Contribution Of Capsaicin-sensitive Sensory Neurons To Stress-induced Increases In Gastric Tissue Levels Of ... - American Journal Of Physiology - Gastrointestinal And Liver Physiology

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Contribution of capsaicin-sensitive sensory neurons to
stress-induced increases in gastric tissue levels
of prostaglandins in rats
Naoaki Harada,1 Kenji Okajima,2 Mitsuhiro Uchiba,2 Takeshi Katsuragi1
1Department of Pharmacology, School of Medicine, Fukuoka University, Fukuoka, 814-0180;
and 2Department of Diagnostic Medicine, Graduate School of Medical Science,
Kumamoto University, Kumamoto, 860-0811, Japan
Submitted 27 August 2002; accepted in final form 21 July 2003
Harada, Naoaki, Kenji Okajima, Mitsuhiro Uchiba,
Takeshi Katsuragi. Contribution of capsaicin-sensitive
sensory neurons to stress-induced increases in gastric tissue
levels of prostaglandins in rats. Am J Physiol Gastrointest
Liver Physiol 285: G1214–G1224, 2003. First published July
31, 2003; 10.1152/ajpgi.00364.2002.—We examined whether
capsaicin-sensitive sensory neurons might be involved in the
increase in the gastric tissue level of prostaglandins, thereby
contributing to the reduction of water immersion restraint
stress (WIR)-induced gastric mucosal injury in rats. Gastric
tissue levels of calcitonin gene-related peptide (CGRP), 6keto-PGF1, and PGE2 were transiently increased 30 min
after WIR. These increases were significantly inhibited by
subcutaneous injection of capsazepine (CPZ), a vanilloid receptor antagonist, and by functional denervation of capsaicin-sensitive sensory neurons induced by the administration
of high-dose capsaicin. The administration of capsaicin
(orally) and CGRP (intravenously) significantly enhanced the
WIR-induced increases in the gastric tissue level of prostaglandins 30 min after WIR, whereas CGRP-(8–37), a CGRP
receptor antagonist, significantly inhibited them. Pretreatment with N-nitro-L-arginine methyl ester (L-NAME), a
nonselective inhibitor of nitric oxide (NO) synthase (NOS),
and that with indomethacin inhibited the WIR-induced increases in gastric tissue levels of prostaglandins, whereas
either pretreatment with aminoguanidine (AG), a selective
inhibitor of the inducible form of NOS, or that with NS-398,
a selective inhibitor of cyclooxygenase (COX)-2, did not affect
them. CPZ, the functional denervation of capsaicin-sensitive
sensory neurons, and CGRP-(8–37) significantly increased
gastric MPO activity and exacerbated the WIR-induced gastric mucosal injury in rats subjected to 4-h WIR. The administration of capsaicin and CGRP significantly increased the
gastric tissue levels of prostaglandins and inhibited both the
WIR-induced increases in gastric MPO activity and gastric
mucosal injury 8 h after WIR. These effects induced by
capsaicin and CGRP were inhibited by pretreatment with
L-NAME and indomethacin but not by pretreatment with AG
and NS-398. These observations strongly suggest that capsaicin-sensitive sensory neurons might release CGRP,
thereby increasing the gastric tissue levels of PGI2 and PGE2
by activating COX-1 through activation of the constitutive
form of NOS in rats subjected to WIR. Such activation of
capsaicin-sensitive sensory neurons might contribute to the
reduction of WIR-induced gastric mucosal injury mainly by
inhibiting neutrophil activation.
calcitonin gene-related peptide; endothelial cells; gastric mucosal injury; neutrophils.
CAPSAICIN-SENSITIVE SENSORY neurons, nociceptive neurons, are activated by a wide variety of noxious stimuli
(7) and they have been shown to play an important role
in gastric cytoprotection by increasing gastric mucosal
blood flow through the release of calcitonin gene-related peptide (CGRP) (21). Because chemical ablation
of the sensory fibers resulted in a marked increase in
the severity of inflammation (39), capsaicin-sensitive
sensory neurons might contribute to the reduction of
tissue injury by regulating the inflammatory responses. PGI2 plays important role in the gastric cytoprotection that prevents gastric mucosal injury induced by various noxious stimuli (24, 34). Activated
neutrophils play critical roles in the development of the
gastric mucosal injury induced by stress (13), nonsteroidal anti-inflammatory drugs (43), and hemorrhagic
shock (27) by inducing local inflammation (39, 44, 45).
Because PGI2 inhibits neutrophil activation by increasing the intracellular concentration of cAMP (22, 38),
PGI2 might play a role in the gastric cytoprotection by
inhibiting neutrophil activation. Consistent with this
hypothesis are our previous findings showing that gastric tissue levels of PGI2 were significantly increased in
rats subjected to water immersion restraint stress
(WIR), which contributed to the prevention of stressinduced gastric mucosal injury mainly by inhibiting
neutrophil activation (15, 16). However, the mechanism(s) by which the gastric tissue level of PGI2 increases in rats subjected to WIR are not known. Because capsaicin reduced the WIR-induced gastric mucosal injury by increasing the gastric tissue level of
CGRP (33) and because CGRP has been shown to increase the endothelial production of PGI2 in vitro (6),
activation of capsaicin-sensitive sensory neurons might
contribute to gastric cytoprotection by increasing the gasAddress for reprint requests and other correspondence: K. Okajima, Dept. of Diagnostic Medicine, Graduate School of Medical
Science, Kumamoto Univ., Kumamoto, 860-0811, Japan (E-mail:
whynot@kaiju.medic.kumamoto-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. Section 1734
solely to indicate this fact.
Am J Physiol Gastrointest Liver Physiol 285: G1214–G1224, 2003.
First published July 31, 2003; 10.1152/ajpgi.00364.2002.
0193-1857/03 $5.00 Copyright © 2003 the American Physiological Society http://www.ajpgi.orgG1214
tric tissue level of PGI2, which might reduce the gastric
mucosal injury by inhibiting neutrophil activation.
PGE2 is a well-known gastric cytoprotective agent
synthesized in endothelial cells from PGH2, a common
precursor of PGI2 (11). Because PGE2 has been shown
to possess biological activities similar to those of PGI2,
(46), activation of capsaicin-sensitive sensory neurons
might increase the gastric tissue levels of PGE2 as well
as PGI2, thereby contributing to the reduction of gastric mucosal injury.
Recent studies have demonstrated that CGRP increases endothelial production of nitric oxide (NO) (3)
and that NO, in turn, activates cyclooxygenase
(COX)-1, thereby increasing endothelial production of
prostaglandins (4). Taken together, these observations
strongly suggest that CGRP might increase endothelial production of PGI2 and PGE2 through NO-mediated activation of COX-1.
In the present study, we examined whether activation of capsaicin-sensitive sensory neurons reduces the
stress-induced gastric mucosal injury by inhibiting
neutrophil activation in rats through increase in the
endothelial production of prostaglandins. Furthermore, possible involvement of NO in the process leading to the increase of prostaglandin production was
also examined.
MATERIALS AND METHODS
Reagents. Capsaicin; capsazepine (CPZ), an antagonist of
capsaicin; N-nitro-L-arginine methyl ester (L-NAME), a nonselective inhibitor of NO synthase (NOS); aminoguanidine, a
selective inhibitor of inducible form of NOS; and indomethacin, a nonselective inhibitor of COX, were purchased from
Sigma (St. Louis, MO). NS-398, a selective inhibitor of
COX-2, was a generous gift from Taisho Pharmaceutical
(Saitama, Japan). Rat -CGRP and human CGRP-(8–37)
were purchased from Peptide Institute (Osaka, Japan). All
other reagents were of analytical grade.
WIR-induced gastric mucosal lesion formation in rats.
Adult male Wistar rats (Nihon, Hamamatsu, Japan) weighing 280–320 g were used in each experiment. The care and
handling of the animals were in accordance with the National Institutes of Health guidelines. All experimental procedures described below were approved by the Kumamoto
University Animal Care and Use Committee. Before each
experiment, rats were deprived of food but not water for 24 h.
The animals were then placed in a restraint cage and immersed up to a level of water the xiphoid process at 22°C as
described previously (40). At the indicated times during WIR,
the animals were anesthetized by an intraperitoneal injection of pentobarbital sodium (50 mg/kg) and exsanguinated
via the abdominal aorta. Their stomachs were removed and
filled with 10 ml of 1% Formalin and immersed in 1% Formalin for 24 h. Their stomachs were then cut along the
greater curvature and examined for mucosal lesions. Because
most of the observed gastric mucosal lesions were linear,
with widths of 2 mm, in almost all lesions, the total length
of each linear hemorrhagic erosion was calculated as the
lesion index (mm) by an independent observer blinded to the
treatment as previously described (35).
Administration of various agents. Capsaicin and CPZ were
dissolved in 10% Tween 20/10% ethanol (10%) with normal
saline. capsaicin (1 mg/kg) was administered orally 1 h before
stress as described previously (2). CPZ (15 mg/kg) was injected subcutaneously 30 min before stress as described previously (31). CGRP (10 g/kg) and CGRP-(8–37) (100 g/kg)
were dissolved in sterile distilled water and injected intravenously immediately before stress as described previously
(23). L-NAME (5 mg/kg) and aminoguanidine (40 mg/kg) were
dissolved in normal saline solution and administered subcutaneously 30 min before the experiment as described previously (12, 29). Indomethacin (5 mg/kg) was suspended in
bicarbonate-buffered saline and injected subcutaneously 30
min before the experiment as described previously (15). NS398 (30 mg/kg) was suspended in a 0.5% carboxymethyl
cellulose aqueous suspension and administered orally 1 h
before the experiment as described previously (1). Solutions
were prepared immediately before the experiments. Each
control animal received the vehicle in these experiments.
However, because results in control experiments using the
vehicle of each solution were not significantly different from
those obtained by using saline (data not shown), we used as
a representative control the data obtained by using saline in
the present study.
Denervation of primary sensory nerves by capsaicin. Ablation of gastric visceral afferent nerves was accomplished by
high-dose capsaicin administration as previously described
(8, 23, 29, 33). Rats received a total dose of 125 mg/kg
capsaicin administered subcutaneously in divided doses over
2 days. Two weeks after treatment with high-dose capsaicin,
animals were subjected to stress. To determine the effectiveness of the sensory afferent nerve denervation procedure, a
drop of 0.001% capsaicin in saline was instilled into either
eye of the rats, and their protective wiping movements were
observed. Capsaicin-treated rats that showed any wiping
movement were excluded from the study. Control animals
were injected subcutaneously with 1 ml of 10% Tween 20/
10% ethanol (10%) with normal saline.
Determination of gastric CGRP level. Gastric levels of
CGRP were determined in animals before and during WIR by
modification of the methods as described previously (10).
Briefly, the stomachs were weighed and then homogenized in
3 ml of 2 N acetic acid. The homogenates were bathed in 90°C
water for 20 min and then centrifuged at 4,500 g for 10 min
(4°C). CGRP was extracted from the supernatant by using
reverse-phase C18 columns (Amersham, Little Chalfont,
UK). Columns were prepared by washing with 5 ml of methanol, followed by 10 ml of water before use. The supernatant
was applied onto the column, followed by washing with 20 ml
of 0.1% trifluoroacetic acid. CGRP was eluted with 3 ml of
60% acetonitrile in 0.1% trifluoroacetic acid, and the solvent
was evaporated under a stream of nitrogen gas. The concentration of CGRP was assayed by using a specific enzyme
immunoassay kit (SPI-BIO, Massey Cedex, France). The
sensitivity of the CGRP assay was 10 pg/ml. The antiserum
cross-reacted 100% of rat - and -CGRP according to the
manufacturer’s data sheet. Results are expressed as micrograms of CGRP per gram of tissue.
Immunohistochemical staining of CGRP in the stomach.
The peroxide-antiperoxide technique was used for immunohistochemical staining of the stomach with anti-CGRP antibody according to methods described previously with slight
modification (38). The unfixed tissue blocks of rat stomach
were frozen in dry ice-cooled optimum cutting temperature
compound (Tissue Tek; Miles, Elkhart, IN). Sections (6–8 m
thick) were mounted on glass slides, immersed in absolute
acetone at 20°C for 5 min, rinsed in PBS five times for 5 min
each, and then incubated for 20 min with 10% porcine serum
in PBS at room temperature. They were incubated for 1 h at
37°C with rabbit anti-CGRP polyclonal antibody at 1:100
dilution. After five rinses in PBS, the sections were treated
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with horseradish peroxidase-conjugated anti-rabbit IgG
(MBL, Nagoya, Japan) at 1:2,000 dilution for 1 h at 37°C.
Reaction products were developed by immersing the sections
in 33-diaminobenzidine tetrahydrochloride solution containing 0.03% hydrogen peroxide. The control for immunostaining was performed by nonimmune rabbit serum as the first
step in place of primary antiserum and omission of the first
step or use of the first antiserum preabsorbed with an excess
of the homologous antigen. Samples were mounted with
Entellan onto glass slides, examined, and photographed under a light microscope.
Determination of gastric tissue levels of 6-keto-PGF1 and
PGE2. Gastric levels of 6-keto-PGF1 and PGE2 were determined in animals before and during WIR according to the
methods described previously (30, 32). Briefly, stomachs
were weighed and then homogenized in 5 ml of 0.1 M PBS
(pH 7.4) at 5°C. The homogenates were centrifuged at 2,000
g for 10 min to remove the tiny amounts of solid tissue debris.
The supernatant was then acidified with 1 M HCl. 6-ketoPGF1 and PGE2 were extracted from the supernatant by
using columns packed with ethyl-bonded silica gel (ethyl C2;
Amersham). Columns were prepared by washing with 2 ml of
methanol, followed by 2 ml of water. The acidified supernatant was applied onto the column followed by washing sequentially with 5 ml of 10% ethanol and 5 ml of hexane.
6-keto-PGF1 and PGE2 were eluted with 5 ml of methyl
formate, and the solvent was evaporated under a stream of
nitrogen gas. The evaporated supernatant was reconstituted
with the buffer equipped in specific enzyme immunoassay
kits for the determination of the concentration of 6-ketoPGF1 and PGE2 (Amersham). The cross reactivities of the
assay for 6-keto-PGF1 with PGE2, PGF2, thromboxane B2,
and arachidonic acid were 2.8, 1.4, 0.03, and 0.01%, respectively, according to the manufacturer’s data sheet. The crossreactivities of the assay for PGE2 with PGE1, PGF2, 6-ketoPGF1, and thromboxane B2 were 7.0, 4.3, 5.4, and  0.1%,
respectively, according to the manufacturer’s data sheet.
Results are expressed as micrograms of 6-keto-PGF1 or
PGE2 per gram of tissue.
Measurement of gastric MPO activity. After animals were
immersed for the indicated period of stress, all were immediately killed. Their stomachs were quickly removed and
opened along the greater curvature. In some experiments,
leukocyte infiltration in gastric mucosa was assessed by
determining tissue activity of MPO, an enzyme used as a
marker for leukocyte infiltration in a variety of tissues including rat gastric mucosa (5, 41, 42). MPO activity was
determined by a modification of the method as described
previously (26). Briefly, the stomachs were weighed and
suspended in 6 ml of 50 mM phosphate buffer (pH 6.0)
containing 1% hexadecyltrimethylammonium bromide. Samples were homogenized; the homogenate was sonicated,
freeze-thawed, and then centrifuged (4,500 g for 15 min at
4°C). MPO activity was determined in the supernatant (0.1
ml) after the addition of 0.6 ml of PBS (pH 6.0) containing
0.05 ml of 1.25 mg/ml of o-dianisidine dihydrochloride and
0.05 ml of 0.05% hydrogen peroxide. The change in absorbance at 460 nm over a 6.5-min period was measured in a
spectrophotometer (model DU-54; Beckman, Irvine, CA). One
unit of MPO activity is defined as the amount of enzyme that
will reduce 1 mol peroxidase/min. Results are expressed as
units of MPO activity per gram of tissue.
Statistical analysis. Data are expressed as means  SD.
Results were compared by using either an ANOVA followed
by Scheffé’s post hoc test for multiple comparisons or a
Student’s t-test for single comparison. A level of P  0.05 was
considered statistically significant.
RESULTS
Changes in gastric tissue levels of CGRP, 6-ketoPGF1, and PGE2 and immunohistochemical staining
of CGRP in the stomach in rats subjected to WIR.
Gastric tissue levels of CGRP were significantly increased 30 min after WIR compared with the pre-WIR
levels (Fig. 1A). These levels were decreased rapidly to
the pre-WIR levels at 1 h of WIR and were not changed
during 1 to 8 h of WIR (Fig. 1A). Immunohistochemical
staining of CGRP in the rat gastric mucosa 30 min
after WIR was increased in the lamina propria of the
gastric mucosa (Fig. 2B) compared with that of nonFig. 1. Changes in gastric levels of calcitonin gene-related peptide
(CGRP; A), 6-keto-PGF1 (B), and PGE2 (C) in rats subjected to water
immersion restraint stress (WIR). Values are expressed as means 
SD derived from 5 animal experiments. *P  0.05 vs. pre-WIR; §P 
0.01 vs. pre-WIR.
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stressed rats (Fig. 2A). Gastric tissue levels of both
6-keto-PGF1 and PGE2 were also significantly increased 30 min after WIR compared with the pre-WIR
levels (Fig. 1, B and C). These levels began to decrease
after 1 h of WIR and were significantly decreased to
less than pre-WIR levels after 6 and 8 h of WIR (Fig. 1,
B and C).
Effects of CPZ, the functional denervation of capsaicin-sensitive sensory neurons and capsaicin on the
increases in gastric tissue levels of CGRP, and immunohistochemical staining of CGRP in the stomach 30
min after WIR. Both CPZ and the functional denervation of capsaicin-sensitive sensory neurons significantly inhibited the increases in gastric tissue levels of
CGRP (Fig. 3) and the increase in the gastric mucosal
immunohistochemical staining of CGRP 30 min after
WIR (Fig. 2, C and D). The administration of capsaicin
significantly enhanced the WIR-induced increase in
the gastric tissue level of CGRP 30 min after WIR (Fig.
3). Each control animal that received the vehicle or
saline as shown in MATERIALS AND METHODS showed the
increase in the gastric mucosal immunohistochemical
staining of CGRP (data not shown).
Effects of CPZ, CGRP-(8–37), functional denervation
of capsaicin-sensitive sensory neurons, capsaicin, and
CGRP on increases in gastric tissue levels of 6-ketoPGF1 and PGE2 30 min after WIR. Gastric tissue
levels of 6-keto-PGF1 30 min after WIR in animals
pretreated with CPZ and CGRP-(8–37) and in those
with the functional denervation of capsaicin-sensitive
sensory neurons (2.03  0.28, 2.06  0.15, and 1.95 
0.22 g/g tissue, respectively; n 5 in each group)
were significantly lower than those in control animals
(2.76  0.21 g/g tissue; n 5, P  0.01). However,
these levels in animals pretreated with capsaicin and
CGRP (6.05  0.90 and 5.90  0.52 g/g tissue, respectively; n 5 in each group) were significantly higher
than those in control animals (P  0.01). Gastric tissue
levels of PGE2 30 min after WIR in animals pretreated
with CPZ or CGRP-(8–37) and in those with the functional denervation of capsaicin-sensitive sensory neurons (2.62  0.40, 2.56  0.31, and 2.66  0.25 g/g
tissue, respectively; n 5 in each group) were significantly lower than those in control animals (5.96  0.48
g/g tissue; n 5, P  0.01). These levels in animals
Fig. 2. Effects of WIR, capsaicin, capsazepine
(CPZ), and functional denervation of capsaicin-sensitive sensory neurons on immunohistochemical staining of CGRP in the stomach.
Immunohistochemical CGRP staining of frozen sections of the stomach were determined
by anti-rat CGRP antibody in intact animals
(A) and in animals 30 min after WIR (B–D)
(original magnification
100). CPZ (15 mg/
kg) was injected subcutaneously 30 min before WIR. Functional denervation of capsaicin-sensitive sensory neurons was performed
as described in MATERIALS AND METHODS. A:
intact; B: WIR; C: WIR CPZ; D: WIR
functional denervation of capsaicin-sensitive
sensory neurons. The administration of each
vehicle did not affect the WIR-induced immunohistochemical staining of CGRP. In each
group, 5 animals were examined; typical results are shown.
Fig. 3. Effects of CPZ, functional denervation of capsaicin-sensitive
sensory neurons, and capsaicin on WIR-induced increase in gastric
CGRP levels after 30 min of WIR. CPZ (15 mg/kg) was injected
subcutaneously 30 min before WIR. Functional denervation of capsaicin-sensitive sensory neurons was performed as described in MATERIALS AND METHODS. Capsaicin (1 mg/kg) was administered orally
1 h before WIR. Values are means  SD. *P  0.01 vs. WIR.
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pretreated with capsaicin and CGRP (12.40  0.38 and
11.15  0.58 g/g tissue, respectively; n 5 in each
group) were significantly higher than those in control
animals (P  0.01).
Effects of L-NAME, aminoguanidine, indomethacin,
and NS-398 on increases in gastric tissue levels of
6-keto-PGF1 and PGE2 30 min after WIR. Gastric
tissue levels of 6-keto-PGF1 30 min after WIR in
animals pretreated with L-NAME and indomethacin
(1.34  0.36 and 1.26  0.28 g/g tissue, respectively;
n 5 in each group) were significantly lower than
those in control animals (2.76  0.21 g/g tissue; n 5,
P  0.01). In contrast, these levels were not affected by
pretreatment with aminoguanidine and NS-398
(2.16  0.46 and 2.40  0.58 g/g tissue, respectively;
n 5 in each group). Gastric tissue levels of PGE2 30
min after WIR in animals pretreated with L-NAME
and indomethacin (1.90  0.25 and 2.11  0.20 g/g
tissue, respectively; n 5 in each group) were significantly lower than those in control animals (5.96  0.48
g/g tissue; n 5, P  0.01). However, these levels
were not affected by pretreatment with aminoguanidine and NS-398 (5.74  0.68 and 5.68  0.75 g/g
tissue, respectively; n 5 in each group).
Effects of L-NAME, aminoguanidine, indomethacin,
and NS-398 on capsaicin- or CGRP-induced increases
in gastric tissue levels of 6-keto-PGF1 and PGE2 30
min after WIR. Pretreatment with L-NAME inhibited
the capsaicin- or CGRP-induced increases in gastric
tissue levels of 6-keto PGF1 (Fig. 4A) and PGE2 (Fig.
4B) 30 min after WIR, whereas that with aminoguanidine did not affect these levels (Fig. 4, A and B).
Although pretreatment with indomethacin inhibited
the capsaicin- or CGRP-induced increases in gastric
tissue levels of 6-keto PGF1 (Fig. 4A) and PGE2 (Fig.
4B) 30 min after WIR, that with NS-398 did not inhibit
these increases (Fig. 4, A and B).
Effects of CPZ, functional denervation of capsaicinsensitive sensory neurons, CGRP-(8–37), capsaicin,
and CGRP on the WIR-induced changes in gastric
accumulation of neutrophils and gastric mucosal injury. Gastric accumulation of neutrophils as evaluated
by measuring the gastric MPO activity increased 8 h
after WIR compared with pre-WIR levels (15). The
gastric lesion index was significantly increased 4 h
after WIR compared with the pre-WIR level, and it
reached the maximum 8 h after WIR (15). To clarify
whether activation of capsaicin-sensitive sensory neurons contributes to the reduction of WIR-induced gastric mucosal injury by limiting gastric neutrophil accumulation, we examined the effects of CPZ, CGRP-(8–
37), and the functional denervation of capsaicinsensitive sensory neurons on the changes in gastric
MPO activity and gastric lesion index 4 h after WIR
when these variables did not reach their maximum
values. Pretreatment with CPZ and CGRP-(8–37), and
the functional denervation of capsaicin-sensitive sensory neurons significantly increased both the gastric
Fig. 4. Effects of pretreatment with
N-nitro-L-arginine methyl ester (LNAME), aminoguanidine (AG), indomethacin (IM), and NS-398 on capsaicin- or CGRP-induced changes in the
gastric tissue levels of 6-keto-PGF1
(A) and PGE2 (B) 30 min after WIR.
Capsaicin (1 mg/kg) was administered
orally 1 h before WIR. CGRP (10 g/kg)
was injected intravenously immediately before WIR. L-NAME (5 mg/kg),
AG (40 mg/kg), and IM (5 mg/kg) were
injected subcutaneously 30 min before
capsaicin or CGRP administration.
NS-398 (30 mg/kg) was administered
orally 1 h before capsaicin or CGRP
administration. Values are expressed
as means  SD. ‡P  0.01 vs. WIR
capsaicin; ¶P  0.01 vs. WIR CGRP.
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MPO activity and the gastric lesion index 4 h after WIR
(Fig. 5, A and B). Although the administration of capsaicin and CGRP had no effect on the gastric MPO
activity, it reduced the gastric lesion index 4 h after
WIR (Fig. 5, A and B). CPZ, CGRP-(8–37), and the
functional denervation of capsaicin-sensitive sensory
neurons did not affect the WIR-induced increases in
either the gastric MPO activity or the gastric lesion
index 8 h after WIR (Fig. 5, C and D), whereas capsaicin and CGRP significantly inhibited these increases
(Fig. 5, C and D).
Effects of L-NAME, aminoguanidine, indomethacin,
and NS-398 on the WIR-induced changes in gastric
accumulation of neutrophils and gastric mucosal injury. Pretreatment with L-NAME and indomethacin
significantly increased gastric MPO activities and gastric mucosal injury 4 h after WIR (Fig. 6); that with
aminoguanidine and NS-398 had no effects on these
values (Fig. 6). Pretreatment with L-NAME, aminoguanidine, indomethacin, and NS-398 did not affect these
variables seen 8 h after WIR (data not shown).
Effects of L-NAME, aminoguanidine, indomethacin,
and NS-398 on the capsaicin- and CGRP-induced effects on gastric accumulation of neutrophils and gastric
mucosal injury. Pretreatment with L-NAME and indomethacin significantly increased gastric MPO activities in animals given capsaicin and CGRP 4 h after
WIR; that with aminoguanidine and NS-398 did not
affect these values (Fig. 7A). Although pretreatment
with L-NAME and indomethacin abrogated both capsaicin- and CGRP-induced reduction of the gastric lesion index 4 h after WIR, that with aminoguanidine
and NS-398 had no affect on these variables (Fig. 7B).
Inhibition of gastric MPO activities induced by capsaicin and CGRP 8 h after WIR were not observed when
animals were pretreated with L-NAME and indomethacin, whereas these inhibitory effects were unaffected
in animals pretreated with aminoguanidine and NS398 (Fig. 7C). Although reduction of the gastric lesion
index induced by capsaicin or CGRP seen 8 h after WIR
was not observed in animals pretreated with L-NAME
and indomethacin, such effects were unaffected by pretreatment with aminoguanidine and NS-398 (Fig. 7D).
DISCUSSION
As shown in the present study, both the gastric
tissue level and the immunohistochemical staining of
CGRP were significantly increased in rats 30 min after
WIR. Although the immunohistochemical staining of
CGRP in the gastric tissue was apparently observed in
Fig. 5. Effects of CPZ, CGRP-(8–37), functional denervation of capsaicin-sensitive sensory neurons, capsaicin, and CGRP on the WIRinduced increase in gastric MPO activity and the gastric lesion index
after 4 h (A and B) or 8 h (C and D) of WIR. CPZ (15 mg/kg) was
injected subcutaneously 30 min before WIR. Functional denervation
of capsaicin-sensitive sensory neurons was performed as described in
MATERIALS AND METHODS. CGRP-(8–37) (100 g/kg) and CGRP (10
g/kg) were injected intravenously immediately before WIR. Capsaicin (1 mg/kg) was administered orally 1 h before WIR. Values are
means  SD. *P  0.01 vs. WIR.
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the surface epithelium as well as in the lamina propria
(Fig. 2), distribution of the sensory neurons was not
shown in the surface epithelium of the stomach (19),
suggesting that staining in the surface epithelium
might be an artifact. Increases in the gastric tissue
level and the immunohistochemical staining of CGRP
were inhibited by pretreatment with CPZ and the
functional denervation, and the increase in the gastric
tissue level of CGRP was enhanced by pretreatment
with capsaicin, suggesting that capsaicin-sensitive
sensory neurons could be activated in the stomach of
rats subjected to WIR.
Ren et al. (33) demonstrated that high-dose capsaicin treatment markedly decreased gastric CGRP levels in rats subjected to WIR to 25% of the levels of
rats subjected to WIR alone. Although we treated rats
with high-dose capsaicin according to the method described by Ren et al. (33), gastric CGRP levels in the
rats subjected to WIR were decreased to 40% of the
levels of rats subjected to WIR alone. In the present
study, we excluded animals that showed protective
wiping movements after instillation of 0.001% capsaicin into either eye to select the animals with successful functional denervation.
Gastric tissue levels of CGRP were not significantly
decreased during WIR compared with the pre-WIR
levels as shown in the present study. However, recent
studies have demonstrated that gastric tissue levels of
CGRP were significantly decreased in rats after 3–4 h
of WIR compared with the pre-WIR levels (8, 33).
Because a range of physical and chemical stimuli can
promote the release of CGRP from the sensory nerve
endings (7), the tissue level of CGRP after the insult
might be dependent on the magnitude of the insult.
Consistent with this hypothesis, the gastric mucosal
lesion indexes after WIR in these two reports were
about three times higher than those observed in the
present study, explaining why the gastric tissue levels
of CGRP were decreased in these two previous reports.
Gastric tissue levels of 6-keto-PGF1, a stable metabolite of PGI2, and PGE2 were increased 30 min after
WIR, which contributed to prevent the WIR-induced
gastric mucosal injury mainly by inhibiting neutrophil
activation (9, 12, 13, 18). However, the mechanisms
underlying the increase in the gastric tissue level of
PGI2 and PGE2 have not been fully clarified.
Because CGRP is released from the nerve endings of
capsaicin-sensitive sensory neurons located around
submucosal arterioles (19), the released CGRP could
interact with endothelial cells. CGRP has been shown
to increase the endothelial production of PGI2 in vitro
(6), suggesting that the released CGRP in the gastric
mucosa might increase the gastric tissue level of PGI2
in rats subjected to WIR. Consistent with this hypothesis are the present observations showing that the
increase in the gastric tissue level of 6-keto-PGF1 was
enhanced by capsaicin and CGRP and was inhibited by
CPZ, the functional denervation of capsaicin-sensitive
sensory neurons and CGRP-(8–37). Furthermore, because the increase in the gastric tissue level of PGE2
was enhanced by capsaicin and CGRP and was inhibited by CPZ, it is possible that WIR-induced activation
of capsaicin-sensitive sensory neurons might also increase the gastric PGE2 production by releasing CGRP.
We previously reported that pretreatment with indomethacin inhibited the WIR-induced increase in the
gastric tissue level of 6-keto-PGF1, whereas that with
NS-398, a selective inhibitor of COX-2, did not affect
the increase (16). Because the WIR-induced increases
in the gastric tissue levels of 6-keto-PGF1 and PGE2
were inhibited by indomethacin, but were not affected
by NS-398, the capsaicin-sensitive sensory neuron-mediated increase in the gastric production of PGI2 and
PGE2 might be mediated by COX-1. Consistent with
these observations are previous studies reporting that
CGRP has been shown to increase the endothelial
production of NO that activates COX-1 selectively,
Fig. 6. Effects of pretreatment with L-NAME, AG, IM, and NS-398
on WIR-induced changes in gastric MPO activity (A) and the gastric
lesion index (B) after 4 h of WIR. L-NAME (5 mg/kg), AG (40 mg/kg),
and IM (5 mg/kg) were injected subcutaneously 30 min before capsaicin or CGRP administration. NS-398 (30 mg/kg) was administered
orally 1 h before capsaicin or CGRP administration. Values are
means  SD. *P  0.01 vs. WIR.
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Fig. 7. Effects of pretreatment with LNAME, AG, IM, and NS-398 on capsaicin- or CGRP-induced changes in gastric MPO activity and the gastric lesion
index after 4 h (A and B) or 8 h (C and
D) of WIR. Capsaicin (1 mg/kg) was
administered orally 1 h before WIR.
CGRP (10 g/kg) was injected intravenously immediately before WIR.
L-NAME (5 mg/kg), AG (40 mg/kg), and
IM (5 mg/kg) were injected subcutaneously 30 min before capsaicin or CGRP
administration. NS-398 (30 mg/kg)
was administered orally 1 h before capsaicin or CGRP administration. Values
are means  SD. ‡P  0.01 vs. WIR
capsaicin; ¶P  0.01 vs. WIR CGRP.
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AJP-Gastrointest Liver Physiol • VOL 285 • DECEMBER 2003 • www.ajpgi.org
thereby inducing the endothelial production of prostaglandin in vitro (3, 4).
WIR-induced increases in gastric tissue levels of
6-keto-PGF1 and PGE2 were inhibited by pretreatment with L-NAME, a nonselective inhibitor of NOS,
but not by pretreatment with aminoguanidine, a selective inhibitor of an inducible isoform of NOS. These
observations suggest that the constitutive form of NOS
could be importantly involved in the capsaicin-sensitive sensory neuron-mediated increases in the gastric
tissue levels of PGI2 and PGE2 in rats subjected to
WIR. Consistent with these observations is our previous report showing that activation of capsaicin-sensitive sensory neurons increased the hepatic production
of PGI2 in rats subjected to hepatic ischemia/reperfusion by activating endothelial NOS (17).
We (15, 16) previously reported that gastric PGI2
contributed to prevent stress-induced gastric mucosal
injury mainly by inhibiting neutrophil activation. Gastric PGE2 has also been shown to prevent indomethacin-induced gastric mucosal injury by inhibiting neutrophil activation (35). Thus activation of capsaicinsensitive sensory neurons might contribute to the
reduction of the gastric mucosal injury by inhibiting
neutrophil activation through increasing the gastric
tissue levels of PGI2 and PGE2. The administration of
capsaicin and CGRP significantly inhibited the 8-h
WIR-induced increases in both the gastric MPO activity and the gastric mucosal injury. Furthermore, neither capsaicin- nor CGRP-induced effects were observed in animals pretreated with L-NAME and indomethacin, but these effects were unaffected in animals
pretreated with aminoguanidine and NS-398. These
observations strongly suggest that activation of capsaicin-sensitive sensory neurons might lead to the reduction of gastric mucosal injury by inhibiting neutrophil
activation through promotion of the constitutive form
of NOS- and COX-1-mediated production of PGI2 and
PGE2 in rats subjected to WIR.
Capsaicin-sensitive sensory neurons in the gastric
mucosa could be activated in rats subjected to WIR as
shown in the present study. However, the mechanisms
by which capsaicin-sensitive sensory neurons could be
activated in rats subjected to WIR are not fully understood. Preliminary studies showed that pretreatment
of rats with omeprazole, a proton pump inhibitor, and
famotidine, an H2-receptor antagonist, both of which
potently inhibit acid secretion, significantly inhibited
the WIR-induced increases in gastric tissue levels of
both CGRP and 6-keto-PGF1. These observations suggest that gastric acid back diffused to the gastric mucosa might play a role in the stimulation of the capsaicin-sensitive sensory neurons in rats subjected to
WIR. These observations are consistent with previous
observations that showed that capsaicin-sensitive sensory neurons could be activated by an acidic environment (19) and that an acid-induced increase in the
gastric mucosal blood flow could be mediated by capsaicin-sensitive sensory neuron activation (20).
Because CGRP, NO, PGI2, and PGE2 have been
shown to increase the gastric mucosal blood flow (15,
19, 21, 25), activation of capsaicin-sensitive sensory
neurons, which leads to CGRP release and a subsequent increase in the endothelial production of NO,
PGI2, and PGE2, might contribute to maintain the
gastric mucosal integrity by increasing gastric mucosal
blood flow as well as by inhibiting neutrophil activation
(14, 19). Thus capsaicin-sensitive sensory neurons in
the gastric mucosa might play a role not only in the
sensory nervous system but in the cytoprotective system, which could increase the gastric mucosal blood
flow and attenuate the local inflammatory responses.
Hecker et al. (18) demonstrated that, although PGE2
inhibited N-formyl-methionyl-leucyl-phenylalanine-induced cytotoxic enzyme release from human neutrophils by increasing intracellular levels of cAMP, neither PGI2 nor iloprost, a stable analog of PGI2, had any
effect. In contrast, Kainoh et al. (22) reported that both
PGE2 and PGI2 inhibited oxygen free radical production by increasing intracellular cAMP levels in rat
polymorphonuclear leukocytes. Although inhibition of
neutrophil activation by PGI2 in vitro is still controversial, iloprost prevented WIR-induced gastric mucosal
injury by inhibiting accumulation of neutrophils in rats
(15). Therefore, PGI2 might contribute to prevent
stress-induced gastric mucosal injury by inhibiting
neutrophil activation in our animal model of gastric
mucosal injury.
Figure 8 shows the possible mechanism by which
activation of capsaicin-sensitive sensory neurons reduced the gastric mucosal injury by increasing the
Fig. 8. Diagram of the possible mechanism by which activation of
capsaicin-sensitive sensory neurons increased gastric tissue levels of
prostaglandins in rats subjected to stress. eNOS, endothelial nitric
oxide synthase; COX-1, cyclooxygenase-1.
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gastric tissue levels of prostaglandins in rats subjected
to WIR.
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