Departments of 1 Biochemistry and 4 Physiology, Kitasato University School of Medicine and 2 Department of Biochemistry, Kitasato University School of Allied Health Sciences, Sagamihara 228-8555; and 3 Department of Anatomy, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
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ABSTRACT |
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We examined the effects of the calcitonin gene-related peptide (CGRP), including the possible participation of nitric oxide (NO), on mucin biosynthesis in the surface epithelium and remaining deep mucosa as well as the entire mucosa and compared the distribution of CGRP and NO synthase (NOS) using a combination of double immunofluorescence labeling and multiple dye filter. Pieces of tissue obtained from the corpus and antrum were incubated in a medium containing [3H]glucosamine and CGRP, with or without the NOS inhibitor. CGRP dose-dependently enhanced [3H]glucosamine incorporation into the corpus mucin but had no effect on antral mucin biosynthesis. The CGRP receptor antagonist, CGRP-(8-37), prevented the increase in 3H-labeled corpus mucin. This stimulation of corpus mucin synthesis disappeared after removal of the surface mucus cell layer. CGRP activated the mucin biosynthesis in the surface mucus cells. In the full-thickness corpus mucosa, CGRP-induced activation was completely blocked by the NOS inhibitor. CGRP-immunoreactive fibers were intertwined within the surface mucus cell layer with type I NOS immunoreactivity. These results show that CGRP-stimulated mucin biosynthesis mediated by NO is limited to surface mucus cells of the rat gastric oxyntic mucosa.
gastric mucin biosynthesis; nitric oxide; calcitonin gene-related peptide
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INTRODUCTION |
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CONSIDERABLE EVIDENCE INDICATES that the calcitonin gene-related peptide (CGRP) is the major transmitter by which afferent nerve fibers within the gastric mucosa counteract mucosal damage caused by ethanol and other injurious factors (11, 13, 25, 32, 40). Exogenous CGRP is very active in increasing blood flow through the rat and rabbit stomach (3, 27), an action that is mediated by CGRP1 receptors (27). This peptide is also shown to inhibit acid secretion in the rat and canine stomach via activation of the same receptors (22, 26). These results demonstrate that CGRP has a significant role in gastric mucosal function through the specific receptors. CGRP-immunoreactive nerve fibers are shown to run parallel and close to gastric glands in the lamina propria, some of which approach the surface epithelial cells, of the rat stomach (10, 39). Prior studies have suggested that CGRP receptors exist on the gastrointestinal epithelial cells (5). Although CGRP was reported to have no effect on mucus secretion from human nasal mucosa (30), information about the effects of CGRP on gastric mucus cells is lacking.
Mucus cells produce a gel-forming, high molecular weight mucus glycoprotein (mucin) (2) and have several cell types in the distinct regions (corpus and antrum) or particular layers (surface mucosa and deep mucosa) of the mammalian gastric mucosa (8). The mucins from surface mucus and gland mucus cells in a single tissue section of the rat and human stomach are individually characterized by a combination of galactose oxidase-cold thionine Schiff staining and paradoxical concanavalin A staining (33). We have clearly demonstrated that the surface mucus and the gland mucus cells from rat corpus and pyloric gland cells from the antrum produce distinct mucins bearing a particular carbohydrate structure, using original monoclonal antibodies (21). Our recent studies have shown that gastrin, a gastrointestinal hormone, accelerates mucin biosynthesis in surface mucus cells, but not in gland mucus cells, of the rat gastric oxyntic mucosa (17). More recently, we found that nitric oxide (NO), produced by type I NO synthase (NOS) in surface mucus cells, stimulates the mucin synthesis of these cells of the corpus region (16). These results strongly indicate that a distinct control mechanism underlies the biosynthesis and accumulation of a mucin present in a specific region and layer of the gastric mucosa.
The first aim of this study was to investigate the effects of CGRP on mucin biosynthesis in distinct sites and in the layers of the rat gastric mucosa using a scraping method to separate the surface mucus cell layer from the remaining deep mucosa of the rat stomach (16, 17). In the second step, we examined whether endogenous NO release contributes to the CGRP-evoked stimulation of gastric mucin biosynthesis in the rat gastric mucosa. In the third part of this study, indirect double-labeling immunofluorescence techniques combined with a multiple dye filter were used to examine the distribution of NOS and CGRP in the rat gastric corpus mucosa.
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MATERIALS AND METHODS |
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Materials.
The following substances were used for this study: rat -CGRP and
human CGRP-(8-37) (Peptide Institute, Osaka, Japan);
NG-nitro-L-arginine
(L-NNA; Wako Pure Chemical, Osaka, Japan);
2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide sodium salt (carboxy-PTIO; Dojindo Laboratories, Kumamoto, Japan); D-[1,6-3H(N)]glucosamine hydrochloride (GlcN)
(1,787 GBq/mmol; New England Nuclear, Boston, MA); guinea pig anti-NOS
polyclonal antibody (Euro-Diagnostica, Malmö, Sweden); rabbit
polyclonal antibody against CGRP (Cambridge Research Biochemicals,
Northwich, UK); rhodamine-conjugated goat anti-guinea pig IgG
(Chemicon, Temecula, CA); and FITC-conjugated goat anti-rabbit IgG
(Cappel, Durham, NC). Rat
-CGRP, CGRP-(8-37), and
carboxy-PTIO were dissolved in PBS and added at appropriate
concentrations to the dishes. L-NNA was prepared in 0.01 N
HCl before being neutralized to pH 7.0. Stock solutions of these drugs
were freshly prepared, and the concentrations reported are final bath
concentrations. To assess the stability of CGRP, the concentrations in
the medium before and after 5-h incubation were determined by a CGRP
enzyme immunometric assay kit (SPI-BIO, Massy, France) under the same conditions used in the present study. At least 90% of CGRP dissolved in the medium of all experimental groups could be recovered in intact
form after 5-h incubation, indicating that CGRP was similarly stable
during contact with each tissue.
Tissue preparation of different sites and layers. Seven-week-old male Wistar rats (SLC, Shizuoka, Japan), each weighing ~170 g, were deprived of food but allowed free access to water for 24 h before the experiments. They were euthanized by inhalation of CO2, and their stomachs were immediately excised and then cut along the greater curvature. The gastric contents were gently rinsed out from the gastric mucosa by immersion in PBS. To obtain the full-thickness layer samples of the glandular tissue, the forestomach was removed from the remaining glandular stomach. The mucosa obtained was separated into the corpus and antrum and then cut into small pieces of 2 × 2 mm. Alternatively, after the stomach was opened and gently washed, the corpus was selected and cut into specimens of 10 × 10 mm. The surface mucosa of these corpus specimens was removed from the remaining deep mucosa with forceps having a J-shaped cusp (A-12-2; Natsume, Tokyo, Japan), according to our previously described method (17). The deep mucosal layer of the corpus was sliced into small pieces of ~4 mm2, similar to the full-thickness layer specimens. The validity of the separating manipulation was confirmed by immunohistochemical staining as previously described (16).
Tissue culture. For cultures of the full-thickness and deep-layer samples, eight tissue fragments were randomly picked out from ~5-6 different stomachs and then placed, with the mucosal surface facing up, on a stainless steel grid in the central well of a plastic culture dish (60 × 15 mm; Falcon, Lincoln Park, NJ) and then treated with 0.75 ml of medium and 0.05 ml of test substances. The tissue culture method of Eastwood and Trier (7) was used with modification (19). The medium consisted of 90% Eagle's minimum essential medium and 10% dialyzed fetal calf serum with 370 kBq/ml of [3H]GlcN. All of the dishes were maintained at 37°C for 5 h in 5% CO2 and 95% air. For culture of the surface layer, a sheet of the surface mucosa of the corpus was randomly picked out from 5 different stomachs and then placed, with the mucosal surface facing up, on a Falcon cell insert in the center of the well of a plate dish with the medium previously described and CGRP solution. All of these dishes were maintained at 37°C, primarily for 5 h and occasionally for 7 or 9 h in 5% CO2 and 95% O2.
Measurement of synthesized mucin. On completion of the culture period, the cultured tissues were harvested from the medium after being gently rinsed with PBS and boiled at 100°C for 3 min in 0.05 M Tris · HCl buffer, pH 7.2. The extraction and isolation of gastric mucins were performed as previously described in detail (17, 19). In brief, the tissue fragments were homogenized with a Physcotron microhomogenizer (Niti-On, Chiba, Japan). After Triton X-100 was added to a 2% (vol/vol) concentration, the homogenate was shaken for 1 h at 37°C and centrifuged at 8,000 g for 30 min to obtain the supernatant. This supernatant was applied onto a Bio-Gel A-1.5 m column (1 × 30 cm; Bio-Rad, Hercules, CA). The fractions eluted with the void volume were collected, and the radioactivity was measured as synthesized mucin (19). To compare the synthesis of mucin, the total radioactivity of these fractions was divided by the tissue protein content of each homogenate and expressed as disintegrations per minute (dpm) per milligram of tissue protein. The protein content in the tissue homogenate was determined by the bicinchoninic acid method with a Pierce protein assay kit (Pierce, Rockford, IL) using bovine serum albumin as the standard.
Immunohistochemical techniques. Male Wistar rats weighing ~170 g were used. After anesthetizing with sodium pentobarbital (0.05 mg/g), they were perfused through the heart with heparinized saline followed by a fixative solution (4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer at pH 7.4). The stomachs were excised from the body, and the corpus mucosa was selected and immersed in the same fixative for an additional 6 h at 4°C. After a brief washing with PBS, the specimens were transferred to 30% sucrose in PBS and kept overnight at 4°C. The specimens were then cut at 10 µm on a cryostat and mounted on poly-L-lysine-coated slides. The sections were processed for indirect double-labeling immunofluorescence as previously described (24). In brief, they were first incubated overnight at 4°C with guinea pig anti-NOS polyclonal antibody (1:1,000) and then incubated under the same conditions with rabbit antiserum directed against CGRP (1:1,500). After they were washed with PBS, they were incubated with a mixture of antisera containing rhodamine-conjugated goat anti-guinea pig IgG (1:100) and FITC-conjugated goat anti-rabbit IgG (1:100) for 2 h at room temperature. The immunostained sections were examined with a Zeiss Axiomat microscope equipped with a dual band filter set (Chroma Technologies) for simultaneous viewing of both red and green fluorescent dyes. To test the specificity of the antisera, the sections treated with NOS antiserum were incubated with FITC-conjugated anti-rabbit IgG, and those treated with CGRP antiserum were reacted with rhodamine-conjugated anti-guinea pig IgG.
Statistical analysis. The results of mucin biosynthesis in Figs. 1, 2, and 5 are expressed relative to the average value of the corresponding control and represent means ± SD. The values presented in Figs. 3 and 4 are given as means ± SD. One-way ANOVA with Dunnett's test was used for statistical analysis, with P < 0.05 being taken as significant.
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RESULTS |
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Effects of -CGRP on [3H]GlcN incorporation into
gastric mucins in full-thickness corpus and antrum.
Figure 1 shows the biosynthetic activity
of mucin in the full-thickness corpus and antrum as measured by
[3H]GlcN incorporation with or without
-CGRP. In the
controls (0 M) of the corpus and antral tissues, 3H
radioactivity incorporated into the gastric mucins was 12,452 ± 2,322 and 16,750 ± 1,425 dpm/mg tissue protein, respectively. The
addition of
-CGRP significantly enhanced [3H]GlcN
incorporation into mucin in the corpus region in a
concentration-dependent manner (Fig. 1A). In contrast, there
was no significant change in antral mucin biosynthesis after
-CGRP
addition (Fig. 1B).
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Effects of CGRP-(8-37) on -CGRP-stimulated
mucin biosynthesis.
The influences of the carboxy-terminal fragment of human
-CGRP,
CGRP-(8-37), a CGRP1 receptor antagonist,
on
-CGRP-stimulated incorporation of [3H]GlcN into the
mucin in the full-thickness corpus mucosa are shown in Fig.
2. Addition of
CGRP-(8-37) (10
6 M) had no significant
effect on mucin biosynthesis in either the corpus or the antrum
(n = 5). The
-CGRP-induced increase in
3H-labeled mucin in the corpus mucosa was completely
blocked by the addition of 10
6 M
CGRP-(8-37).
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Effects of -CGRP on [3H]GlcN incorporation into
gastric mucins in the deep layer of corpus mucosa.
To examine whether the stimulative effects of
-CGRP on corpus mucin
biosynthesis are limited to specific mucus cells of the rat gastric
mucosa, the deep-layer tissues of the corpus, including the gland mucus
cells but with removal of a large portion of the surface mucus cells
(16), were cultured in the presence of
[3H]GlcN. Figure 3 shows
the biosynthetic activity of mucin in the full-thickness and deep
mucosal layer tissues of corpus mucosa with or without the addition of
-CGRP. In the full-thickness corpus tissue, 3H-labeled
mucins were significantly increased with the addition of
10
7 and 10
6 M
-CGRP. In the control
situation with no added
-CGRP, the deep corpus mucosa synthesized
75% of the 3H-labeled mucin of the full-thickness corpus
mucosa. In the deep-layer tissues of the corpus, mucin biosynthesis was
not susceptible to the addition of
-CGRP (Fig. 3).
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Effects of -CGRP on [3H]GlcN incorporation into
gastric mucins in the surface layer of corpus mucosa.
Figure 4 shows the biosynthetic activity
of mucin in the surface layer, largely consisting of the surface mucus
cells, of corpus mucosa with or without the addition of
10
6 M
-CGRP. In the control situation without
-CGRP, ~10% of the 3H-labeled mucin synthesized in
the full-thickness corpus mucosa was recovered by incubation of the
surface mucus cell layer. The addition of
-CGRP enhanced
[3H]GlcN incorporation into mucin by 203%
(P < 0.01) after the 5-h incubation period. The
effects of
-CGRP on the incorporation of [3H]GlcN into
the mucin were essentially the same, even when the incubation time was
extended up to 9 h. 3H-labeled mucin in the surface
layer of corpus mucosa linearly increased with time up to 9 h
(Fig. 4).
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Effects of an NOS inhibitor or an NO antidote on
-CGRP-stimulated mucin biosynthesis.
The effects of the L-arginine analog L-NNA, an
NOS inhibitor, on
-CGRP-stimulated incorporation of
[3H]GlcN into the mucin in the full-thickness corpus
mucosa are shown in Fig. 5A.
Addition of L-NNA (10
5 M) had no significant
effect on mucin biosynthesis in the full-thickness corpus mucosa. The
-CGRP-induced increase in 3H-labeled mucin in the corpus
mucosa was completely blocked by the addition of 10
5 M
L-NNA. Concurrent pretreatment with L-arginine
(5 × 10
3 M) entirely reversed the stimulation of
mucin biosynthesis to the level observed after
-CGRP alone (Fig.
5A), whereas D-arginine (5 × 10
3 M) had no significant effect (radioactivity relative
to the control level, 102 ± 9%, n = 5).
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Immunolocalization with antibodies against CGRP and NOS.
As shown in Fig. 6a,
CGRP-immunoreactive fibers were detected in the lamina propria,
submucosal layer, and smooth muscle layer of the rat gastric corpus
mucosa. Figure 6b shows the double
immunofluorescence labeling for CGRP (FITC) and NOS (rhodamine) in the
surface layer of the corpus mucosa examined with the multiple dye
filter system. NOS immunoreactivity was detected in the surface mucus
cells. CGRP-immunoreactive fibers reached into the surface mucus cell layer, which is immunoreactive for NOS (Fig. 6b). The
yellowish parts of the CGRP fibers within the surface mucus cell layer
indicate overlap between CGRP and NOS at different depths. Neither CGRP nor NOS immunoreactivity was found in the corpus sections incubated with the corresponding preabsorbed antiserum.
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DISCUSSION |
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The gastric mucosa receives a very dense nerve supply, including
peptide-containing nerve fibers (9, 10).
Nervous mechanisms are important for the regulation of gastric
functions. Cholinergic stimulation has been shown to modulate mucus
metabolism in rabbit and canine gastric mucosal explants
(35, 36). We have found that acetylcholine
accelerates mucin biosynthesis in the corpus gland mucus cells and
antral mucus cells of the rat stomach, using an experimental method
similar to that in this study (17). Neuroactive peptide
CGRP has been extensively studied by numerous laboratories (13, 15, 27, 28) to
be the major transmitter by which afferent nerve fibers within the
gastric mucosa cause vasodilatation. The findings of this study show
that CGRP exerts stimulatory effects on mucin biosynthesis in rat
gastric corpus mucosa without vascular functions, because the influence
of gastric blood flow is generally ignored in our experimental model.
This activation of mucin synthesis has been completely prevented by the
CGRP1 receptor antagonist CGRP-(8-37).
Although the concentrations of CGRP required for the stimulation of
mucin synthesis in this study,
~107-10
5 M, were rather higher than
those in the normal gastric mucosa (20), our data indicate
that exogenous CGRP acts on the gastric mucus cells through the
specific receptors. Because stimulation of corpus mucin synthesis is
closely related to mucosal protective activity (18), these
results suggest that gastric mucosal protection elicited by CGRP
(6, 28) is due not only to a
hyperemia-dependent mechanism (14) but also to the
hyperemia-independent activation of mucus cell function.
In this study, the antral tissue separated from the corpus was also cultured. In contrast to acetylcholine, CGRP had no significant effect on mucin biosynthesis in this region of the rat stomach. Although the distribution of CGRP immunoreactive fibers in the antrum is very similar to that in the corpus (10), our data show that responsiveness of the gastric mucus cells to CGRP varies at different regions of the stomach. Similarly, NO, known to be an important mediator of CGRP-evoked vasodilatation, significantly accelerated mucin biosynthesis only in the oxyntic region but yielded no significant change in the antral region of the rat stomach (16). These findings support the assumption that mucin production is differently regulated in the corpus and antrum of the gastric mucosa.
Mucus-producing cells of the mammalian gastric corpus mucosa have been classified mainly into surface mucus and gland mucus cells (23, 38). Recent evidence demonstrates that mucus from these two types of human cells has distinct roles in the colonization of Helicobacter pylori, bacteria implicated in the etiology of gastric disease (37). Elucidation of the regulatory mechanism for these different mucus cells may help clarify the complicated defense mechanism of the gastric mucosa. Recently, we devised a scraping method to separate the surface mucosal layer from the remaining deep mucosa of the rat stomach (17). Immunohistochemical staining with RGM21, an original monoclonal antibody that strongly reacts with the surface mucus cell-derived mucin (21), has clearly shown that the deep mucosa remaining after scraping with forceps lacks the surface mucus cells but shows no observable damage to the underlying gland mucus cells (16). With the use of this scraping method, we have already demonstrated the acetylcholine-stimulated [3H]GlcN incorporation into the mucin in the deep corpus mucosa, indicating the stimulatory effect of acetylcholine on the gland mucus cells (17). In the present study, we surveyed the effect of CGRP on the mucin biosynthesis in the deep corpus mucosa without the surface mucus cell layer. Unlike the situation with the full-thickness layer of the corpus mucosa, CGRP failed to stimulate the mucin synthesis in the deep corpus mucosa. These findings strongly suggest that CGRP has distinct effects on the mucin biosynthesis between the surface mucus and the gland mucus cells in the rat corpus mucosa.
We examined the effect of CGRP on mucin biosynthesis in the surface mucus cell-rich layer peeled from the corpus mucosa. We have already demonstrated that the surface mucosal layer of the corpus, chiefly composed of surface mucus cells, can be easily removed mechanically from the remaining deep mucosa of the rat (16). In this study, incorporation of [3H]GlcN into the surface layer mucin linearly increased with time up to 9 h, indicating that it is feasible to estimate the biosynthetic activity of mucin in the surface mucosal layer. In contrast to the deep corpus layer, mucin biosynthesis of the surface layer was stimulated by the addition of CGRP. Although the ratio of 3H radioactivity in the surface corpus mucin to that in the full-thickness corpus mucosa was somewhat smaller than the ideal value calculated by the radiolabeled deep corpus mucin, the most striking finding in this study was that the stimulant effect of CGRP is limited to the specific region and layer of the gastric mucosa.
Price et al. (34) reported that the immunoreactivities of the NOS antiserum and the monoclonal antibody directed against NOS localized at the surface layer of gastric mucosa, except for the myenteric plexus. Recently, we found that NO, produced by NOS in the surface mucus cells, stimulates the mucin synthesis of these cells of the corpus region (16). To clarify the participation of endogenous NO in the CGRP-induced increase in gastric mucin synthesis, we examined the susceptibility to the NOS inhibitor L-NNA (31). The CGRP-induced increase in 3H-labeled corpus mucin was completely suppressed by the addition of L-NNA, and this blockade was reversed with the coaddition of L-arginine. Our present results on the effects of carboxy-PTIO, a new class of NO antidotes that can react only with NO (1), also indicated that NO itself plays an important role in mediating the activation of the corpus mucin synthesis elicited by CGRP. Recently, L-NNA has been shown to inhibit the gastroprotective effect of CGRP against ethanol-induced gross and histological damage (25). Similarly, inhibition of NO biosynthesis blunted the gastric hyperemic reaction to CGRP (4, 28). From the results of this study, it can be concluded that the CGRP-induced activation of mucin biosynthesis critically involves NO synthesis.
Here we show that exogenous CGRP makes a functional contact with NOS. Although immunohistochemical observation has shown that NOS localizes at some cell types, such as neuronal cells, endothelial cells, and surface mucus cells, in rat gastric mucosa (34), our recent study indicated that NOS, distributed in the surface layer, contributed to surface mucus cell functions of the gastric corpus mucosa (16, 17). The precise localization of CGRP within the gastric corpus mucosa, particularly with regard to whether or not a CGRP-containing fiber is in contact with the surface mucus cells that have NOS, is required to define the role played by CGRP in gastric mucin synthesis. In this study, the combination of double immunolabeling and a multiple dye filter system clearly demonstrated that NOS immunoreactivity actually existed in the surface mucus cells of the rat gastric corpus and contacted nerve fibers containing CGRP. These immunohistochemical findings support the assumption that CGRP modulates the surface mucus cell function of the rat stomach. Our results also confirmed and extended the recent work of Suzuki et al. (39), who reported that CGRP immunoreactive nerve fibers approached the surface layer of gastric corpus mucosa.
Although the species specificity of CGRP should be generally kept in
mind, pharmacological comparisons of human -CGRP and rat
-CGRP
have demonstrated that these peptides exert the same action as coronary
vasodilator transmitters in the rat (12). In our gastric
tissue culture system, the biosynthetic responses to human
-CGRP
were essentially the same as those to rat
-CGRP (data not shown),
indicating that these peptides exhibit qualitatively similar biological
properties. It has already been shown that rats and human subjects have
at least two subspecies of CGRP, namely
-CGRP and
-CGRP. Most of
the CGRP present in afferent neurons is
-CGRP, whereas the only form
of CGRP in enteric neurons is
-CGRP (13). Mulderry et
al. (29) showed that
-CGRP concentrations were three to
six times higher than
-CGRP concentrations in the rat stomach. In
contrast,
-CGRP is present predominantly in the intestine and colon.
Although further studies are needed to clarify the role of
-CGRP on
mucus cells, our data suggest that CGRP may be a controlling factor for
gastric mucus cell functions.
In summary, the present findings demonstrate that CGRP has distinct effects on mucin biosynthesis in a specific region and layer of rat gastric mucosa, suggesting different regulatory mechanisms underlying the mucus metabolism of distinct mucus-producing cells. The CGRP-induced stimulation of mucin biosynthesis is mediated by NO and occurs in the surface mucus cells, but not in gland mucus cells, of the gastric oxyntic mucosa.
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ACKNOWLEDGEMENTS |
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This study was supported in part by Grants-in-Aid for Scientific Research from the Japanese Ministry of Education, Science and Culture, by the Academic Frontier Project of the Japanese Ministry of Education, Science, Sports and Culture, by grants from Terumo Life Science Foundation, and by Kanagawa Academy of Science and Technology Research Grants.
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. Ichikawa, Dept. of Biochemistry, School of Medicine, Kitasato Univ., 1-15-1, Kitasato, Sagamihara, Kanagawa 228-8555, Japan (E-mail: t.ichika{at}kitasato-u.ac.jp).
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. §1734 solely to indicate this fact.
Received 24 September 1999; accepted in final form 27 January 2000.
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