Expression of intrinsic factor and pepsinogen in the rat
stomach identifies a subset of parietal cells
J.-S.
Shao1,
W.
Schepp2, and
D. H.
Alpers1
1 Department of Medicine,
Washington University School of Medicine, St. Louis, Missouri 63110;
and 2 Department of Internal
Medicine II, Technical University of Munich, 81675 Munich,
Germany
 |
ABSTRACT |
Morphological and functional heterogeneity of
parietal cells has been thought to be due to different maturation
positions within the gastric gland. Morphodynamic studies
have shown that 2% of parietal cells in mice derive from a pre-neck
(chief) cell precursor. Intrinsic factor (IF) and pepsinogen, markers
of rat chief cells, were used to determine if these proteins identified a subset of parietal cells that might reflect origin from the pre-neck
cell lineage. The zymogenic region of the rat stomach and
gradient-isolated fractions enriched in parietal and chief cells were
fixed in 10% buffered Formalin or in Bouin's solution. Immunostaining
was performed using indirect immunoperoxidase histochemistry and
double-labeled immunofluorescence with antibodies raised against human
IF, pepsinogen II, and
H+-K+-adenosinetriphosphatase
(H+-K+-ATPase). In intact tissue, parietal
(H+-K+-ATPase-positive)
cells were found starting at the upper edge of the isthmus, but
parietal cells positive for IF and pepsinogen were only found from just
below the isthmus and neck region to the base of the gastric gland.
Three to four percent of isolated parietal cells were positive for
these ectopic markers. This subset of cells was also positive for
H+-K+-ATPase.
Thus products of rat chief cells are expressed in a subset of parietal
cells. The percentage of positive cells is similar to that predicted to
be derived from the pre-neck (chief) precursor lineage in the mouse.
The distribution of these cells to the lower neck and base of the gland
suggests that the expression of chief cell products is consistent with
either predetermination by lineage or parietal cell maturation or with
both processes.
gastric glands; gastric mucosal cell lineage
 |
INTRODUCTION |
GASTRIC UNITS IN THE zymogenic zone of the mouse and
rat contain five principal epithelial lineages (11): mucous surface cells, parietal cells, zymogenic cells that contain intrinsic factor
(IF) and pepsinogen, enteroendocrine cells, and brush or caveolated
cells. The gastric gland is divided vertically into three regions: the
pit, roughly corresponding with the upper third; the isthmus and neck,
corresponding to the middle third; and the base or lower third of the
gland (11). In the rat, the neck and isthmus regions contain four cell
types: nondifferentiated cells, immature surface cells, mucous neck
cells, and neck parietal cells (4, 15). The neck parietal cell appears
to have features intermediate between the immature surface cell and the
mature parietal cell found at the base of the gastric gland (4). The neck parietal (pre-parietal) cells do not appear to divide but acquire
differentiated functions [e.g.,
H+-K+-adenosinetriphosphatase
(H+-K+-ATPase), canaliculi] as they
migrate from the isthmus to the neck and eventually to the basal region
of the gastric gland (15). In the mouse, the cell dynamics appear to be
similar, but the quantitation of the precursor cell types has been
better delineated. When pre-neck precursors arrive at the upper portion
of the base region of the gland, they differentiate into pre-neck
(pre-zymogenic) cells with granules (12). Transformation of most (98%)
pre-neck cells into zymogenic cells occurs as the cells continue to
descend into the lower portion of the gastric gland. Murine
pre-parietal cells, on the other hand, arise from all three major
granule-free precursor cell types (pre-pit, pre-parietal, pre-neck),
with an estimated 2% of parietal cells deriving from the pre-neck
precursors (12).
IF production in the rat (3) and mouse (18) has been thought to occur
exclusively in chief cells. This finding was confirmed originally in
the rat, using parietal cell preparations >80% pure isolated by
Percoll gradients (23). However, work from our laboratory (16), using
antiserum raised against native rat IF, first suggested that some
parietal cells expressed IF, particularly in the region of the isthmus
and neck, although the reactivity was not strong. Moreover, analysis of
the rat stomach by in situ hybridization revealed weak reactivity over
some parietal cells (5). A more recent study reported no signal over
rat parietal cells using radioactive probe RNA (19). In contrast, in
the mouse IF is found only in differentiated zymogen cells, despite the
fact that pepsinogen is also found in mucous neck cells in the
mucoparietal section of the gastric mucosa (18). Finally, IF has been
demonstrated in multiple cell types in human gastric mucosa (9), in
which the parietal cell is the usual site for immunocytochemical
localization (17). Subsets of positive chief and enteroendocrine cells
contained immunoreactive IF, both at the light and electron microscopic level (9).
To resolve these discrepant findings and to test if IF could be used as
a marker for a subset of parietal cells that might derive from chief
cell precursors, we localized IF and another chief cell marker,
pepsinogen, by single- and double-labeled immunocytochemistry in the
rat glandular stomach and in isolated parietal cells from that tissue.
We used monospecific, high-titer antisera raised against recombinant
human IF, a peptide from rat
H+-K+-ATPase,
and human pepsinogen II that recognizes rat pepsinogen. Because IF has never been found in ectopic locations in gastric mucosa
(18), the mouse was not chosen for these studies, despite the fact that
cellular proliferation kinetics are better understood in that model.
 |
MATERIALS AND METHODS |
Animals.
Male rats (180 g) obtained from Sasco (Omaha, NE) were fasted
overnight. After rats were killed by cervical dislocation, we removed
the glandular stomach. Next, sections of the body
(containing the zymogenic and parietal cells) and antrum to be used for
tissue immunocytochemistry were fixed in 10% buffered Formalin for 2 h
at room temperature before exposure to 70% ethanol overnight at
4°C. For gastric mucosal cell isolation, the rat mucosal cells were
first released by enzymatic digestion (Pronase E). Then the cells were
separated according to size and density by sequential use of
counterflow elutriation and density gradient centrifugation to yield a
highly enriched chief cell fraction containing <1% parietal cells
and a parietal cell fraction enriched almost to purity (24). The purity
of the parietal cell preparation was judged by the addition of nitro
blue tetrazolium (Sigma Chemical, St. Louis, MO) at a concentration of
1 mg/ml to a fresh parietal cell suspension
(106 cells/ml) air dried on a
slide. After incubation at 37°C for 30 min, the slides were washed
with phosphate buffer. Under the light microscope the parietal cells
appear dark blue to purple, and nonparietal cells are not stained. Cell
pellets derived from both parietal and chief cell fractions were
prepared by fixing an aliquot of the fraction in Bouin's solution for
2-4 h, followed by centrifugation at 6,000 g for 10 min and storage in 70%
ethanol at 4°C until used. Three separate groups of isolated cells
were examined.
Immunocytochemistry.
Enriched chief and parietal cell pellets or gastric mucosal tissue
blocks were embedded in paraffin, and 5-µm-thick sections were cut
and mounted on slides. The sections were subsequently treated with
xylene to remove the paraffin and dehydrated in graded ethanol. The
standard avidin-biotin-peroxidase complex method was used (26).
Sections were treated with 1%
H2O2
in methanol for 20 min, followed by preincubation for 20 min at
37°C in 0.1 M phosphate-buffered saline (PBS) containing 5% bovine
serum albumin and 10% normal goat serum. The primary antisera used
were rabbit anti-H+-K+-ATPase
-subunit, raised against amino acids 2-23 of the rat protein
(1:500) [kindly supplied by Michael Caplan, Yale University (Ref.
18)], rabbit polyclonal anti-human IF (1:200), raised against
recombinant human IF produced in baculovirus-infected Sf9 cells (9),
and rabbit anti-human pepsinogen II (1:1,000) (the latter provided
courtesy of Dr. Michael Samloff, University of California Los Angeles
School of Medicine). Anti-rat IF had been shown previously to
cross-react with human IF (16), but the availability of anti-human IF
was now much greater, because of the markedly increased yield of human
IF over rat IF in the baculovirus expression system. Human and rat IF
share 80% amino acid identity (8), and as expected, rat and human IF
both react strongly with anti-human IF (D. H. Alpers and M. M. Gordon,
unpublished observations). The anti-pepsinogen II identifies one of the
major pepsinogens in humans (22) and the only pepsinogen found in the
rat (14).
These antisera were diluted in preincubation buffer and applied to
sections. Sections were incubated for 1 h at 37°C with biotinylated
goat anti-rabbit immunoglobulin G (1:200), followed by
streptavidin-peroxidase for 30 min at 37°C (Vector Laboratories, Burlingame, CA). Between each application of antiserum, the sections were rinsed extensively with PBS. The presence of antibody was revealed
using 3,3'-diaminobenzidine (DAB; Sigma Fast DAB) and H2O2
(Sigma Fast Urea
H2O2;
Sigma Chemical) at room temperature for 3-5 min. In some studies,
the biotinylated Griffonia
simplicifolia lectin 1 (GS1 lectin,
Vector Laboratories) was used to identify rat parietal cells (13). We
added 200 µl of a 1:150 dilution of the lectin (0.5 mg/ml) to each
section and incubated the sections for 1 h at 37°C. The sections
were then rinsed and treated with streptavidin-peroxidase and DAB as
noted above. In control preparations, the primary antiserum used was
normal rabbit serum (1:200). Routine hematoxylin and eosin staining was
performed after immunocytochemistry using DAB staining. When IF and
pepsinogen-positive cells were quantitated in tissue or cell sections,
the slide was examined at ×400, and 5,000-6,000 cells were
examined manually.
For immunofluorescence, the secondary antibodies were labeled with Cy3
(Jackson Immunoresearch Laboratories, West Grove, PA) and fluorescein
isothiocyanate (FITC) (Sigma Chemical) and used at 1:100 dilutions.
After overnight incubation with the first primary antibody at 4°C,
secondary antibody labeled with Cy3 (red fluorescence) was added for 1 h at room temperature. The second primary antiserum and the secondary
antibody labeled with FITC (green immunofluorescence) were each added
for 1 h at room temperature. Between each application of antiserum, the
sections were extensively rinsed with PBS, the effectiveness of which
was checked by parallel incubation and washing of a section treated
with normal rabbit serum. Sections were mounted with a 1:1 dilution of
PBS and glycerol under coverslips and examined in a Zeiss Axioskop
photomicroscope, using filters of the appropriate wavelength.
 |
RESULTS |
Parietal cell distribution in the rat gastric body.
Sections of the rat gastric body were examined for the distribution of
parietal cells by using GS1 lectin, which binds to parietal cells but
not to chief cells (13). Figure
1A shows that the stained cells occupied the lower two-thirds of the gastric glands, including the isthmus and neck (middle third) and base (lower
third). Occasional GS1 lectin-stained cells were found in the lower
portion of the upper third or pit region. This distribution fits well
with the fact that parietal cells originate in the isthmus region from
pre-parietal cells (10), but within the isthmus and neck region soon
acquire
H+-K+-ATPase-rich
tubulovesicles, rudimentary canaliculi, and long microvilli
characteristic of maturing parietal cells (12). When the rat gastric
body was immunostained for
H+-K+-ATPase
(Fig. 1B), the distribution of
positive cells was virtually identical with that seen using GS1 lectin
(Fig. 1A). This result is
consistent with the early appearance of tubulovesicles (the intracellular site of
H+-K+-ATPase)
in parietal cells found in the isthmus and neck region (4, 12, 15).

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Fig. 1.
Histocytochemical localization of parietal cells in the rat glandular
stomach. Tissue was stained as described in MATERIALS
AND METHODS, using avidin-biotin peroxidase complex
(ABC) techniques. A: biotinylated
Griffonia simplicifolia lectin 1. B: antiserum against human
H+-K+-ATPase.
Note that parietal cells are distributed in lower 2/3 of the mucosa,
comprising the isthmus and neck and basal regions of gastric glands.
A and
B: original magnification,
×100.
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Immunostaining of the rat gastric body using chief cell markers.
In contrast to the distribution of the total parietal cell population
(Fig. 1), the distribution of IF and pepsinogen was confined largely to
the basal third of the gastric glands (Fig. 2, A and
C). However, occasional cells with
the shape and size of parietal cells were found scattered rather evenly
throughout the basal third of the gland; such cells are identified in
Fig. 2, B and
D (arrows). None of these cells had
mucus secretory granules visible by light microscopy. Between 1.5% and
3.1% of the parietal cells in this region stained positively for IF
and/or pepsinogen. Most chief (zymogenic) cells were positive
using these markers. When double-labeled immunofluorescence was
performed with these two antisera, only occasional cells outside the
basal third of the gland stained positively with both antisera (Fig.
2E). These cells, similar to all the
others, stained yellow in color prints (see Fig. 3), consistent with
the presence of both antigens in the cells. Sections stained with
normal rabbit serum were consistently negative (Fig.
2F).

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Fig. 2.
Immunocytochemical localization of intrinsic factor (IF) and pepsinogen
(Pep) in the rat glandular stomach. Immunocytochemistry was performed
as described in MATERIALS AND METHODS,
using antisera against human IF and pepsinogen II.
A-D
and F: ABC method utilized.
E: double-labeled immunofluorescence
used. A and
B: anti-IF.
C and
D: antipepsinogen.
E: double-labeled with second antisera
labeled with Cy3 (red) for IF and with fluorescein isothiocyanate
(FITC) (green) for pepsinogen. F:
normal rabbit serum. Note that cell staining is largely confined to
lower 1/3 of gastric glands. Most chief cells and some parietal cells
(see arrows) are positive for IF and pepsinogen, and no cells are
clearly positive for each protein independently.
A, C,
E, and
F: original magnification, ×100.
B and
D: original magnification,
×400.
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Identification of these large triangular-shaped cells as parietal cells
was confirmed by double-labeled immunofluorescence using anti-IF and
anti-H+-K+-ATPase
(Fig. 3). Figure
3A shows IF distributed among cells
within the lower half of the basal region (lower third) of the gastric gland and in cells concentrated at the base of the glands.
H+-K+-ATPase
was not found in these basal cells (Fig.
3B). The double-labeled image (Fig.
3C) shows that some cells (stained
yellow) contained both
H+-K+-ATPase
and IF. These cells amounted to 3-10% of parietal cells in any
given gastric gland. Figure 3,
A-C,
is taken from a region of the glandular stomach in which these
dual-labeled cells were more concentrated. In the upper half of the
basal region of the gastric glands (Fig. 3,
D-F),
cells shaped like parietal cells stained for both chief cell markers,
IF and pepsinogen, but were much less frequent than in the lower
portions of the glands. In all the antral tissues examined, only a rare
parietal cell contained IF and pepsinogen. The antral mucous and
enteroendocrine cells were uniformly negative.

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Fig. 3.
Double-labeled immunofluorescent localization of
H+-K+-ATPase,
IF, and pepsinogen in the rat glandular stomach. Immunofluorescence was
performed as described in MATERIALS AND
METHODS using same dilutions used for
immunocytochemical studies.
A-C:
lower half of basal region (lowest third) of gastric glands. Note that
parietal cells [PC; stained green for
H+-K+-ATPase
(H/K)] occur singly throughout this region, even up to the base
of the glands. Chief cells (CC; stained red) containing IF but not
H+-K+-ATPase
are more concentrated at the base of these glands. Double-labeled
parietal cells (stained yellow) are fairly frequent in this region.
D-F:
upper half of basal region of gastric glands. Here, IF-containing
parietal cells are less frequent, and these cells also contain
pepsinogen, confirming single-labeled study shown in Fig. 2.
A-C:
original magnification, ×200.
D-F:
original magnification, ×400.
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Immunostaining of isolated parietal and chief cell preparations.
To better characterize and document the subset of parietal cells
expressing IF and pepsinogen, we prepared rat gastric mucosal cell
fractions enriched for chief and parietal cells. In the chief cell
fraction, 87-89% of the cells were positive for IF and pepsinogen (Fig. 4, A
and B). In double-labeled
fluorescence studies, no cells were identified that were positive for
one but not the other protein (see Fig. 5). No freshly isolated cells
stained positively with nitro blue tetrazolium as expected for parietal
cells, but only a small number (<1%) of the fixed chief cell
population stained positively using antibodies against
H+-K+-ATPase
or using GS1 lectin, both markers for parietal cells in the rat. When
almost pure (>95%) parietal cell preparations were examined in the
same way the results were quite different. In one preparation, 3.5% of
the parietal cells that were identified by positive nitro blue
tetrazolium staining were positive for IF and/or pepsinogen
(Fig. 4, C and
D). Under higher power
magnification, the cells that were positive for IF did not appear to be
morphologically different from the other cells (Fig. 4,
E and
F). In other preparations, 4% and
11% of the isolated parietal cells were positive. Normal rabbit serum
applied to chief cells (Fig. 4G) and
parietal cells (not shown) displayed no reactivity.

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Fig. 4.
Immunocytochemical staining of isolated rat chief and parietal cells.
A: chief cells (CC),
anti-IF, no counterstain. B: chief
cells, antipepsinogen (PEP), no counterstain.
C: parietal cells (PC),
anti-IF counterstained with hematoxylin and eosin.
D: parietal cells, antipepsinogen
counterstained with hematoxylin and eosin.
E: parietal cells, anti-IF, no
counterstain. F: parietal cells,
anti-IF counterstained with hematoxylin and eosin.
G: chief cells, normal rabbit serum.
Most isolated chief cells, but only 3-4% of isolated parietal
cells, are positive for both IF and pepsinogen. Arrow in
A marks a chief cell that stained very
positively for IF. Other arrows mark parietal cells staining positively
for IF (C,
E) and for pepsinogen
(D).
E and
F: * indicates a parietal cell
unstained (E) and stained
(F) with hematoxylin and eosin.
A-D
and G: original magnification,
×200. E and
F: original magnification,
×800.
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Using the lower frequency of positive reaction in the parietal cell
preparation, we found it easy with double-labeled immunofluorescence to
identify individual double-labeled cells using IF and pepsinogen antisera. As shown in Fig. 5,
A-C,
the same cells that were positive for one chief cell marker were
positive for the other. Thus, although there was heterogeneity in
expressing chief cell markers among parietal cells, there did not
appear to be subsets of parietal cells that produced only IF or only
pepsinogen. These data confirmed the findings in intact rat gastric
mucosa.

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Fig. 5.
Double-labeled immunofluorescence of isolated parietal cells.
Immunofluorescent staining was carried out as described in
MATERIALS AND METHODS.
A and
D: cells reactive with anti-IF first
antibody that were detected with Cy3 (red)-labeled second antibody.
B: cells reactive with antipepsinogen
first antibody and detected with FITC (green)-labeled second antibody.
C: use of both anti-IF and
antipepsinogen first antibodies. Note that all positive parietal cells
react with antibody against both chief cell markers.
E:
anti-H+-K+-ATPase
with FITC (green)-labeled second antibody.
F: anti-IF and
anti-H+-K+-ATPase.
Note that all IF positive cells are also positive for the parietal cell
marker.
A-C:
arrows mark parietal cells positive for the chief cell markers IF and
pepsinogen.
D-F:
upper arrows mark parietal cells positive for IF and
for the parietal cell marker
H+-K+-ATPase;
lower arrows identify a cell that is
H+-K+-ATPase
positive but IF negative. A,
D, and
F: original magnification, ×400.
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Although the purity of the parietal cell preparation exceeds 95% as
judged by the uptake of the supravital dye nitro blue tetrazolium, the
parietal cells were subjected again to double-labeled immunofluorescence using antisera against IF and
H+-K+-ATPase
to eliminate the possibility that the IF- and pepsinogen-positive cells
were contaminating chief cells. Figure 5,
D-F,
demonstrates that the subset of parietal cells that were positive for
IF also contained the parietal cell marker
H+-K+-ATPase
and were not chief cells.
 |
DISCUSSION |
The findings in the present study confirm the original observations of
Lee et al. (16) that some parietal cells in the rat express IF. We have
extended this observation by demonstrating pepsinogen expression in the
same cells and by estimating the percentage of parietal cells that
express these chief cell markers in the glandular portion of rat
stomach and in a purified preparation of parietal cells. Double-labeled
experiments using the parietal cell marker
H+-K+-ATPase
confirmed that the cells expressing IF and pepsinogen were indeed
parietal cells. Moreover, the isolated parietal cells expressing the
chief cell markers were morphologically the same as the rest of the
parietal cell preparation. These data are somewhat different from those
reported by Maeda et al. (19), in which in situ hybridization with rat
IF mRNA was used. Maeda et al. (19) report no signal over parietal
cells, although some low level signal does appear to be present in
their study. IF mRNA was seen at low levels in rat parietal cells in
another study from our laboratory (5). Maeda et al. (19) conclude that "the primary transcription site of the rat IF gene is gastric chief
cells," a conclusion with which the present data agree. It seems
likely that expression of IF in a small percentage of parietal cells
might not have been detected, especially if the protein concentration
in parietal cells was less than in chief cells (16).
The percentage of parietal cells that express chief cell markers
appears to range from 1.5% to 10%. This is the range of positive cells found in gastric glands both in whole tissue sections
(1.5-3.1%), where the prevalence differs from one gland to
another (3-10%), and in preparations of isolated parietal cells
(4-11%). There may be several explanations for this variability.
First, there could be biological variation from one set of animals to
another. Second, there could be differences in sampling from different regions of the stomach. Third, local tissue factors could stimulate production of IF and pepsinogen in parietal cells. These latter two
possibilities are supported by the variability in positive cells from
gland to gland and the increased abundance of IF-expressing parietal
cells toward the base of the glands. Fourth, it is possible that during
cell isolation, a process that takes a number of hours, IF synthesis
might remain active, while IF secretion is impaired. This
discrepancy might increase the sensitivity of IF detection in parietal
cells that do not appear to contain the protein in situ.
Such a phenomenon has been seen with isolated enterocytes producing
apolipoprotein B (apo B) (7). Although it was thought from this study
(7) that enterocyte content of apo B increased after fat feeding, in
fact the content fell in tissue (1). However, that the
variability was seen both in tissue and in isolated cells somewhat
opposes this methodological explanation. The important point is that
there does exist a subset of parietal cells in the rat that express
chief cell markers, and these cells are a small percentage of the total
parietal cell population. There are other examples of subsets of
epithelial cells in the gastrointestinal tract that express a product
unique to that cell type. Such examples would include the cystic
fibrosis transmembrane regulator (2).
A pyloric glandular region is present in fish, adult amphibians,
reptiles, and mammals, and in amphibians the mucosal structure is less
complex because it only includes three cell types (mucous, oxynticopeptic, and endocrine) instead of the four (mucous, parietal, zymogenic, and endocrine) found in mammals (6). The fact that oxyntic
(parietal) and peptic (zymogenic) functions have been combined in a
single cell during phylogeny makes the ectopic location of IF and
pepsinogen seem more logical. Pepsinogen (but not IF) is produced in
the oxynticopeptic cell. One can speculate that the ability to produce
pepsinogen (and by inference IF) is retained in the parietal and
zymogenic cells, which appear to derive from their ancestral cell in
amphibians.
The significance of the IF and pepsinogen-containing parietal cell
subset and its origin in the rat are not clear. It is intriguing to
think that this subset may correspond with that proportion of parietal
cells that derive from the pre-neck lineage. In the mouse
this lineage supplies ~2% of parietal cells (12). The regulatory
factors that would lead to expression of IF in this subset of cells are
not known. Nucleotides from
1029 to +55 in the 5' upstream
region of the mouse IF gene direct IF expression only in parietal cells
(18). By Harr analysis, the first 400 base pairs of the 5'
upstream sequence of the rat IF gene were highly conserved compared
with the mouse sequence. Thus, in both mouse and rat, it seems that
there are unidentified cis-acting elements that affect the promoter activity and help to direct IF
expression in chief cells. Regulation of these elements may allow
expression in adult parietal cells, and this regulation may occur in
the subset of parietal cells that derive from the pre-neck lineage.
However, expression of IF and pepsinogen does not occur in the isthmus
and neck region in which pre-neck cells are first identified, but only
when the cells have migrated into the basal region. Thus IF and
pepsinogen expression may be the result of maturation of the pre-neck
lineage of parietal cells. In fact, a vertical expression gradient
consistent with increasing cell maturation is apparent; IF-positive
cells increase in frequency toward the base of the gastric glands. It
seems possible that local factors may play a role in IF and pepsinogen
expression in this subset of parietal cells. The continuous presence of
glucocorticoids is necessary for active transcription of pepsinogen
mRNA in the adult rat gastric mucosa (25). Many cytokines, especially
interferon-
and interleukin-1
, are important in stimulating gene
expression in epithelial cells. Local factors may account for the
mosaicism seen in epithelial mucosal cells in the intestine (20, 21).
It should be possible to examine the role of local factors in IF and
pepsinogen expression in organ explants or in isolated preparations of
parietal cells.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institutes of Health Grant P01
DK-33487 (D. H. Alpers) and Deutsche Forschungsgemeinschaft Grant DFG
Sche 229/7-2 (W. Schepp).
 |
FOOTNOTES |
Address for reprint requests: D. H. Alpers, Gastroenterology Division,
Washington Univ. School of Medicine, 660 S. Euclid Ave., Box 8124, St.
Louis, MO 63110.
Received 5 March 1997; accepted in final form 29 September 1997.
 |
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