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INTRODUCTION |
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 (1-3), a 114-kDa 
-subunit (gene locus Atp4a) and a 35-kDa (protein moiety) 
-subunit (gene locus
Atp4b). The -subunit contains ATP and cation binding
sites and carries out the catalytic and transport functions of the
enzyme (1), and it also contains sequences responsible for apical
membrane localization (4). The heavily glycosylated -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) and may also contribute to proper folding and membrane localization
of the enzyme (1).
When the resting parietal cell is stimulated by acid secretogogues, the
tubulovesicles are transformed into the secretory canaliculus (6). 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 -subunit (7), 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) and possibly for the differentiation of chief
cells (9). 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, 10-12). 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, 9).
Expression of diphtheria toxin (13) or simian virus 40 large T antigen
(14) 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) 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 -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
-subunit and that observed after treatment with H,K-ATPase
inhibitors or elimination of the H,K-ATPase -subunit (7).
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EXPERIMENTAL PROCEDURES |
Preparation of Targeting Construct--
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). A polymerase chain reaction strategy was used to obtain fragments for insertion into the MJK+KO (16) 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.
Gene Targeting and Generation of Mutant
Animals--
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). 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.
Analysis of Blood Gases and Electrolytes--
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).
Acid-Base Equivalents and pH of Stomach Contents--
pH and
acid-base equivalents of the gastric contents were measured as
described previously with slight modifications (16, 17). 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.
Serum Gastrin Determination--
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).
Northern Blot Analysis--
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): rat gastric H,K-ATPase -subunit, rat
gastric H,K-ATPase -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).
Immunohistochemistry--
Antibodies and lectin and the
dilutions for each used in immunohistochemical analysis of stomach
sections were as follows: rabbit anti--subunit of porcine gastric
H,K-ATPase (1:100; Calbiochem-Novabiochem); rabbit anti--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.
Microscopy and Morphometry--
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) 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.
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RESULTS |
Generation of Atp4a/ Mice--
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. 1C)
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.
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Fig. 1.
Gastric H,K-ATPase
-subunit gene targeting and Southern blot
analysis. A, gene targeting strategy. Top,
genomic structure showing exons 4-13; middle, targeting
construct, with neomycin resistance gene replacing 31 codons in exon 8;
bottom, structure of gene after homologous recombination.
tk, herpes simplex virus thymidine kinase gene used for
negative selection. H, HindIII; E,
EcoRI. The 6.9-kb HindIII and 8.8-kb
EcoRI fragments present in the wild-type allele are shown
above, and the 8.5-kb HindIII and 6.2-kb
EcoRI fragments present in the mutant allele are shown
below. The probes used to distinguish between wild-type and
mutant alleles after digestion with HindIII (probe a) or
EcoRI (probe b) are indicated. B, Southern blot
analysis of DNA isolated from ES cells after electroporation with
targeting construct and selection with gancyclovir and G418. DNA was
digested with HindIII and hybridized with probe a.
C, Southern blot genotyping of offspring from a heterozygous
mating. DNA from tail biopsies was digested with EcoRI and
hybridized with probe b.
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Systemic Acid/Base and Electrolyte Status--
The -subunit of
the gastric H,K-ATPase is expressed in mouse kidney (19), and it has
been suggested that it might function in renal control of acid-base or
potassium homeostasis (20). 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.
Atp4a/ Mice Are Achlorhydric and
Hypergastrinemic--
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 -subunit gene leads to a
reduction in net acid secretion, we measured the pH and acid-base content of gastric secretions from histamine-treated
Atp4a+/+, Atp4a+/, and
Atp4a/ mice. The contents of
Atp4a+/+ and Atp4a+/
stomachs were similar with respect to both pH (3.17 ± 0.17 and 3.13 ± 0.21, respectively; Fig.
2A) and acid equivalents
(35.4 ± 9.0 µeq/g and 32.5 ± 5.2 µeq/g, respectively;
Fig. 2B). In contrast, the pH of the gastric contents of
Atp4a/ mice was close to neutrality
(6.9 ± 0.1; Fig. 2A) and contained net base (4.5 ± 2.3 µeq base/g; Fig. 2B).
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Fig. 2.
Impaired gastric acid secretion in
Atp4a/ mice. A, pH of
gastric secretions; B, acid-base content (expressed as
microequivalents per g of wet stomach weight) of the stomachs of
8-10-week-old mice (n = 7 Atp4a+/+, 6 Atp4a+/,
and 7 Atp4a/). Samples were collected 45 min
after subcutaneous injection of histamine HCl. The genotypes are
indicated below, and the mean for each measurement is
indicated by a horizontal bar. *, p < 0.001; **, p < 0.005 for difference between
Atp4a/ and Atp4a+/+
mice as determined by analysis of variance-protected Bonferroni's
t test.
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The peptide hormone gastrin is known to play an important role in the
regulation of acid secretion (21) and is induced in several mouse
models of achlorhydria (7, 16) or hypochlorhydria (22). Northern blot
analysis showed that gastrin mRNA was increased ~4-fold in
Atp4a/ stomachs compared with
Atp4a+/+ or Atp4a+/
stomachs (Fig. 3A). In
addition, serum gastrin concentrations (Fig. 3B) 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).
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Fig. 3.
Gastrin mRNA in stomach and serum gastrin
levels. A, Northern blot analysis of gastrin mRNA
(top) from stomachs of three mice of each genotype
(indicated above each lane). After hybridization
with a rat gastrin cDNA probe, the blot was stripped and hybridized
with a mouse L32 ribosomal subunit cDNA probe as a loading control
(bottom panel). Each lane contained 10 µg of
total stomach RNA from an 8-week-old mouse. B, sera from
8-week-old mice were analyzed by radioimmune assay to determine gastrin
concentrations. n = 6 Atp4a+/+,
5 Atp4a+/, and 12 Atp4a/ mice. Data are expressed as mean ± S.E. *, p < 0.03 for difference between
Atp4a/ and either
Atp4a+/ or Atp4a+/+ as
determined by single-factor analysis of variance.
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Northern Blot Analysis of H,K-ATPase, Intrinsic Factor, and
Pepsinogen mRNA--
High levels of the gastric H,K-ATPase
-subunit mRNA were present in Atp4a+/+
stomachs, and there was little, if any, decrease in
Atp4a+/ stomachs (Fig.
4, top panel). Although trace
levels of an ~1-kb mRNA were detected in samples from
Atp4a/ 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 -subunit was ~1.8-fold more abundant in the
Atp4a/ samples than in the
Atp4a+/+ or Atp4a+/
samples (Fig. 4, second panel).
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Fig. 4.
Northern blot analysis of stomach RNA.
Total stomach RNA (10 µg) from 8-week-old
Atp4a+/+, Atp4a+/, and
Atp4a/ mice was hybridized with probes for
mRNAs expressed in parietal cells (H,K-ATPase and subunit
mRNAs) and chief cells (intrinsic factor and pepsinogen mRNAs).
The L32 ribosomal subunit mRNA served as loading control. Note that
H,K-ATPase -subunit (HKA) mRNA was eliminated in
Atp4a/ stomachs and that expression of
H,K-ATPase -subunit (HKA) mRNA was up-regulated
1.8-fold (determined by PhosphorImager analysis) in the
Atp4a/ stomachs. The amount of intrinsic
factor mRNA was approximately the same in all three genotypes,
while pepsinogen mRNA was markedly decreased in
Atp4a/ mice.
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In the rodent stomach, pepsinogen and intrinsic factor are expressed
almost exclusively in chief cells (23, 24), making them excellent
molecular markers for this cell type. In the
Atp4a/ stomach, intrinsic factor mRNA
was detected at levels comparable with those observed in
Atp4a+/+ 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).
Immunohistochemical Analyses Reveal -Subunit Protein,
Pepsinogen, and Abundant Parietal and Chief Cells in
Atp4a/ Stomachs--
As noted above, the -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.
5A), but no specific staining was detected in Atp4a/ parietal cells (Fig.
5B).
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Fig. 5.
Immunofluorescent detection of gastric
H,K-ATPase -subunit in
Atp4a+/+ stomachs but not
Atp4a/ stomachs. Sections from
stomachs of 10-week-old Atp4a+/+ (A)
and Atp4a/ (B) mice were
incubated with a polyclonal antibody directed against the N-terminal
sequence of the gastric H,K-ATPase -subunit. Binding of the primary
antibody was detected using Texas Red-conjugated secondary antibody.
Cells in the neck and base of the gastric gland were strongly stained
in Atp4a+/+ stomachs (A) but not in
Atp4a/ stomachs (B).
Scale bar, 50 µm.
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To determine whether -subunit protein was present in
Atp4a/ 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 -subunit and with D. biflorus agglutinin, both
of which have been used as parietal cell markers (23). In both
Atp4a+/+ and Atp4a/
stomachs, a comparable number of cells were stained with the -subunit antibody (Fig. 6,
B and F) and with D. biflorus
agglutinin (Fig. 6, D and H). Staining with the
-subunit antibody seemed less intense in
Atp4a/ sections compared with
Atp4a+/+ sections (Fig. 6, B and
F), suggesting that -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, 11, 12). While these cells
did not exhibit normal parietal cell morphology, their staining with
both the -subunit antibody and D. biflorus agglutinin
confirmed that they were parietal cells; this conclusion was
supported by analysis of their ultrastructure.
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Fig. 6.
Immunofluorescent identification of parietal
cells in Atp4a+/+ and
Atp4a/ stomachs. Sections from
stomachs of 10-week-old Atp4a+/+
(A-D) and Atp4a/
(E-H) mice were examined for reactivity to
parietal cell markers. Control sections from
Atp4a+/+ (A) and
Atp4a/ (E) stomachs incubated
with the secondary antibody alone exhibited no specific staining. When
incubated with a rabbit antibody against porcine gastric H,K-ATPase
-subunit and Texas Red-conjugated donkey anti-rabbit IgG secondary
antibody, cells in both Atp4a+/+ (B)
and Atp4a/ (F) sections stained
positively for the gastric H,K-ATPase -subunit. When incubated with
blocking serum alone, no specific staining was observed in control
sections from Atp4a+/+ (C) and
Atp4a/ (G) stomachs; some degree
of autofluorescence was observed. When incubated with 40 µg/ml
D. biflorus agglutinin, both Atp4a+/+
(D) and Atp4a/ (H)
sections stained positively for parietal cells. The arrows
indicate gastric H,K-ATPase -subunit-positive cells (F)
and D. biflorus agglutinin-positive cells (H)
with vacuole-like dilations. Scale bar, 50 µm.
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To determine if mature chief cells containing pepsinogen stores were
present in Atp4a/ stomachs, despite the
sharp decrease in pepsinogen mRNA, stomach sections from
Atp4a+/+ and Atp4a/
mice were stained with an anti-pepsinogen antibody. As shown in Fig.
7, both Atp4a+/+
(Fig. 7B) and Atp4a/ (Fig.
7D) 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.
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Fig. 7.
Immunofluorescent detection of pepsinogen in
Atp4a+/+ and
Atp4a/ stomachs. Control sections
from stomachs of 10-week-old Atp4a+/+
(A) and Atp4a-/- (C) mice
incubated with Texas Red-conjugated secondary antibody alone exhibited
no specific staining. When incubated with a primary antibody directed
against human pepsinogen II and then with the Texas Red-conjugated
secondary antibody, sections from both Atp4a+/+
(B) and Atp4a/ (D)
stomachs stained positively for pepsinogen. Scale
bar, 50 µm.
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Histopathologic Changes in the Gastric Mucosa of
Atp4a/ Mice--
Sections from juvenile (17-day-old)
and adult (10-12-week-old) Atp4a+/+ and
Atp4a/ 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.
8A) and comprised 39.0 ± 1.8% of the epithelial cells
in the gland (Table II). In
Atp4a/ 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 -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. In
Atp4a+/+ 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%; Table
II).
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Fig. 8.
Histology of toluidine blue-stained sections
of oxyntic gastric glands from Atp4a+/+
and Atp4a/ stomachs.
A, composite image of gastric glands from the corpus of an
adult wild-type stomach. Typical parietal cells are indicated by
arrows. Chief cells with characteristic secretory granules
were present at the base of the glands (arrowheads).
B, chief cells (arrowheads) and parietal cells
(arrows) were also present in adult
Atp4a/ gastric glands. Parietal cells had
either numerous vacuole-like dilations (top arrow) or a
single large dilation (bottom arrow). C, severely
dysplastic gastric glands from an adult
Atp4a/ mouse, with chief cells
(arrowhead), parietal cells with cytoplasmic dilations
(arrow), and a large cyst surrounded by a single layer of a
low cuboidal epithelium (double arrow). Scale
bar, = 50 µm.
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Table II
Epithelial cell populations of gastric glands
Data were collected from toluidine blue-stained plastic sections.
Values for each cell type are means ± S.E. and represent the
percentage of the total epithelial cell population. n = 4 for Atp4a+/+; n = 4 for
Atp4a/.
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As anticipated on the basis of immunohistochemical staining of adult
stomachs with anti-pepsinogen antibodies (Fig. 7), chief cells with
darkly staining secretory granules were visible in toluidine
blue-stained sections from both Atp4a+/+ (Fig.
8A) and Atp4a/ (Fig. 8,
B and C) gastric glands. As in juvenile mutant
mice, chief cells in adult mutants appeared to have a reduced number of
granules, and the lighter shading of the cytoplasm observed in
toluidine blue-stained sections suggested a reduction in the amount of
rough endoplasmic reticulum. Chief cells comprised 15.2 ± 2.1 and
12.0 ± 1.8% of the epithelial cells in
Atp4a+/+ and Atp4a/
gastric glands, respectively (Table II). Although the mean value for
the number of chief cells in Atp4a/ mice
was slightly less than in Atp4a+/+ mice, the
difference was not statistically significant. Also, it should be noted
that the apparent reduction in the number of granules in
Atp4a/ chief cells could have led to an
undercount of these cells in the null mutant, since possible chief
cells containing less than six birefringent granules were included in
"other" cell types.
Neither the proportion of mucous cells
(Atp4a+/+, 43.5 ± 1.9%;
Atp4a/, 41.2 ± 3.5%) nor the
proportion of mitotic cells (Atp4a+/+, 0.10 ± 0.06%; Atp4a/, 0.14 ± 0.06%)
differed significantly between the two genotypes (Table II). The
proportion of enteroendocrine cells was increased significantly in the
Atp4a/ glands
(Atp4a+/+, 0.61 ± 0.15%;
Atp4a/, 1.91 ± 0.43%).
Undifferentiated cells and cells not identifiable as a specific gastric
cell type (classified as "other") were increased by ~3.5-fold in
Atp4a/ glands
(Atp4a+/+, 2.6 ± 1.4%;
Atp4a/, 9.1 ± 2.1%; Table II). At
least part of the increase in "other" cell types was due to the
presence of what may have been immature parietal cells, since some of
these cells contained numerous mitochondria but lacked canalicular membranes.
No evidence of hyperplasia was observed in stomachs of the
10-12-week-old Atp4a/ mice, consistent with
the lack of a significant increase in mitotic cells. Morphometric
analysis of the gastric glands from adult Atp4a+/+ and Atp4a/
mice showed that the glandular thickness was similar in the two genotypes (400 ± 21 and 417 ± 35 µm, respectively; Table
II). However, the thickness of the epithelial cell layer was
significantly thinner in Atp4a/ glands
(Atp4a+/+, 14.6 ± 0.6 µm;
Atp4a/, 8.9 ± 1.2 µm;
p < 0.05; Table II). The decrease in the thickness of
the epithelial layer in the Atp4a/ glands
was most likely attributable to the large dilations in the gastric
gland lumen (Fig. 8, B and C), which pressed the
cells against the basement membrane.
Ultrastructure of Parietal and Chief
Cells--
Atp4a+/+ parietal cells had numerous
mitochondria and extensive intracellular canaliculi densely packed with
microvilli (Fig. 9A), and
tubulovesicles were observed immediately adjacent to the canaliculi. In
contrast, the Atp4a/ parietal cells did not
contain normal canaliculi; rather, they contained dilated canaliculi
(Fig. 9B) with few, if any, of the microvilli typical of
Atp4a+/+ canaliculi (Fig. 9A).
Present on the apical membrane of mutant parietal cells was a sparse
population of short, dense microvilli (Fig. 9B), which were
similar in appearance to the microvilli on the apical membrane of
mucous and chief cells. Spherical vesicles, rather than normal
tubulovesicles, were occasionally observed in
Atp4a/ parietal cells (Fig. 9B).
Large cytoplasmic glycogen pools in Atp4a/
parietal cells were also observed (Fig. 9B). Interestingly,
some mitochondria in the Atp4a/ parietal
cells appeared enlarged and filled with concentric cristae (Fig.
9C) rather than the normal transverse cristae seen in
mitochondria of Atp4a+/+ parietal cells.
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Fig. 9.
Electron micrographs of parietal cells in
Atp4a+/+ and
Atp4a/ stomachs. A,
parietal cell from an Atp4a+/+ mouse, with well
developed secretory canaliculus (c), nucleus (N),
and many mitochondria. B, in the
Atp4a/ parietal cell, canaliculi were
dilated, and microvilli of the canaliculus (arrowheads) were
less abundant, shorter, and more densely stained than in wild-type
cells. Atp4a/ parietal cells contained a few
rounded vesicles (arrows) rather than numerous
tubulovesicles and accumulated glycogen (g). C,
Atp4a/ parietal cells often contained large
mitochondria with concentric cristae. Scale bars,
5 µm (A and B) or 1 µm (C).
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Electron micrographs showed chief cells with both secretory granules
and rough endoplasmic reticulum in Atp4a/
stomachs (Fig. 10). Subjectively, the
numbers of granules and the amount of rough endoplasmic reticulum
appeared to be decreased relative to those of wild-type cells,
consistent with observations by light microscopy. Nevertheless, it was
clear that mature chief cells with well developed secretory granules
were produced in Atp4a/ glands and that
their numbers were relatively normal.
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Fig. 10.
Electron micrograph of chief cell in
Atp4a/ stomach. An example is
shown of a well differentiated chief cell present in
Atp4a/ stomach. It should be noted that not
all chief cells of Atp4a/ stomachs were as
well developed as the one shown here; in general, they had fewer
secretory granules (SG) and less abundant rough endoplasmic
reticulum (RER) than those found in wild-type stomachs.
Scale bar, 5 µm.
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Metaplasia in Atp4a/ Stomachs--
In both
toluidine blue- (data not shown) and in hematoxylin and eosin-stained
sections of adult Atp4a/ stomachs, cells
with numerous distinct cilia located on the apical membrane and
extending into the lumen of the gland were observed (Fig.
11A). Ciliated cells
comprised 1.08 ± 0.45% of the epithelial cells counted in
Atp4a/ glands (Table II). In contrast,
ciliated cells were seen only rarely in juvenile
Atp4a/ glands and were not observed in
gastric glands of Atp4a+/+ mice (Table II).
Ciliated cells were observed in small cystically dilated regions of the
gastric gland (Fig. 11A) and in the base of the gland and
occurred both in clusters and singly. Ultrastructural analysis (Fig.
11B) revealed that the cilia had the typical structure of
motile cilia. A central pair of microtubules, nine peripheral microtubule doublets, radial spokes connecting the central and peripheral microtubules, and dynein arms were apparent in
cross-sections of the ciliary shaft (see inset of Fig.
11B), and the basal body contained the typical nine triplet
microtubules (not shown).
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Fig. 11.
Ciliated metaplasia of
Atp4a/ stomach. A,
hematoxylin- and eosin-stained section of
Atp4a/ mouse stomach, showing cells with
cilia on their apical membrane (arrows). Scale bar, 10 µm.
B, electron micrograph of a ciliated cell from an
Atp4a/ stomach. Numerous cilia
(arrows) project from the apical surface into the lumen
of the gastric gland. The inset shows a cross-section
of a cilia. Scale bar, 4 µm.
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Large irregularly shaped cysts were sometimes observed near the apex of
the adult Atp4a/ gastric gland in both
toluidine blue-stained (Fig. 8C) and hematoxylin- and
eosin-stained (data not shown) sections. These large cysts were lined
with a single layer of epithelial cells, which were not immediately
identifiable as a gastric epithelial cell type but had the appearance
of a low cuboidal undifferentiated epithelial cell type. In hematoxylin
and eosin-stained thick sections, cysts were often filled with cellular
debris and mucous (data not shown), leading to the tentative
identification of the surrounding epithelial cell layer as mucous cells.
Stomachs from 12-week-old Atp4a+/+ and
Atp4a/ mice were stained with PAS/Alcian
blue, which stains neutral carbohydrates red (PAS-positive) and acidic
carbohydrates blue (Alcian blue-positive) and were examined by light
microscopy. Mucous pit cells from Atp4a+/+ mice
stained red at the apical membrane (Fig.
12A). Most of the pit cells
from Atp4a/ mice stained red, but a
few stained purple (Fig. 12B), indicating that they
contained both neutral and acidic carbohydrates, and epithelial cells
lining the cysts in the Atp4a/ glands also
stained dark purple (Fig. 12B). Fig. 12C shows a
small dilation at the base of a Atp4a/ gland
lined with PAS-positive (red), Alcian blue-positive (blue), and
PAS/Alcian blue-positive (purple) cells. These data indicate that some
mucous cells in Atp4a/ gastric glands
produce and secrete modified mucosubstances.
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Fig. 12.
PAS/Alcian blue-stained stomach sections
from Atp4a+/+ and
Atp4a/ gastric glands.
Paraffin-embedded sections from adult mouse stomachs were stained with
PAS and Alcian blue to analyze carbohydrate moieties. A,
mucous cells in the apex of Atp4a+/+ oxyntic
glands stained bright red (arrows),
indicating the presence of neutral polysaccharides. B,
mucous cells in the apex of Atp4a/ oxyntic
glands exhibited the normal red staining
(arrows), but mucous cells surrounding cystically dilated
regions often stained purple (arrowheads),
indicating the presence of both neutral and acidic polysaccharides.
C, in addition to cells staining red or
purple, a small cyst at the base of an
Atp4a/ gastric gland contained cells that
stained blue (arrows), indicating the presence of
acidic polysaccharides and the absence of neutral polysaccharides.
Scale bars, 50 µm (A and
B) or 20 µm (C).
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DISCUSSION |
In preparing the gastric H,K-ATPase -subunit-deficient mouse,
we deleted sequences in exon 8 encoding the catalytic phosphorylation site, which is critical for enzyme activity, and inserted the neomycin
resistance gene. Northern blot analysis demonstrated that the mutation
eliminated expression of the -subunit mRNA, and immunostaining
of sections from Atp4a/ stomachs showed that
-subunit protein was also absent. These data confirmed that the
targeting procedure had produced a null mutation. Homozygous mutant
mice were born in a normal Mendelian ratio and appeared healthy,
indicating that the gastric H,K-ATPase -subunit is not required for
embryonic development or for viability of the animal during the time
period studied.
On the basis of its expression in mouse kidney and the occurrence of
HCO3 reabsorption in the collecting
duct that is sensitive to SCH28080, an inhibitor of the gastric
H,K-ATPase, it seemed possible that this enzyme might play a role in
the maintenance of systemic acid-base homeostasis (19, 20). However,
analysis of blood from adult mutant and wild-type mice revealed no
significant perturbation of acid-base balance. Normal acid-base status
was observed previously in mice lacking the colonic H,K-ATPase (25),
which is also expressed in kidney. Thus, neither of the known
H,K-ATPase isoforms is necessary for systemic acid-base homeostasis
when mice are maintained under normal conditions. In contrast, the B1
subunit of the V-type H+-ATPase clearly functions in renal
control of acid-base homeostasis, since null mutations in its gene have
been shown to cause distal renal tubular acidosis in humans (26).
Whether gastric H,K-ATPase activity in the collecting duct can provide
partial compensation when an animal is subjected to systemic acidosis
remains to be determined.
Expression of the H,K-ATPase -subunit was eliminated in
Atp4a/ stomachs, but mRNA encoding the
-subunit was increased and -subunit protein was easily detected
in Atp4a/ parietal cells. Thus, the
H,K-ATPase-deficient mouse described here differs from that prepared by
van Driel and colleagues (7), in which the -subunit gene was
targeted. In that model, expression of the -subunit was eliminated,
and expression of the -subunit was reduced to very low levels.
A stomach phenotype similar to that reported for mice lacking the
H,K-ATPase -subunit (7) (i.e. achlorhydria,
hypergastrinemia, and histopathologic changes in the gastric mucosa)
was observed in -subunit-deficient mice. Achlorhydria and
hypergastrinemia were expected, since achlorhydria is consistent with
the established role of the H,K-ATPase -subunit in acid secretion,
and hypergastrinemia is known to occur as a result of achlorhydria. In
both knockouts, parietal cells develop and are present at normal
numbers in adult stomachs, but they lack tubulovesicles and contain
dilated canaliculi rather than a normal secretory canaliculus. These
observations suggest that both subunits are necessary for biogenesis of
the secretory membranes.
Although the phenotypes of the two H,K-ATPase-deficient mice are
similar in many respects, there appear to be significant differences as
well. Gastric mucosal hypertrophy, mononuclear cells in the lamina
propria and submucosal regions, and a reduction in the number of chief
cells were observed in mice lacking the -subunit (7) but not in
those lacking the -subunit. In both mice, the dilated canaliculi
become more prominent with age; however, this defect did not seem as
severe in mice lacking the -subunit as in those lacking the
-subunit. Also, in -subunit-deficient mice, the dilated
canaliculi, which were often massive and caused the cells to be
severely swollen, were reported to be invariably intracellular (7),
whereas the dilated canaliculi in the -subunit knockout were not as
prominent and appeared to be continuous with the lumen of the gland.
Whether these differences are due to the loss of different subunits or
are due to differences in genetic background or other factors is
unclear. It is known that the -subunit is necessary for delivery of
the -subunit to the plasma membrane (7, 27) and that it has a
sequence involved in endocytic retrieval of the pump (5). Furthermore,
the -subunit can attain cell surface expression in the absence of
the -subunit (27), and a population of -subunit monomers has been
identified in the parietal cell (28). Thus, it is conceivable that the
-subunit is necessary not only for endocytic retrieval, which is
intimately involved in the transition from the stimulated to the
resting state (5), but also for recruitment of secretory membranes to
the apical membrane of the parietal cell. If this hypothesis is
correct, then the mechanism for incorporating secretory membranes into
the apical membrane of the parietal cell may be deficient in the
-subunit knockout but largely intact in the -subunit knockout,
which expresses significant levels of -subunit. With stimulated
secretion occurring via K+ and Cl channel
activities, the dilated canaliculi would tend to become enlarged in
-subunit-deficient parietal cells, due to the lack of an outlet for
accumulated fluid, but less distended in those lacking the -subunit,
in which the canaliculi appear to be continuous with the lumen of the gland.
Abnormally large mitochondria with concentric cristae and massive
glycogen stores were observed in a subset of parietal cells from
10-12-week-old -subunit-deficient mice. Mitochondria with concentric cristae have been observed in respiratory chain disorders (29-31) or after treatment with toxins (32) that inhibit mitochondrial activity. The absence of H,K-ATPase activity and reduced
transepithelial transport in -subunit-deficient parietal cells would
be expected to cause a sharp reduction in cellular ATP utilization. It
is possible that the occurrence of abnormal mitochondria in
Atp4a/ parietal cells is due to low
mitochondrial activity secondary to sharply reduced cellular ATP
utilization, which would be expected to occur as a result of the loss
of H,K-ATPase activity and reduced transepithelial transport. The
accumulation of glycogen could also be due to decreased energy
utilization by the parietal cell. This interpretation must be
considered with caution because altered mitochondria and increased
glycogen stores were not reported in -subunit-deficient parietal
cells, which should have the same deficit in energy utilization. This
difference, however, may be due to the relatively young age (35 days)
of the -subunit-deficient mice that were studied (7).
When animals are treated with omeprazole, an H,K-ATPase inhibitor,
their parietal cells develop dilated canaliculi and undergo an
increased rate of degeneration, invasion by macrophages occurs, and the
preparietal cell population increases (8). Karam and Forte (8)
suggested the possibility that complete and extended inhibition of acid
secretion might be sufficient to cause parietal cell degeneration. A
potential mechanism is that a block in the secretion of acid across the
apical membrane might be detrimental to cells in which the transport
processes on the basolateral membrane are being stimulated, since such
an imbalance might cause severe perturbations of intracellular pH and
volume homeostasis. Along these lines, it has been proposed that the
parietal cell degeneration observed in mice lacking the NHE2
Na+/H+ exchanger might be due to cell volume
perturbations secondary to a deficiency in
Na+/H+ exchange across the basolateral membrane
during active acid secretion (16). Atp4a/
mice developed dilated canaliculi that are similar in appearance to
those of omeprazole-treated animals; however, there was no evidence of
an increased rate of degeneration or production of parietal cells. The
number of parietal cells was essentially the same in mutant and
wild-type mice; there appeared to be little, if any, increase in the
number of preparietal cells or inflammatory cells; and the number of
mitotic cells was essentially the same. These results suggest that the
viability of -subunit-deficient parietal cells is not seriously
affected by an imbalance between apical and basolateral transport
processes; they also demonstrate that parietal cells containing dilated
caniculi and lacking the ability to secrete gastric acid can,
nevertheless, remain viable. Thus, the degeneration of the modified
parietal cells observed previously after treatment with omeprazole must
be due to factors other than the loss of acid secretion. One
possibility is that covalent modification of the pump might lead to
cell injury (discussed in Ref. 8), as observed in Clara cells of the
lung after covalent binding of 4-ipomeanol to cellular proteins
(29).
A second major difference between the gastric mucosa of
Atp4a/ mice and that of animals treated with
omeprazole is that long term omeprazole treatment leads to a sharp
reduction in the number of mature chief cells (9), whereas only a small
decrease in chief cells, which was not statistically significant, was
observed in adult Atp4a/ mice. An apparent
block in the maturation of chief cells has been observed in several
achlorhydric mouse lines, including NHE2 Na+/H+
exchanger-deficient mice (16), mice in which mature parietal cells were
ablated by expression of diphtheria toxin (13), and mice in which the
simian virus 40 T antigen was expressed in parietal cells (14). In
addition to the loss of acid secretion, other comm
-Subunit Have
Achlorhydria, Abnormal Parietal Cells, and Ciliated Metaplasia*
,
TOP
ABSTRACT
INTRODUCTION
