Regulation of gastric epithelial cell development revealed in H+/K+-ATPase beta -subunit- and gastrin-deficient mice

Teo V. Franic1, Louise M. Judd1, David Robinson1, Simon P. Barrett1, Katrina L. Scarff1, Paul A. Gleeson1, Linda C. Samuelson2, and Ian R. Van Driel1

1 Department of Pathology and Immunology, Monash University Medical School, Alfred Hospital, Melbourne, Victoria, Australia 3181; and 2 Department of Physiology, University of Michigan, Ann Arbor, Michigan 48109-0622


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The gastric H+/K+-ATPase is essential for normal development of parietal cells. Here we have directly assessed the role of the H+/K+-ATPase beta -subunit (H/K-beta ) on epithelial cell development by detailed quantitation of the epithelial cell types of the gastric mucosa of H/K-beta -deficient mice. H/K-beta -deficient mice had a 3.1-fold increase in the number of immature cells per gastric unit; however, the numbers of surface mucous and parietal cells were similar to those in the gastric units of wild-type mice. The effect of elevated gastrin levels in the H/K-beta -deficient mice was determined by producing mice that are also deficient in gastrin. We demonstrated that the increased production of immature cells and resulting hypertrophy is caused by the overproduction of gastrin. However, the depletion of zymogenic cells, which is another feature of H/K-beta -deficient mice, is independent of hypergastrinemia. Significantly, parietal cells of H/K-beta - and gastrin-deficient mice had abnormal secretory membranes and were devoid of resting tubulovesicular membranes. Together these data suggest a homeostatic mechanism limiting the number of immature cells that can develop into end-stage epithelial cells and indicate a direct role for H/K-beta in the development of mature parietal cells.

parietal cell, gastric mucosa, membrane biosynthesis


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE GASTRIC H+/K+-ATPase, present in gastric parietal (oxyntic) cells, exchanges luminal K+ for cytoplasmic H+ and is the enzyme principally responsible for gastric luminal acidification (6, 24, 30). The gastric H+/K+-ATPase is composed of two noncovalently linked subunits, an alpha  (H/K-alpha )- and a beta  (H/K-beta )-subunit, with apparent molecular masses of 95 and 60-90 kDa, respectively (1, 24). H/K-alpha (encoded by the mouse gene Atp4a) contains the catalytic sites of the enzyme and has 10 transmembrane domains. The highly glycosylated H/K-beta (2, 29) (encoded by the mouse gene Atp4b) has one transmembrane domain (28), is required for the transport of an active enzyme complex from the endoplasmic reticulum to the apical membranes (8, 11), and is absolutely required for gastric acid secretion (25).

Gastric parietal cells contain an extensive network of secretory membranes. These membranes contain the gastric H+/K+-ATPase protein and are the site of gastric acid production (6, 23, 26). In the resting state the membranes appear as a network of cytoplasmic helically coiled tubules often termed tubulovesicles (6, 22). On stimulation with secretagogues the membranes are transformed into a secretory canaliculus, which is an expansion of the apical membrane and is composed of long microvilli. These actively secreting membranes are associated with the actin cortical cytoskeleton, probably via associations mediated by a member of the ERM family of proteins, ezrin (5, 10).

The majority of the gastric mucosa is glandular epithelium. The glands or units are composed of three major differentiated cell types (12). Pit (surface mucous) cells occur in the upper pit zone of the units and secrete protective mucus. Acid-secreting parietal cells are found in the middle and lower regions of the units. Zymogenic (chief) cells secrete pepsin and, in some species, intrinsic factor and predominate toward the base of units. The cells of the gastric units are in continuous turnover. Mature epithelial cells develop from stem cells via a series of well-defined cellular intermediates (15).

Recently, mice in which the genes encoding the gastric H+/K+-ATPase subunits were mutated have been produced (25, 27). Analysis of the H/K-beta -deficient mice indicated that this protein was required for gastric acid secretion and correct biosynthesis of parietal cell membranes. In addition, the gastric mucosa was greatly hypertrophied and significant perturbations were observed in the cell types present in the mucosa. In this paper we quantify the mucosal cell perturbations. We also examine the role that the constitutively elevated levels of gastrin found in H/K-beta -deficient mice play in determining phenotype (gastrin levels were 6.7-fold higher than normal). To do this, we have used mice deficient in the hormone gastrin (Ref. 7; encoded by the mouse gene Gast). The gastrin-deficient mice are achlorhydric, and their parietal cells are unable to secrete acid in response to histamine or acetylcholine, the two other stimuli that can induce gastric acid secretion.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. H/K-beta -deficient [Atp4bo/o; 2 mutant (o) Atp4b alleles] and gastrin-deficient (Gasto/o; 2 mutant Gast alleles) mice are as described previously (7, 25). To generate mice deficient in both H/K-beta and gastrin, Atp4bw/o [BALB/c strain; 1 wild-type (w) and 1 mutant allele] and Gastw/o (129 strain) mice were bred together to generate mice of the four genotypes analyzed in this work.

In this paper the genotypes of mice used in experiments are as follows: wild-type, Atp4bw/w Gastw/w; H/K-beta -deficient, Atp4bo/o Gastw/w; H/K-beta - and gastrin-deficient, Atp4bo/o Gasto/o; gastrin-deficient, Atp4bw/w Gasto/o. All mice were housed at the Monash University Animal Facility under conventional conditions.

Histological and quantitative analysis. Quantitative histological analysis was as previously described (13). Briefly, stomachs from 35-day-old mice were washed in PBS, fixed in 10% formalin in phosphate buffer, and embedded in paraffin wax. Sections (4 µm) were cut, fixed, dewaxed, and stained with hematoxylin and eosin. In all analyses, only the oxyntic mucosa was examined. Morphological criteria used to identify individual cell types are described in Fig. 2. Apoptotic cells were identified by strict morphological criteria of cell shrinkage, condensed nuclear chromatin, and loss of contact with adjacent cells. We have found this method to be at least as reliable in identifying apoptotic cells in sections of gastrointestinal tissue as other methods such as labeling of nicked DNA (TUNEL; L. M. Judd, I. R. van Driel, T. De Jong, and P. O'Brien, unpublished observations). For quantitative purposes, five sections from each stomach at least 30 µm apart were analyzed. From each section, 6 or 7 complete longitudinal profiles of units were selected at random, totaling 32 gastric units per mouse. Images were collected at high magnification using a Leica DRM 300 light microscope and Leica Image Capture software for analysis. Statistical analysis involved a repeated-measures ANOVA test.

Immunohistochemical analysis of mitosis. Proliferating cells were detected by two techniques. First, anti-proliferating cell nuclear antigen (PCNA) staining was used. Stomachs from 35-day-old mice were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4 and embedded in paraffin wax. Dewaxed and cleared sections (4 µm) were incubated in DAKO antigen retrieval solution (DAKO S1700) at 98°C for 25 min. Sections were allowed to cool for 20 min before incubation with an anti-PCNA antibody (Ref. 33; DAKO M0879, 0.48 mg/l), followed by a streptavidin-horseradish peroxidase complex (no. 140169, Amersham). Bound horseradish complex was detected by incubation with PBS containing 0.05% diaminobenzidine and 0.03% nickel chloride for 12 min. Sections were then counterstained for 30 s with hematoxylin.

Proliferating cells were also detected by staining chromatin that had incorporated 5-bromo-2'-deoxyuridine (BrdU). Thirty-five-day-old H/K-beta -deficient mice and wild-type littermates were injected with 100 mg of BrdU (no. B5002; Sigma)/kg body wt 6 h before death. Stomachs were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4, and sections were cut as described in Histological and quantitative analysis. Dewaxed and cleared sections were incubated (15 min) with 0.8 mg/ml proteinase K (Amresco) before depurination with 2 M HCl at 37°C for 20 min. Endogenous peroxidase activity was inactivated by incubation with 1% H2O2 in PBS. BrdU incorporation was detected using a biotinylated anti-BrdU conjugate (2 µg/ml; no. 03-3940, Zymed) followed by a streptavidin-horseradish peroxidase complex (no. 140169, Amersham). Bound horseradish peroxidase was detected by incubation with the diaminobenzidine containing nickel chloride for 15 min. Sections were then counterstained with hematoxylin and eosin. Image capture and analyses were performed as described in Histological and quantitative analysis.

Electron microscopy. Stomachs from 35-day-old mice were washed in cold PBS and fixed in 4% paraformaldehyde, 4% sucrose, and 2% glutaraldehyde in 0.1 M phosphate buffer pH 7.4 at 4°C overnight. The tissue was postfixed in 1% osmium tetroxide, dehydrated in graded acetone, and embedded in hard Spurr's resin. Sections (90 nm) were examined after they were stained with 2% uranyl acetate and lead citrate in a Phillips 400T transmission electron microscope operating at 80 kV. At least 50 parietal cells from at least two animals of each genotype were analyzed.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Gastric mucosae of H/K-beta -deficient mice are hypertrophic because of an accumulation of immature cells but are deficient in zymogenic cells. Figure 1 represents hematoxylin and eosin-stained sections of gastric mucosae from mice of various genotypes. Figure 1, A and B, shows mucosae from 35 day-old wild-type and H/K-beta -deficient mice, respectively. The units of the gastric mucosae of the H/K-beta -deficient mice were approximately twice the length of the mucosae from wild-type littermates. The mucosae of the H/K-beta -deficient animals (Fig. 1, B and F) contained abnormal "vacuolated" parietal cells that are absent from normal mucosae (Fig. 1, A and E) and are discussed below.


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Fig. 1.   The gastric mucosae of 35-day-old mice of varying genotypes. Stomachs of mice of the genotypes indicated were removed fixed, sectioned, stained with hematoxylin and eosin, and examined by light microscopy. The lumen of the stomach is at the top of each panel. Asterisks in F and G indicate "vacuoles" in parietal cells. Bar in D applies to panels A-D; bar = 100 µm. Bar in H applies to panels E-H; bar = 25 µm. H/K-beta , H+/K+-ATPase beta -subunit.

We determined the cell types present in gastric mucosae using the method previously described (Ref. 13; see MATERIALS AND METHODS). Briefly, sections of paraffin-embedded stomachs were stained with hematoxylin and eosin and viewed by high-magnification light microscopy. We have found that the distinct morphological characteristics of the cell types of the gastric mucosa allow this to be a reliable method for quantitation. The structure of the cell types as viewed under high magnification is shown in Fig. 2. Parietal cells have been categorized into cells with a structure that is indistinguishable from parietal cells found in wild-type animals (Fig. 2A) and those that contain larger vacuole-like structures (Fig. 2, B and C). Immature cells were defined as cells that did not contain characteristic features of mature cells. They were either small cells with a large nuclear-to-cytoplasmic ratio that most likely represent the self-renewing stem cells (Fig. 2D) or larger cells that were probably cell-committed precursors such as pre-pit and pre-parietal cells (Fig. 2E; Ref. 17). Examples of zymogenic, pit, and apoptotic cells are also shown in Fig. 2, F-H, respectively. These cells were identified by their well-defined morphological features.


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Fig. 2.   Cell types present within gastric units of 35-day-old mice. Stomachs of mice were removed fixed, sectioned, stained with hematoxylin and eosin, and examined by light microscopy. Arrows indicate the nuclei of the following cell types. Normal parietal cells were very large cells that displayed weak cytoplasmic staining with eosin (A). "Vacuolated" parietal cells from H/K-beta -deficient mice showed >= 1 "vacuole(s)" within the cytoplasm. The vacuoles did not stain with hematoxylin or eosin (B and C). Immature cells did not contain characteristic features of mature cells. Small immature cells had almost no visible cytoplasm and likely represent the self-renewing stem cells (D). Larger immature cells displayed weak cytoplasmic staining with eosin and moderate staining with hematoxylin but were clearly smaller than parietal cells and were probably committed precursors such as pre-pit and pre-parietal cells (E). Zymogenic cells were located toward the base of the units and stained strongly with hematoxylin (F). Pit cells were distinctly columnar with lightly stained apical cytoplasm that contained granules (G). Apoptotic cells displayed cell shrinkage, condensed nuclear chromatin, and loss of contact with adjacent cells (H). Bar in A = 5 µm and applies to all panels.

Figure 3 depicts the quantitation analyses. The H/K-beta -deficient mice had approximately twofold more cells in total per gastric unit section (Fig. 3H) than wild-type littermates (66.2 ± 13.7 in wild-type mice compared with 132.2 ± 25.9 in H/K-beta -deficient mice; all numbers in the text are the mean ± SD number of cells per gastric unit section calculated from the data of the 3 animals shown in Fig. 3). This increase in cellularity was primarily caused by an elevated number of immature cells (Fig. 3B; 32.6 ± 9.1 in wild-type mice compared with 101.6 ± 23.7 in H/K-beta -deficient mice).


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Fig. 3.   Quantitative analysis of the epithelial cells of 35 day-old mice. Stomachs of mice of the genotypes indicated were removed fixed, sectioned, stained with hematoxylin and eosin, and examined by high-magnification light microscopy. Cell types in the gastric mucosa were quantitated as described in MATERIALS AND METHODS. A: pit cells; B: total immature cells; C: apoptotic cells; D: normal parietal cells; E: vacuolated parietal cells; F: total parietal cells; G: zymogenic cells; H: total cells. Each data point represents mean ± SD from 1 animal. Absence of an error bar indicates an SD of 0. C-E and H: *significant difference (P < 0.0001) between the mean value of the mice in the marked genotype and that of the 3 other genotypes. G: *2 genotypes that have mean values that are significantly different (P < 0.0001) from the nonasterisked genotype means but not from each other. B and F: significant differences in mean values: *P < 0.0001, **P < 0.001, ***P < 0.01.

The number of zymogenic cells was greatly reduced in H/K-beta -deficient mice. Only 0.36 zymogenic cells/gastric unit section were found in gastric units analyzed in the H/K-beta -deficient mice compared with an average of 13.2 zymogenic cells/gastric unit section in wild-type animals (37-fold reduction; P < 0.0001).

The total number of parietal cells in each gastric unit was not vastly different between the H/K-beta -deficient (17.2 ± 4.2) and wild-type (13.8 ± 4.6) mice, although this difference was statistically significant (P < 0.001). We divided parietal cells into different groups based on their structure (see above). The mucosae of wild-type animals only contained parietal cells that did not have vacuole-like structures, whereas all of the parietal cells in the H/K-beta -deficient mice contained visible vacuoles. No significant differences were found in the number of pit cells in any of the genotypes.

Hypergastrinemia in H/K-beta -deficient mice is responsible for mucosal cell hypertrophy but not for depletion of zymogenic cells. One of the features observed in the H/K-beta -deficient mice is highly elevated serum gastrin levels that result from the neutral gastric pH (25). We wanted to determine which aspects of the phenotype of these mice were attributable to this chronic hypergastrinemia. To do this, we bred the H/K-beta -deficient mice with gastrin-deficient mice described previously (7). These mice have a null mutation in the gastrin gene and produce no gastrin protein. Mice with null mutations in both the Atp4b and Gast genes (H/K-beta and gastrin deficient) are viable and fertile and are grossly indistinguishable from littermates (data not shown).

The mucosae of gastrin-deficient mice (Fig. 1, D and H) were indistinguishable from the mucosae of wild-type animals (Fig. 1, A and E). Furthermore, quantitative analyses (Fig. 3) indicated that there were no significant differences between the numbers of individual gastric cell types in these animal strains.

The length of the mucosa (Fig. 1C) and the total cell number (Fig. 3H) in the gastric unit sections of H/K-beta - and gastrin-deficient mice were comparable to those of the wild-type and gastrin-deficient mice. The lack of hypertrophy apparent in the H/K-beta - and gastrin-deficient mice compared with the H/K-beta -deficient mice was caused by the absence of the greatly elevated number of immature cells (1.4-fold higher than wild-type compared with 3.1-fold in H/K-beta -deficient mice; numbers of immature cells: 45.3 ± 10.6 in H/K-beta - and gastrin-deficient mice, 101.6 ± 23.8 in H/K-beta -deficient mice, and 32.6 ± 9.1 in wild-type mice). A small but significant decrease in total parietal cell number compared with wild-type animals was observed (Fig. 3F; 9.7 ± 3.1 in H/K-beta - and gastrin-deficient mice compared with 13.8 ± 4.6 in wild-type mice; P < 0.0001). Large vacuolated parietal cells (Fig. 1, C and G) were evident in the H/K-beta - and gastrin-deficient mice, although by light microscopy approximately one-half of the total number of parietal cells were indistinguishable from parietal cells in wild-type mucosae (however, it should be noted that by electron microscopy the secretory membranes had an abnormal structure; see below). Like the H/K-beta -deficient mice, mucosae of mice of this genotype are almost devoid of zymogenic cells (Fig. 3G; 1.04 ± 2.6), indicating that the elevated level of gastrin is not responsible for this phenotype in H/K-beta -deficient mice.

Cell proliferation and death. The number of apoptotic cells in the gastric mucosa was quantitated by histological analysis (Fig. 3C) and identified by strict morphological criteria of cell shrinkage, condensed nuclear chromatin, and loss of contact with adjacent cells. Only H/K-beta -deficient mice had a significantly increased number of apoptotic cells in the gastric mucosa (6.7 ± 3.5 in H/K-beta -deficient mice compared with 0.7 ± 1.2 in wild-type mice; P < 0.0001). In the H/K-beta - and gastrin-deficient mice the numbers of immature cells and apoptotic cells were moderately increased relative to wild-type animals [1.4-fold higher for immature cells (for numbers see above); 2.5-fold increase for apoptotic cells: wild type, 0.7 ± 1.2; H/K-beta - and gastrin-deficient, 1.8 ± 1.8, not significant].

The proliferation of gastric mucosal cells in mice of all four genotypes was assessed by staining with an anti-PCNA/cyclin antibody that stains cells in the G1 phase of the cell cycle (Fig. 4). This antibody has been widely used to identify cells in mitosis (33). As noted previously, most proliferating cells in the mucosa of wild-type animals were located in the isthmus region (Fig. 4A). In the H/K-beta -deficient animals, proliferating cells were also located in more basal regions of the gastric units and were present in much greater quantity (Fig. 4B). In gastrin-deficient mice the number and location of PCNA-expressing cells were very similar to those in wild-type mice (Fig. 4D). The number of proliferating cells observed in the H/K-beta - and gastrin-deficient mice was similar to that seen in the wild-type and gastrin-deficient mice (Fig. 4C). However, the location of proliferating cells was not restricted to isthmal regions but, as with the H/K-beta -deficient mice, they were also located toward the base of the mucosa. Hence, these data support the conclusion that elevated gastrin levels in the H/K-beta -deficient mice lead to hyperproliferation of mucosal cells and thus hypertrophy.


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Fig. 4.   Immunohistochemical detection of mitosis. Stomach sections from 35-day-old mice of the genotypes indicated were sequentially incubated with an anti-proliferating cell nuclear antigen/cyclin antibody, a biotinylated anti-mouse antibody-bound antibody, and a streptavidin-horseradish peroxidase complex. Bound complexes were detected using diaminobenzidine, which results in a dark nuclear precipitate. Sections were counterstained with hematoxylin. Bar = 100 µm; applies to all panels.

We quantitated the increased proliferation of gastric mucosal cells in H/K-beta -deficient animals. Mice were injected with BrdU and killed 6 h later, and sections were stained with an anti-BrdU antibody. The number of stained nuclei was quantitated (Fig. 5). This revealed that, as expected, there were far more proliferating cells in gastric units of H/K-beta -deficient mice than in wild-type animals (14.1 ± 7.2 in H/K-beta -deficient mice compared with 4.7 ± 2.7 in wild-type mice; P < 0.0001).


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Fig. 5.   Immunohistochemical detection of 5-bromo-2'-deoxyuridine (BrdU) incorporation. Thirty-five-day-old mice were injected with BrdU and killed 6 h later. Sections were incubated with a biotinylated anti-BrdU antibody and then with horseradish peroxidase conjugated with streptavidin. Binding of the conjugate was detected by incubation with diaminobenzidine. Sections were counterstained with hematoxylin and eosin, and the number of stained nuclei was quantitated as described in MATERIALS AND METHODS. Each data point represents mean ± SD from 1 animal. The means of the 2 groups of mice are significantly different (P < 0.001).

Absence of parietal cell tubular membranes in H/K-beta -deficient mice is not caused by hyperstimulation. Previously, we had observed (25) that the parietal cells of H/K-beta -deficient mice had a complete absence of tubular secretory membranes. Instead, an altered canaliculus was observed. One possible explanation for this observation was that the hypergastrinemia resulted in a chronic stimulation of the parietal cells and all resting tubular membranes were converted to the active canalicular form. The H/K-beta - and gastrin-deficient mice were used to examine this issue. Parietal cells in gastrin-deficient mice were refractory to stimulation via either H2 or acetylcholine receptors (7). Hence, the membranes in the H/K-beta - and gastrin-deficient mice should be in a resting state. The ultrastructure of parietal cells from mice of all four genotypes was examined (Fig. 6). Large numbers of cells (>50) from at least two animals of each genotype were observed, and representative images are shown in Fig. 6. The secretory membranes of the parietal cells from wild-type (Fig. 6A) and gastrin-deficient (Fig. 6B) animals were very similar. Canaliculi and tubular membranes were evident as observed previously. All of the parietal cells from H/K-beta -deficient mice were devoid of normal tubular membranes (Fig. 6C) and contained large, dilated intracellular canaliculi with relatively short microvilli, as we had found previously. The number and size of these canaliculi varied between cells. Some cells contained large vesicles, but we determined previously (25) that these are an artifact induced by aldehyde fixation. The parietal cell secretory membranes of H/K-beta -deficient (Fig. 6C) and H/K-beta - and gastrin-deficient mice (Fig. 6, D and E) appeared very similar. Again, tubular membranes were absent and abnormal canaliculi with short microvilli were found in all cells examined. This confirms that the absence of the normal tubular membranes is not due to hyperstimulation of parietal cells. In many parietal cells of the H/K-beta - and gastrin-deficient mice the abnormal canaliculi were relatively small and more numerous compared with parietal cells of H/K-beta -deficient mice, which accounts for the apparently normal appearance of many parietal cells in these mice by light microscopic analysis.


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Fig. 6.   Electron microscopic examination of parietal cells. Stomach tissue was fixed in aldehyde fixative before embedding, sectioning, and examination as described in MATERIALS AND METHODS. A: Wild type, B: gastrin deficient. C: H/K-beta deficient. D and E: H/K-beta and gastrin deficient. The secretory canaliculus (c) and tubulovesicular membranes (t) are indicated. Asterisks denote abnormal secretory canaliculi. Bar = 3 µm; applies to all panels.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the H/K-beta -deficient mice we found a twofold increase in the total number of gastric mucosal cells and a significant perturbation in the proportions of epithelial cells found in the gastric units. In particular, we found that there was an accumulation of immature epithelial cell precursors, a depletion of zymogenic cells, and an increase in apoptotic and dividing cells. The number of immature cells in the gastric mucosa of the H/K-beta -deficient mice was 3.1-fold higher than in the units of wild-type animals (101.6 ± 23.7 in H/K-beta -deficient compared with 32.6 ± 9.1 in wild-type mice). Staining with antibodies that detected cells in mitosis revealed that this increase in cell number was due to an increase in the rate of cell division in the mucosae of H/K-beta -deficient animals. Furthermore, the proliferating cells were no longer limited to the isthmal region of the gastric units as in normal gastric units but were also found distributed toward the base of the units. This was to be expected, because the immature cells were more widely distributed in the gastric units. The increased production of immature cells and proliferation appeared to be largely the result of elevated levels of gastrin found in H/K-beta -deficient mice, because H/K-beta - and gastrin-deficient mice, which lacked both functional Atp4b and Gast genes, did not display these phenotypes. Gastrin is a hormone that does not appear to be essential for normal proliferation and development of the gastric mucosal lineages but in elevated quantities is able to stimulate hyperproliferation (7, 31). In H/K-beta - and gastrin-deficient mice the total cell number and the number of immature cells were only 0.98-fold and 1.4-fold, respectively, those present in wild-type animals, and this difference was not statistically significant.

The cells of the gastric mucosa are in a continuous state of turnover. New cells are generated by the division of immature cell populations that include pluripotent stem cells and partially differentiated, committed precursors of pit, parietal, and zymogenic cell lineages (14). The rate of production of these cells and their development into end-stage cells must be regulated to maintain cellular homeostasis. The total number of parietal cells and pit cells in H/K-beta -deficient gastric units was only slightly greater than normal despite the enormously elevated number of potential precursors (immature cells). These data suggest the existence of a homeostatic mechanism that limits the number of immature cells that can progress to more mature cells and that immature cells accumulate if production of immature cells exceeds this limit. In other words, production of end-stage cells may be determined not only by the number of immature cells present but also by other factors that permit this further development. These factors could include the availability of growth factors or appropriate "niches" that can be occupied by mature cell types. This suggestion is supported by previous studies. Wang et al. (32) and Konda et al. (18) produced transgenic mice that overproduced gastrin. In both studies, the gastric mucosa was hypertrophic and there appeared to be an overabundance of immature cells. This was substantiated in the latter work with a quantitative analysis that demonstrated that the "proliferative zone" of the gastric units was increased whereas the number of parietal cells was not. Mice with autoimmune gastritis also have elevated gastrin levels and mucosal hypertrophy due to an abundance of immature cells, yet the number of end-stage cells is reduced (13).

H/K-beta -deficient mice also had elevated numbers of apoptotic cells. It would appear likely that the majority of these apoptotic events are the result of cell death of the accumulated immature cells. Apoptotic cells were observed more frequently in the H/K-beta -deficient mice in which immature cells were also more prevalent. Furthermore, we (13) and others (20) have noted an abundance of apoptotic cells in regions of the mucosa that contain immature mucosal cells blocked in development.

We observed a 36.7-fold reduction in the number of zymogenic cells in the gastric mucosa of the H/K-beta -deficient mice. A relationship between parietal and zymogenic cell lineages was first noted in mice that lack parietal cells because of genetic ablation (3, 20, 21). These mice also lack zymogenic cells, as do mice deficient in Na+/H+ exchanger isoform 2, under autoimmune attack (13), treated chronically with anti-secretagogues (16), or infected with Helicobacter sp. (19). Interestingly, H/K-alpha -deficient mice do not have a deficiency in zymogenic cells (27). Examination of the phenotype of these various situations does not reveal an obvious common trait that would explain the absence of zymogenic cells. Achlorhydria is not the cause because gastrin-deficient and H/K-alpha -deficient mice that lack stomach acid have normal numbers of zymogenic cells. The absence of parietal cells per se also does not appear to be a requirement because animals with normal numbers of parietal cells (H/K-beta deficient and H/K-beta and gastrin deficient, omeprazole treated) are deficient in zymogenic cells. On the other hand, the parietal cells in these mice are structurally abnormal and perhaps unable to produce a factor that normally nurtures zymogenic cells. Many of these mice have highly elevated gastrin levels, but even those that do not (H/K-beta - and gastrin-deficient mice) are zymogenic cell deficient, and, furthermore, some mice that are constitutively hypergastrinemic (gastrin-transgenic mice, H/K-alpha -deficient mice) have normal numbers of zymogenic cells. Many of these mice have abnormally high numbers of immature gastric epithelial cells that may be supplying a signal that inhibits zymogenic cell development (20). However, not all of them do (H/K-beta and gastrin deficient). In short, the explanation for the failure of genesis of zymogenic cells does not appear to be as simple as the surfeit or deficit in another cell type.

Parietal cells in H/K-beta -deficient mice have an abnormal membrane structure. The tubular membranes corresponding to the resting state of the membranes were absent. Instead, an abnormal canaliculus was present. The canaliculus appeared to be totally intracellular and had fewer, shorter microvilli than normal. In some cells the canaliculus was vast, occupying almost all of the cytoplasm and easily visible by light microscopy. On the basis of these data and other reports (4, 9), we suggested that H/K-beta is required for the formation of the tubular membranes, perhaps by supplying an endocytosis signal. One caveat to this interpretation was that the high levels of gastrin in the H/K-beta -deficient mice meant that the parietal cells were constitutively receiving a stimulatory signal that resulted in the conversion of all of the tubular membrane to the active canalicular form. To rule out this possibility here we have produced mice that were deficient in both H/K-beta and gastrin. Mice deficient in gastrin are achlorhydric, and, furthermore, parietal cells in these mice appear refractory to stimulation via cholinergic and histamine receptors (7). The ultrastructure of the parietal cells of H/K-beta - and gastrin-deficient mice as well as wild-type, H/K-beta -deficient, and gastrin-deficient littermates was examined. The structure of the secretory membranes of the wild-type and H/K-beta -deficient mice was as we had observed previously (25). The structure of secretory membranes in the gastrin-deficient mice was very similar to that seen in the wild-type animals. The membranes of the H/K-beta - and gastrin-deficient mice were very similar to those of the H/K-beta -deficient mice. Typical tubular membranes were absent, and abnormal dilated canaliculi were apparent. These data indicate that the absence of the tubular membranes is not due to hyperstimulation of the parietal cells and further supports the hypothesis that H/K-beta is required for the correct formation of parietal cell membranes. The parietal cells of mice deficient in H/K-alpha have been reported to be very similar to those of the H/K-beta -deficient mice; in particular, tubulovesicles were absent (25). This raises the possibility that both subunits and an intact H+/K+-ATPase heterodimer are required for tubular membrane formation. Although in heterologous expression systems a proportion of H/K-beta monomer can be transported to the cell surface, it is not clear where the "orphaned" H/K-beta subunit is located in the parietal cells of H/K-alpha -deficient mice, and it is possible that the putative endocytosis signal of H/K-beta does not function as efficiently in a monomeric form.

In this paper we have further elucidated a critical role for H/K-beta in normal development of the gastric mucosa and have determined the role of hypergastrinemia in the phenotype of H/K-beta -deficient mice. The mice deficient in both H/K-beta and gastrin will be very useful tools in the investigation of gastric inflammatory diseases, particularly autoimmune gastritis, because they do not display the severe hypertrophy and inflammation observed in the H/K-beta -deficient gastric mucosae. We have also shown an essential role of H/K-beta in the normal formation of the parietal cell secretory membranes.


    ACKNOWLEDGEMENTS

We thank Dr. Michael Bailey, Department of Epidemiology and Preventative Medicine, Monash University, for guidance in statistical analyses.


    FOOTNOTES

The National Health and Medical Research Council of Australia supported this work.

Current address of T. V. Franic, P. A. Gleeson, I. R. van Driel: The Russell Grimwade School of Biochemistry and Molecular Biology, The University of Melbourne, Melbourne, Victoria, Australia 3010.

Current address of L. M. Judd: Dept. of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, 231 Bethesda Ave., Cincinnati, OH 45267.

Current address of K. L. Scarff: Dept. of Biochemistry and Molecular Biology, Monash University, Clayton, Melbourne, Victoria, Australia 3800.

Address for reprint requests and other correspondence: I. R. van Driel, Dept. of Biochemistry and Molecular Biology, The University of Melbourne, Melbourne, VIC 3010 Australia (E-mail: ian.vandriel{at}unimelb.edu.au

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.

Received 11 April 2001; accepted in final form 11 September 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastrointest Liver Physiol 281(6):G1502-G1511
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