Extract
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visualized by using the TA-Fe staining method, and could
be observed under a light microscope. The density and
distribution of the vessels were measured and analyzed by
using the CMIAS image analysis system, and photographed
using an Olympus PM-10AD photomicrographic system
according to the method of Kong et
al[13].
Data analysis Data are expressed as mean±SD, and the
two-tailed c2 test was used to examine the correlation
between ANP-synthesizing cells and microvessel density.
Statistical significance was estimated by using the
t-test. Differences were considered significant when the
P-value was less than 0.05. All the calculations were performed by
using SPSS 11.0.
Results
Expression of ANP-synthesizing cells in the rat
stomach In the positive control, ANP-synthesizing cells were
present at high density in the cytoplasm of the atrial myocytes
(as red-brown granules; Figure 1A). ANP-synthesizing cells
were also expressed in the gastric mucosa, and the
positively-stained granules were localized in the cytoplasm in
cells in the basal portion of the fundus glands (Figure 1B,
2A). In the negative control, for which normal rabbit serum
was substituted for primary antiserum, no positive staining
for ANP-synthesizing cells was observed (Figure 2B). The
shape of the individual ANP-synthesizing cells was variable:
round, pyramidal and flask shapes were all found. The
general epithelial appearance of these cells was typically
endocrine, and most immunoreactivity was localized in the
basal portion of the stomach. No ANP-synthesizing cell was
detected in the lamina propria, submucosa or smooth muscle.
Distribution of ANP-synthesizing cells in rat gastric
mucosa The distribution of ANP-synthesizing cells (EC cells)
in the different regions of the gastric mucosa was
investigated by using histochemical techniques. Consecutive
serial sections of rat gastric mucosa were stained for chromaffin,
and the chromaffin granules (brown granules) were found to
be localized in the EC cell cytoplasm. No chromaffin
granules were detected in the lamina propria, submucosa, or
smooth muscle, or in the negative control. The distribution
of EC cells in gastric mucosa was further examined by
staining for chromaffin. Three histologically distinct regions
(fundus, body and antrum) were found in the distribution of
EC cells in rat gastric mucosa. EC cells are found in the
mucosa layer, and their density was greatest in the fundus
(46.7%±5.3%; Mean±SD), intermediate in the antrum
(40.1%±4.5%), and lowest in the body (21.6%±3.6%) (Figure
3; n=18).
Identification of ANP-synthesizing cells in the rat
stomach Immunogold labeling was localized in the endocrine
granules of EC cells, which belong to the disperse or diffuse
neuroendocrine system (DNES) in the gastric glands (Figure
4B). These results indicate that EC cells synthesize and
secrete ANP in rat gastric mucosa. In the negative control,
in which normal rabbit serum was substituted for anti-ANP
antiserum, no positive staining was observed (Figure 4A,
4C).
Relationship between ANP-synthesizing cells and
microvessel density The gastric mucosa was cut into
cross-sections, and microvessels were stained successfully by
using the TA-Fe method[12,13]. The microvessels were exhibited
in winding state, and they could be clearly observed in 3
dimensions (Figure 5A). Some microvessels were found
scattered in the antral mucosa, and some branch arteries from the
large vessels ran into the basal glands of the gastric mucosa
(Figure 5B). The density of microvessels varied depending
on location; in the areas in which more basal glands existed,
more microvessels were distributed in the rat gastric mucosa.
In order to investigate the relationship between the
distribution of ANP-synthesizng cells and microvessel density in
the gastric mucosa, data were analyzed by using SPSS 11.0.
There was a significant positive relationship between the
percentage of ANP-synthesizing cells and microvessel
density in the antral mucosa of rats (r=0.53,
P<0.05, Figure 5C), but there was a negative relationship in the body mucosa
(Figure 6A; n=18, r =_0.2914,
P>0.05) and fundus mucosa (Figure 6B;
n=18, r=0.3880, P>0.05).
Discussion
In our previous study, the distribution of natriuretic
peptide receptors (NPR-A and B) in different regions of rat
stomach was investigated by using radioautograph
techniques[9,10]. Furthermore, we found that natriuretic peptide inhibited
gastric smooth muscle contractions in humans, rats and
guinea-pigs[9,10]. NPR exists in both the mucosal and muscle layers,
and the density of NPR in the muscle layer is greatest in the
antrum, intermediate in the body, and lowest in the fundus.
In the present study, ANP-synthesizing cells were localized,
and we verified by using immunohistochemistry and postembedding immunoelectron microscopy techniques that
that EC cells synthesized ANP in rat gastric mucosa.
ANP-synthesizing cells exist in different regions of the stomach,
and their density in the mucosal layer was the greatest in the
gastric fundus, intermediate in the antrum, and lowest in the
body. There was a positive relationship between the
percentage of ANP-synthesizing cells and the density of
microvessels in the antral mucosa, but there was a negative
relationship in the fundus and body mucosa. Our study
suggests that ANP synthesized by EC cells may play an
important role in the inhibitory regulation of gastrointestinal
motility, perhaps not only as a regional autocrine and/or
paracrine regulator, but also as an endocrine regulatory
peptide.
ANP-expressing myoendocrine cells are most densely
distributed in the right atrium, are found to a lesser extent in
the left atrium, and are almost absent in the left
ventricle[14]. Gower et
al[15] and Vuolteenaho et
al[16] demonstrated that the gene for ANP was expressed in specific regions of the rat
gastrointestinal tract. It has been found immunohisto-chemi
cally that EC cells in the antral and small and large intestinal
mucosa, besides being serotonin positive, are also ANP
positive[7,8]. However, it was not clear which cells in the gastric
body and fundus mucosa manufactured ANP. In the present
study we demonstrated immunohistochemically that all EC
cells in the gastric fundus, antrum and body mucosa were
ANP positive. EC cells are an abundant type of
enteroendo-crine cell that contain serotonin and occur throughout the
gastrointestinal tract[17]. There are two types of EC cell: the
open type and the closed type. Open-type EC have a large
basolateral compartment in contact with the basal lamina,
and a narrow apical process that allows access to the lumen.
The present study demonstrated that at least some of the
ANP-synthesizing cells were exposed to both basal lamina
and lumen in the gastric mucosa, and, by using
immuno-electron microscopy, they were identified as EC cells that
synthesized ANP. Immunoreactive ANP has been reported
in immune-type cells in lymphatic nodules in the lamina
propria and the submucosa in guinea pig and rat
intestine[18]. However, in the human stomach immunoreactive ANP has
been not found in the lamina propria or
submucosa[19]. In the present study, no staining for ANP-synthesizing cell was
detected in the lamina propria, submucosa or smooth muscle.
However, there were some microvessels scattered in the
antral mucosa, and some branch arteries from the large vessels
ran into the basal glands of the gastric mucosa. The density
of microvessels was different in different positions: in the
areas where more basal glands were distributed, more
microvessels were found in the gastric mucosa. There was a
significant positive relationship between EC cell density and
microvessel density in the antral mucosa of rats. However,
there was a negative relationship between density of
micro-vessels and ANP-synthesizing cells in the gastric fundus
and body mucosa. These results suggest that the basolateral
plasma membrane of EC cell in antral gastric mucosa may be
adjacent to microvessels by which ANP could enter the
circulation from EC cell. Because the EC cells in the gastric
mucosa are open-type endocrine cells, they can receive
chemical stimulation from the gastric lumen and also from
microvessels, by which process ANP secretion can be
regul-ated. ANP generated by EC cells may enter the circulation
from the gastric mucosa and regulate gastric motility via NPR
in smooth muscles.
Our results are in agreement with and extend the
observations of Li and Goy[20] and Rambotti
et al[21], who demonstrated
the presence of NPR-A and NPR-B transcripts in
extracts of gastric fundus, and localized natriuretic
peptide-induced cGMP production to parietal cells, mucus secreting
cells in the fundus, and pyloric glands, as well as gastric
smooth muscle cells. ANP is known to stimulate gastric acid
secretion and relax gastric smooth
muscle[18,10]. Further evidence for the existence of functional receptors in
gastric tissues comes from reports that ANP stimulates the
production of cGMP in guinea pig chief cells, and that ANP induces
the relaxation of cultured gastric smooth muscle
cells[22,23]. ANP released locally into the gastric
lumen could target these luminally directed receptors, according to the finding by
Rambotti et al[21] that there is ANP-induced
guanylate cyclase activity in both the apical and basolateral surfaces of
mucosal cells within the pyloric glands of rat stomach. This
suggests that ANP may help control a "negative feedback"
system within the stomach, whereby mucus production is
enhanced in response to increasing acid secretion to protect
the lining of the stomach from the effects of acid. This would
provide a regulatory mechanism to ensure that the acid
produced after a meal does not injure the mucosal surface of
the stomach.
In conclusion, our results demonstrated that EC cells
synthesized ANP in rat gastric mucosa, and that the density
of ANP-synthesizing cells in the gastric mucosa was
greatest in the gastric fundus, intermediate in the antrum, and the
lowest in the body. Because there is a positive relationship
between ANP-synthesizing cells and microvessel density
only in the gastric antral mucosa, ANP may regulate gastric
acid and mucus secretion by paracrine methods and gastric
motility by endocrine methods; that is, ANP may be
transported from EC cells to smooth muscle through microvessels.
EC cells may receive chemical stimulation from the gastric
lumen and also from microvessels, by which methods ANP
secretion could be regulated.
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