: 1-8Crossref PubMed Scopus (30) Google Scholar, 12.Helander H.F. Mattsson H. Elm G. Ottosson S. Scand. J. Gastroenterol. 1990; 25: 799-809Crossref PubMed Scopus (23) Google Scholar). While these cells did not exhibit normal parietal cell morphology, their staining with both the β-subunit antibody and D. biflorus agglutinin confirmed that they were parietal cells; this conclusion was supported by analysis of their ultrastructure. To determine if mature chief cells containing pepsinogen stores were present in Atp4a −/− stomachs, despite the sharp decrease in pepsinogen mRNA, stomach sections fromAtp4a +/+ and Atp4a −/−mice were stained with an anti-pepsinogen antibody. As shown in Fig.7, both Atp4a +/+(Fig. 7 B) and Atp4a −/− (Fig.7 D) stomachs stained positive for pepsinogen, and both genotypes displayed an abundance of positively staining cells. These data, along with the normal expression of intrinsic factor mRNA, indicated that mature chief cells were present at relatively normal numbers in adult Atp4a −/− stomachs. Sections from juvenile (17-day-old) and adult (10–12-week-old) Atp4a +/+ andAtp4a −/− stomachs were stained with toluidine blue and examined by light microscopy, and morphometry was performed to determine whether there were significant alterations in the numbers of gastric epithelial cell types. Alterations in stomachs of young null mutants were mild when compared with those of adult mice. Occasional dilated gastric glands and parietal cells with vacuole-like dilations were observed in stomachs of 17-day-old Atp4a −/− mice, and enteroendocrine cells were significantly greater in numbers and in size. Both parietal and chief cells appeared to be slightly reduced in numbers (data not shown), although this may have been due to a reduced rate of maturation. The chief cells that were observed seemed to have fewer granules than those of Atp4a +/+ stomachs, and the only parietal cells that could be clearly identified as such were those few that had dilated canaliculi. As discussed below, a significant reduction in parietal and chief cell numbers was not observed in adult mice, although most parietal cells were clearly abnormal, and chief cells appeared to have fewer granules. Histopathological alterations in adult null mutants were considerably more severe than in young mice and included metaplasia (described in detail below) and a serious disruption of the architecture of the gastric gland (Fig. 8). Parietal cells in adult Atp4a +/+ gastric glands were typical of those normally seen in toluidine blue-stained sections (Fig.8 A) and comprised 39.0 ± 1.8% of the epithelial cells in the gland (Table II). InAtp4a −/− gastric glands, cells identified as parietal cells comprised 37.8 ± 6.4% of the epithelial cells counted (Table II); however, rather than having the typical parietal cell morphology, they contained dilated caniliculi (Fig. 8,B and C), as first observed in sections stained with the β-subunit antibody and D. biflorus agglutinin (Fig. 6) and noted above in juvenile mice. A subset of these altered parietal cells contained massive stores of cytoplasmic glycogen. InAtp4a +/+ gastric glands, glycogen-containing parietal cells comprised 0.03 ± 0.03% of the epithelial cells, while Atp4a −/− gastric glands had significantly more glycogen-containing cells (5.6 ± 0.9%; TableII).Table IIEpithelial cell populations of gastric glandsCell type+/+−/−Total Parietal39.0 ± 1.837.8 ± 6.4 GlycogenaA subset of parietal cells with visible pools of glycogen.0.03 ± 0.035.6 ± 0.9bp < 0.05.Chief15.2 ± 2.112.0 ± 1.8Mucous43.5 ± 1.941.2