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
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ABSTRACT |
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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 -subunit (H/K-
) on
epithelial cell development by detailed quantitation of the epithelial
cell types of the gastric mucosa of H/K-
-deficient mice.
H/K-
-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-
-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-
-deficient mice, is independent of
hypergastrinemia. Significantly, parietal cells of H/K-
- 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-
in the development of mature parietal cells.
parietal cell, gastric mucosa, membrane biosynthesis
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INTRODUCTION |
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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 (H/K-
)- and a
(H/K-
)-subunit, with
apparent molecular masses of 95 and 60-90 kDa, respectively
(1, 24). H/K-
(encoded by the mouse gene
Atp4a) contains the catalytic sites of the enzyme and has 10 transmembrane domains. The highly glycosylated H/K-
(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--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-
-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.
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MATERIALS AND METHODS |
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Animals.
H/K--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-
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.
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-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.
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RESULTS |
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Gastric mucosae of H/K--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-
-deficient mice, respectively. The
units of the gastric mucosae of the H/K-
-deficient mice were
approximately twice the length of the mucosae from wild-type littermates. The mucosae of the H/K-
-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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Hypergastrinemia in H/K--deficient mice is responsible for
mucosal cell hypertrophy but not for depletion of zymogenic cells.
One of the features observed in the H/K-
-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-
-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-
and gastrin deficient) are viable and
fertile and are grossly indistinguishable from littermates (data not shown).
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--deficient mice
had a significantly increased number of apoptotic cells in the
gastric mucosa (6.7 ± 3.5 in H/K-
-deficient mice compared with
0.7 ± 1.2 in wild-type mice; P < 0.0001). In the
H/K-
- 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-
- and gastrin-deficient, 1.8 ± 1.8, not significant].
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Absence of parietal cell tubular membranes in H/K--deficient
mice is not caused by hyperstimulation.
Previously, we had observed (25) that the parietal cells
of H/K-
-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-
-
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-
- 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-
-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-
-deficient (Fig. 6C) and H/K-
- 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-
- and gastrin-deficient mice the abnormal
canaliculi were relatively small and more numerous compared with
parietal cells of H/K-
-deficient mice, which accounts for the
apparently normal appearance of many parietal cells in these mice by
light microscopic analysis.
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DISCUSSION |
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In the H/K--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-
-deficient mice was 3.1-fold higher than in the units of wild-type animals (101.6 ± 23.7 in H/K-
-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-
-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-
-deficient mice, because H/K-
- 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-
- 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--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--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-
-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--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-
-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-
-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-
deficient and H/K-
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-
- and gastrin-deficient mice) are zymogenic cell
deficient, and, furthermore, some mice that are constitutively
hypergastrinemic (gastrin-transgenic mice, H/K-
-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-
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--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-
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-
-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-
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-
-
and gastrin-deficient mice as well as wild-type, H/K-
-deficient, and
gastrin-deficient littermates was examined. The structure of the
secretory membranes of the wild-type and H/K-
-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-
- and
gastrin-deficient mice were very similar to those of the
H/K-
-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-
is
required for the correct formation of parietal cell membranes. The
parietal cells of mice deficient in H/K-
have been reported to be
very similar to those of the H/K-
-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-
monomer can be transported to the cell surface,
it is not clear where the "orphaned" H/K-
subunit is located in
the parietal cells of H/K-
-deficient mice, and it is possible that
the putative endocytosis signal of H/K-
does not function as
efficiently in a monomeric form.
In this paper we have further elucidated a critical role for H/K- in
normal development of the gastric mucosa and have determined the role
of hypergastrinemia in the phenotype of H/K-
-deficient mice. The
mice deficient in both H/K-
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-
-deficient gastric
mucosae. We have also shown an essential role of H/K-
in the normal
formation of the parietal cell secretory membranes.
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ACKNOWLEDGEMENTS |
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We thank Dr. Michael Bailey, Department of Epidemiology and Preventative Medicine, Monash University, for guidance in statistical analyses.
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FOOTNOTES |
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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.
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