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Roles of Cyclooxygenase-2 and Prostacyclin/IP Receptors in
Mucosal Defense against Ischemia/Reperfusion Injury in
Mouse Stomach
Tohru Kotani, Atsushi Kobata, Eiji Nakamura, Kikuko Amagase, and Koji Takeuchi
Department of Pharmacology and Experimental Therapeutics, Kyoto Pharmaceutical University, Misasagi, Yamashina, Kyoto,
Japan
Received July 24, 2005; accepted October 17, 2005
ABSTRACT
We examined the roles of cyclooxygenase (COX) isozymes,
prostaglandins (PGs), and their receptors in the mucosal de-
fense against ischemia/reperfusion (I/R)-induced gastric lesions
in mice. Male C57BL/6 mice, including wild-type animals and
those lacking prostaglandin E
2
(EP)1, EP3, or prostaglandin I
2
(IP) receptors, were used after 18 h of fasting. Under urethane
anesthesia, the celiac artery was clamped (ischemia) for 30 min,
and then reperfusion was achieved for 60 min through the
removal of the clamp, and the stomach was examined for
lesions. I/R produced hemorrhagic gastric lesions in wild-type
mice. The severity of lesions was significantly increased by
pretreatment with indomethacin (a nonselective COX inhibitor)
and rofecoxib (a selective COX-2 inhibitor) but not 5-(4-
chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-
pyrazole (SC-560; a selective COX-1 inhibitor). The expression
of COX-2 mRNA was up-regulated in the stomach following I/R
but not by sham operation or ischemia alone. The ulcerogenic
response was markedly aggravated in IP receptor knockout
mice but not those lacking EP1 or EP3 receptors. I/R increased
the levels of 6-keto-PGF
1
␣
and PGE
2
in the stomach of wild-
type mice, and this response was attenuated by indomethacin
and rofecoxib but not SC-560. Pretreatment of wild-type mice
with iloprost, a prostacyclin (PGI
2
) analog, significantly pre
-
vented the I/R-induced gastric lesions in the absence and
presence of indomethacin or rofecoxib. PGE
2
also reduced the
severity of I/R-induced gastric lesions, yet the effect was much
less pronounced than that of iloprost. These results suggest
that endogenous PGs derived from COX-2 play a crucial role in
gastric mucosal defense during I/R, and this action is mainly
mediated by PGI
2
through the activation of IP receptors.
The damage caused by an interruption of blood supply to
an organ or tissue followed by the reintroduction of blood into
the affected area is called ischemia/reperfusion (I/R) injury.
The phenomenon of I/R injury is a major clinical problem
after stroke, infarction, shock, and organ transplantation.
The depletion of adenosine triphosphate (ATP) and distur-
bance of intracellular calcium homeostasis have been sug-
gested as the major pathophysiological mechanisms during
ischemia, leading to loss of cell viability (Farber et al., 1981;
Cheung et al., 1986). Reperfusion of ischemic tissues para-
doxically exacerbates the injury process and leads to the
release of reactive oxygen species and proinflammatory me-
diators and the attraction of inflammatory cells infiltrating
the tissues (Chamoun et al., 2000; Piper et al., 2003). In the
gastrointestinal tract, I/R injuries are known to be associated
with significant morbidity and mortality during the course of
hemorrhagic shock, abdominal aortic aneurysm repair, isch-
emic bowel disease, and necrotizing enterocolitis (Yasue et
al., 1992; Riaz et al., 2002; Dimmitt et al., 2003).
Nonselective cyclooxygenase (COX) inhibitors damage the
gastrointestinal mucosa in patients as an adverse reaction
(Soll et al., 1991). By contrast, selective COX-2 inhibitors
such as rofecoxib and celecoxib do not induce gastric lesions
in rats (Vane and Botting, 1995; Laudanno et al., 2001).
Hence, these COX-2 inhibitors were expected to be anti-
inflammatory and chemopreventive drugs devoid of gastro-
intestinal toxicity, although chronic treatment with a COX-2
inhibitor delayed the healing of gastric ulcers (Mizuno et al.,
1997; Miura et al., 2004). Recently, it was reported that a
selective COX-2 inhibitor aggravated gastric lesions induced
This research was supported in part by the Kyoto Pharmaceutical Univer-
sity’s “21st Century COE” program and the “Open Research” Program from the
Ministry of Education, Science and Culture of Japan.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.105.093195.
ABBREVIATIONS: I/R, ischemia/reperfusion; COX, cyclooxygenase; PG, prostaglandin; EP, prostaglandin E
2
receptor; PGI
2
, prostacyclin; IP,
prostaglandin I
2
receptor; MPO, myeloperoxidase; PCR, polymerase chain reaction; SC-560, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(triflu
-
oromethyl)-1H-pyrazole; NS-398, N-[2-(cyclohexyloxy)-4-nitrophenyl]methanesulfonamide; DFU, 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methyl sul-
fonyl)phenyl-2(5II)-furanone.
0022-3565/06/3162-547–555$20.00
T
HE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 316, No. 2
Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 93195/3073009
JPET 316:547–555, 2006 Printed in U.S.A.
547
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by I/R in rats, suggesting the involvement of endogenous
prostaglandins (PGs) in the mucosal defense during I/R
(Maricic et al., 1999).
PGs, especially PGE
2
, have been shown to exert a protec
-
tive action in the stomach through the activation of EP1
receptors, although the effects on various functions are me-
diated by different EP receptor subtypes; cf., acid inhibition
by EP3 receptors, an increase of mucus secretion by EP4
receptors, an increase of mucosal blood flow by EP2/EP4
receptors, and an inhibition of gastric motility by EP1 recep-
tors (Takeuchi et al., 2002b). Likewise, prostacyclin (PGI
2
), a
prostanoid mainly synthesized in the endothelium, exerts
various physiological actions at the interface between blood
and tissue (Konturek and Robert, 1982; Whittle et al., 1984).
Since PGI
2
enhances the gastric mucosal microcirculation
through vasodilation and inhibition of platelet aggregation, it
is possible that this prostanoid contributes to the mainte-
nance of the mucosal integrity of the stomach during I/R
(Granger and Kubes, 1994; Saika et al., 1999). However, it
remains unknown which type of prostanoid plays a role in
mucosal defense of the stomach under I/R conditions. In the
present study, we examined the effects of various COX in-
hibitors on I/R-induced gastric lesions in mice and further
investigated which type of prostanoid receptor is involved in
mucosal defense under I/R-induced conditions using animals
lacking EP1, EP3, or IP receptors.
Materials and Methods
Animals. Male C57BL/6 mice (3 months old; SLC, Shizuoka,
Japan) were used. Mice lacking EP1, EP3, or IP receptors were
generated as described previously (Sugimoto et al., 1992; Ushikubi et
al., 1998). In brief, the genes encoding the EP1, EP3, and IP recep-
tors were individually disrupted, and chimeric mice were generated.
These animals were then backcrossed with C57BL/6 mice, and the
resulting heterozygous littermates [EP1 (⫺/⫺), EP3 (⫺/⫺), or IP
(⫺/⫺)] were bred to produce homozygous EP1 (⫺/⫺), EP3 (⫺/⫺), or IP
(⫺/⫺) mice. Homozygous mice were born at the predicted Mendelian
frequency, grew normally, lived longer than 1 year, and were fertile.
Distribution of the EP1, EP3, and IP receptor genes was verified by
Northern blot hybridization, which failed to detect messenger RNAs
encoding the respective receptors in EP1 (⫺/⫺), EP3 (⫺/⫺), and IP
(⫺/⫺) mice. These knockout mice were deprived of food but allowed
free access to tap water for 18 h before the experiments. All experi-
ments were carried out using four to eight mice under urethane
anesthesia unless otherwise specified. The experimental procedures
described here were approved by the Experimental Animal Research
Committee of Kyoto Pharmaceutical University.
Induction of Gastric Mucosal Lesion by Ischemia and
Reperfusion. Acute gastric mucosal lesions were produced by I/R
(Wada et al., 1996). Briefly, under urethane anesthesia (1.25 g/kg
i.p.), the celiac artery was clamped with a small clamp (disposable
vascular clip, holding force of 40 g; BEAR Medical Corporation,
Chiba, Japan), and 30 min later, reperfusion was achieved through
removal of the clamp. After reperfusion for 60 min, the stomach was
excised, inflated by injecting 0.4 ml of 2% formalin for 10 min to fix
the tissue walls, and opened along the greater curvature. In wild-
type mice, the effects of COX inhibitors on the I/R-induced gastric
lesions were examined. Indomethacin (5 mg/kg), rofecoxib (a selec-
tive COX-2 inhibitor, 5 mg/kg), or SC-560 (a selective COX-1 inhib-
itor, 5 mg/kg) was administered p.o. 60 min before ischemia. The
doses of these COX inhibitors were selected to show nonselective
inhibition of both COX-1 and COX-2 or selective inhibition of COX-1
or COX-2, respectively (Takeeda et al., 2003). In addition, the effects
of iloprost, an analog of PGI
2
, and PGE
2
on the I/R-induced gastric
lesions were examined also in both wild-type and IP receptor knock-
out mice. Iloprost (0.3⬃3
g/kg) or PGE
2
(0.1⬃1 mg/kg) was given i.v.
5 min before reperfusion. The area (millimeters
2
⫻10). The person
measuring the lesions did not know the treatments given to the
animals.
In some cases, the gastric mucosa was examined with a light
microscope following I/R. The animals were killed after I/R treat-
ment, and the stomachs were excised. The tissue samples were then
immersed in 2% formalin-saline, embedded in paraffin, sectioned at
4
m, and stained with hematoxylin and eosin (H&E).
Determination of Myeloperoxidase Activity. Myeloperoxi-
dase (MPO) activity in the gastric mucosa was measured after I/R
treatment in wild-type and IP receptor knockout mice, according to a
modified version of the method of Krawisz et al. (1984). After 60 min
after I/R treatment, the animals were sacrificed by withdrawal of
blood from the heart by perfusing with saline, and the stomach was
excised. After rinsing of the tissue with ice-cold saline, the mucosa
was scraped with glass slides, weighed, and homogenized in a 50 mM
phosphate buffer containing 0.5% hexadecyltrimethylammonium
bromide (pH 6.0; Sigma-Aldrich, St. Louis, MO). The homogenized
samples were subjected to freezing and thawing three times and
centrifuged at 2000 rpm for 10 min at 4°C. MPO activity in the
supernatant was determined by adding 100
l of the supernatant to
1.9 ml of 10 mM phosphate buffer (pH 6.0) and 1 ml of 1.5 M
o-dianisidine hydrochloride (Sigma-Aldrich) containing 0.0005%
(w/v) hydrogen peroxide. The changes in absorbance at 450 nm of
each sample were recorded on a Hitachi spectrophotometer (U-2000;
Hitachi, Ibaraki, Japan). Sample protein content was estimated by
spectrophotometric assay (Pierce protein assay kit; Rockford, IL),
and the MPO activity was obtained from the slope of the reaction
curve, based on the following equation: specific activity (
mol H
2
O
2
/
min/mg of protein) ⫽ (OD/min)/OD/
mol H
2
O
2
⫻ mg of protein).
Measurement of Mucosal PGE
2
and 6-Keto-PGF
1
␣
Levels.
Levels of PGE
2
and 6-keto-PGF
1
␣
, the stable metabolite of PGI
2
,in
the gastric mucosa were measured after I/R treatment in wild-type
mice. The animals were killed under deep ether anesthesia after the
60-min reperfusion period, and the gastric mucosa was isolated,
weighed, and placed in a tube containing 100% methanol plus 0.1
mM indomethacin (Futaki et al., 1994). Then the tissues were ho-
mogenized by a Polytron homogenizer (IKA, Tokyo, Japan) and cen-
trifuged at 12,000 rpm for 10 min at 4°C. After the supernatant of
each sample had been evaporated with N
2
gas, the residue was
resolved in assay buffer and used for determination of PGE
2
and
6-keto-PGF
1
␣
. The concentrations of PGE
2
and 6-keto-PGF
1
␣
were
measured using a PGE
2
or 6-keto-PGF
1
␣
enzyme immunoassay kit
(GE Healthcare, Little Chalfont, Buckinghamshire, UK).
Analysis of COX-1, -2, EP1, EP3, and IP mRNA Expression
by Reverse Transcription-PCR. Wild-type animals were killed
under deep ether anesthesia after I/R treatment, and the stomachs
were removed, frozen in liquid nitrogen, and stored at ⫺80°C prior to
use. Tissue samples were pooled from two to three rats for extraction
of total RNA, which was prepared by a single-step acid phenol-
chloroform extraction procedure by use of TRIzol (Invitrogen, Carls-
bad, CA). Total RNA primed by random hexadeoxy ribonucleotide
was reverse-transcribed with the SuperScript preamplification sys-
tem (Invitrogen). The sequences of sense and antisense primers for
the mouse COX-1, COX-2, EP1, EP3, and IP are shown in Table 1. An
aliquot of the reverse transcription reaction product served as a
template in 35 cycles of PCR with 1 min of denaturation at 94°C, 0.5
min of annealing at 56°C, and 1 min of extension at 72°C on a
thermal cycler. A portion of the PCR mixture was electrophoresed in
1.5% agarose gel in Tris-EDTA-acetic acid buffer, and the gel was
stained with ethidium bromide and photographed.
Determination of Gastric Secretion. Acid secretion was mea-
sured in wild-type mice provided with an acute gastric fistula under
urethane anesthesia (1.25 g/kg i.p.). Briefly, the abdomen was in-
cised, and both the stomach and duodenum were exposed. An acute
fistula (inside diameter, 2 mm) made with a polyethylene tube was
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inserted into the stomach from a small incision made in the fore-
stomach and was held in place by a ligature. The stomach was filled
with 0.4 ml of saline (154 mM NaCl) through the fistula, and the
solution was changed every 20 min. The collected samples were
centrifuged at 3000 rpm for 15 min and titrated to pH 7.0 against 10
mM NaOH using an autoburette (Hiranuma Comtite-8, Tokyo, Ja-
pan). Indomethacin (5 mg/kg), SC-560 (5 mg/kg), or rofecoxib (5
mg/kg) was given i.d. 1 h before instillation of saline in the stomach
whereas iloprost (0.1⬃3
g/kg) was given i.v. 5 min before.
Preparation of Drugs. The drugs used were urethane (Tokyo
Kase, Tokyo, Japan), indomethacin (Sigma-Aldrich), SC-560 (Cay-
man Chemical, Ann Arbor, MI), rofecoxib (synthesized in our labo-
ratory), iloprost (Nacalai Tesque, Kyoto, Japan), and PGE
2
(Funa
-
koshi, Tokyo, Japan). All COX inhibitors were suspended in a
hydroxy propyl cellulose solution (Wako, Osaka, Japan). Iloprost was
dissolved in saline, whereas PGE
2
was first dissolved in absolute
ethanol and diluted with saline to the desired concentrations. Each
agent was prepared immediately before use and administered p.o.,
i.d., i.p., or i.v. in a volume of 0.5 ml/100 g of body weight. Control
animals received saline as the vehicle.
Statistics. Data are presented as the mean ⫾ S.E.M. for four to
eight mice per group. Statistical analyses were performed using a
one-way analysis of variance and Student’s t test or Dunnett’s mul-
tiple comparison test where appropriate, and values of P ⬍ 0.05 were
considered significant.
Results
I/R-Induced Gastric Lesions in Wild-Type Mice. Lap-
arotomy without clamping of the gastric artery (sham oper-
ation) did not produce any damage in the gastric mucosa of
wild-type mice. In the animals subjected to I/R treatment
(30-min ischemia followed by reperfusion for 60 min), how-
ever, multiple hemorrhagic lesions were observed in the gas-
tric mucosa, the lesion score being 7.2 ⫾ 4.9 mm
2
(n ⫽ 5).
Ischemia for 30 min did not induce any macroscopically vis-
ible damage in the mucosa. Histologically, most of the dam-
age induced by I/R was restricted to the surface epithelium,
but some damage occurred deep in the mucosa, extending to
the region of pits and glands (Fig. 1, A–D). In sham-operated
animals, no damage was detected even by histological obser-
vation.
Effect of COX Inhibitors on I/R-Induced Gastric Le-
sions in Wild-Type Mice. Following I/R treatment in wild-
type mice, the gastric mucosa developed multiple hemor-
rhagic lesions, the lesion score being 9.9 ⫾ 3.6 mm
2
.
Pretreatment of the animals with indomethacin (5 mg/kg
p.o.) significantly aggravated these lesions, the lesion score
being 22.3 ⫾ 4.5 mm
2
(Fig. 2
A). The severity of I/R-induced
gastric lesions was also significantly increased by prior ad-
ministration of the selective COX-2 inhibitor rofecoxib (5
Fig. 2. A, effects of COX inhibitors on the I/R-induced gastric lesions in
wild-type mice. Indomethacin (5 mg/kg), SC-560 (5 mg/kg), or rofecoxib (5
mg/kg) was given p.o. 60 min before ischemia. Data are presented as
mean ⫾ S.E.M. from five to seven mice. ⴱ, significant difference from
control at p ⬍ 0.05. B, COX mRNA expression in the gastric mucosa of
wild-type (WT) mice subjected to a sham operation or I/R treatment. I,
ischemia for 30 min.
TABLE 1
PCR primer sequences and product size
Molecule Primer Sequence Product Size
bp
GAPDH 5⬘-AACGACCCCTTCATTGAC-3⬘ 191
5⬘-TCCACGACATACTCAGCAC-3⬘
COX-1 5⬘-CTGCATGTGGCTGTGGATGTCATC-3⬘ 389
5⬘-GGTCTTGGTGAGGCAGACCAG-3⬘
COX-2 5⬘-GTCTGATGATGTATGCCACAATCTG-3⬘ 276
5⬘-GATGCCAGTGATAGAGGGTGTTGAA-3⬘
EP1 5⬘-AATACATCTGTGGTGCTGCCAACA-3⬘ 829
5⬘-CCACCATTTCCACATCGTGTGCGT-3⬘
EP3 5⬘-GGTATGCCAGCCACATGAAGAC-3⬘ 529
5⬘-CAAGATCTGGTTCAGCGAAGCC-3⬘
IP 5⬘-GGCACGAGAGGATGAAGTTTAC-3⬘ 408
5⬘-GTCAGAGGCACAGCAGTCAATGG-3⬘
GAPDH, glyceraldehyde-3-phosphate dehydrogenase; bp, base pair(s).
Fig. 1. Gross appearance and histological observations of the gastric
mucosa in wild-type mice subjected to a sham operation or I/R treatment.
A and C, sham; B and D, I/R. Bar, 100
m
PGI
2
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mg/kg p.o.), and the lesion score was almost equivalent to
that observed in indomethacin-pretreated animals. However,
the COX-1-selective inhibitor SC-560 (5 mg/kg p.o.) had no
effect on the development of gastric lesions induced by I/R.
The gene expression of glyceraldehyde-3-phosphate dehy-
drogenase, the housekeeping gene, as well as COX-1 was
clearly detectable in the stomach of control wild-type mice
and was not affected by either ischemia or I/R (Fig. 2B).
Although the expression of COX-2 mRNA was negligible in
the gastric mucosa of control wild-type mice, it was markedly
up-regulated following I/R but not ischemia alone.
I/R-Induced Gastric Lesions in EP1, EP3, and IP Re-
ceptor Knockout Mice. To further investigate which pro-
stanoid receptor is involved in the mucosal defense against
I/R-induced gastric lesions, we compared the gastric ulcero-
genic response to I/R in wild-type mice and the animals
lacking EP1, EP3, or IP receptors. As shown in Fig. 3A, the
expression of EP1, EP3, and IP receptor mRNAs was clearly
detectable in the stomach of wild-type mice.
Following I/R treatment, wild-type mice in each group
developed hemorrhagic lesions in the gastric mucosa, the
lesion score being 7.6 ⫾ 2.8 to 8.0 ⫾ 3.2 mm
2
. Development of
gastric lesions was observed in the animals lacking EP1,
EP3, or IP receptors, although the severity of the lesions
differed in these groups of mice. As shown in Fig. 3B, the
gastric ulcerogenic response to I/R was significantly in-
creased in IP receptor knockout mice, the lesion score reach-
ing roughly 2 times that in wild-type mice. However, the
severity of these lesions in EP1 or EP3 receptor knockout
animals was not significantly different compared with wild-
type mice.
Effect of Iloprost on I/R-Induced Gastric Lesions in
Wild-Type Mice. Since the severity of the I/R-induced gas-
tric lesions was found to increase in IP receptor knockout
animals, we examined the effect of a stable PGI
2
analog,
iloprost, on the ulcerogenic response to I/R in wild-type mice,
in the absence or presence of COX inhibitors.
Pretreatment of the animals with iloprost (0.3⬃3
g/kg
i.v.) dose-dependently prevented the development of I/R-in-
duced gastric lesions in wild-type mice, the degree of protec-
tion being 72.4% (Fig. 4A). However, this agent, even at 3
g/kg, had no effect on these lesions in the animals lacking IP
receptors (Fig. 4B). On the other hand, the I/R-induced gas-
Fig. 3. A, expression of mRNA for prostanoid receptors (EP1, EP3, and
IP) in the mouse stomach. M, marker. B, gastric lesions induced by I/R in
wild-type mice and those lacking EP1, EP3, or IP receptors. Under
urethane anesthesia, the celiac artery was clamped, and then reperfusion
was achieved 30 min later by removal of the clamp. After a 60-min
reperfusion period, hemorrhagic lesions were induced in the gastric mu-
cosa. Data are presented as mean ⫾ S.E.M. from five to seven mice. ⴱ,
significant difference from each group of wild-type mice at p ⬍ 0.05.
Fig. 4. A, effect of iloprost, the IP agonist, on I/R-induced gastric lesions
in wild-type mice. Iloprost (0.3⬃3
g/kg) was given i.v. 5 min before
reperfusion. Data are presented as mean ⫾ S.E.M. from six to eight mice.
ⴱ, significant difference from control at p ⬍ 0.05. B, effect of iloprost on
I/R-induced gastric lesions in IP receptor knockout mice. Iloprost (1
g/kg) was given i.v. 5 min before reperfusion. Data are presented as
mean ⫾ S.E.M. from six to eight mice.
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tric lesions were markedly worsened by prior administration
of either indomethacin (5 mg/kg p.o.) or rofecoxib (5 mg/kg
p.o.), the degree of aggravation being 52.2 or 67.3%, respec-
tively (Fig. 5A). The aggravating effect of these COX inhibi-
tors was significantly abrogated by pretreatment with ilo-
prost (1
g/kg i.v.; Fig. 5B).
Mucosal MPO Activity during I/R in Wild-Type and
IP Receptor Knockout Mice. The severity of I/R-induced
gastric lesions was increased in IP receptor knockout mice
and reduced by supplementation of PGI
2
, respectively. To
confirm that the mucosal inflammatory response during I/R
is also affected by these treatments, we measured MPO ac-
tivity in the gastric mucosa of both wild-type and IP receptor
knockout mice after I/R in the presence or absence of iloprost.
Tissue-associated MPO activity in the gastric mucosa of
sham-operated mice was less than 0.3
mol H
2
O
2
/min/mg of
tissue. Gastric MPO activity in wild-type mice was markedly
increased after I/R, reaching about 4 times over the control
levels, the values being 1.11 ⫾ 0.2
mol H
2
O
2
/min/mg of
tissue (Fig. 6A). The MPO activity was further increased in
IP receptor knockout animals in response to I/R, the values
being 2.56 ⫾ 0.41
mol H
2
O
2
/min/mg of tissue, which is
significantly greater than that observed in wild-type mice.
On the other hand, the increase in gastric MPO activity
following I/R in wild-type mice was dose-dependently sup-
pressed by prior administration of iloprost (0.3⬃3
g/kg), and
a significant effect was observed at 1 and 3
g/kg, the inhi-
bition being 44.1 and 44.0%, respectively (Fig. 6B).
Gastric Mucosal 6-Keto-PGF
1
␣
and PGE
2
Contents in
Wild-Type Mice. Levels of 6-keto-PGF
1
␣
, the stable metab
-
olite of PGI
2
, in the gastric mucosa were significantly in
-
creased in wild-type mice following I/R treatment compared
with a sham operation (Fig. 7A). This increase was signifi-
cantly prevented by prior administration of indomethacin (5
mg/kg p.o.) and rofecoxib (5 mg/kg p.o.) but not SC-560 (5
mg/kg p.o.). Likewise, the mucosal PGE
2
content was also
significantly increased by I/R treatment, and the response
was significantly attenuated by both indomethacin and rofe-
coxib but not SC-560 (Fig. 7B).
Effect of Various COX Inhibitors and Iloprost on
Gastric Acid Secretion. The stomach of wild-type mice
secreted acid at a rate of about 3.01 to 3.42
Eq for 2 h.
Fig. 5. Effect of iloprost (1
g/kg) on the I/R-induced gastric lesions in
wild-type mice pretreated with COX inhibitors. Indomethacin (5 mg/kg)
or rofecoxib (5 mg/kg) was given p.o. 60 min before ischemia. Iloprost (1
g/kg) was given i.v. 5 min before reperfusion. Data are presented as the
mean ⫾ S.E.M. from five to six mice. Significant differences at p ⬍ 0.05:
ⴱ, from the corresponding control; #, from treatment with (A) indometh-
acin or (B) rofecoxib.
Fig. 6. A, I/R-induced changes in gastric MPO activity in wild-type and IP
receptor knockout mice. Under urethane anesthesia, the celiac artery was
clamped (ischemia), and then reperfusion was achieved 30 min later by
removal of the clamp. After a 60-min reperfusion period, gastric MPO
activity was measured. Data are presented as the mean ⫾ S.E.M. from six
mice. Significant difference at p ⬍ 0.05: ⴱ, from sham; #, from control. B,
the effect of iloprost on I/R-induced gastric MPO activity in wild-type
mice. Iloprost (0.3⬃3
g/kg) was given i.v. 5 min before reperfusion. Data
are presented as mean ⫾ S.E.M. from six mice. ⴱ, significant difference
from control at p ⬍ 0.05.
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2
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Neither indomethacin, SC-560, nor rofecoxib had any effect
on basal acid secretion in wild-type mice, the values being
equivalent in all groups (Table 2). Likewise, iloprost (0.3⬃3
g/kg) given i.v. did not significantly affect acid output at any
dose, although a slight decrease (12.6%) was observed at the
highest dose, 3
g/kg. In addition, no difference in basal acid
secretion was observed between wild-type and IP receptor
knockout mice, the values being 3.01 ⫾ 0.40
Eq/2 h and
3.51 ⫾ 0.43
Eq/2 h, respectively (Table 3).
Effect of PGE
2
on I/R-Induced Gastric Lesions in
Wild-Type Mice. The severity of I/R-induced gastric lesions
remained unaltered in EP1 or EP3 receptor knockout mice.
However, because the levels of PGE
2
content in the gastric
mucosa were found to increase following I/R with the up-
regulation of COX-2 expression, there is a possibility that
PGE
2
plays some role in mucosal defense of the stomach
during I/R through a different EP receptor subtype. We
therefore examined the effect of PGE
2
on I/R-induced gastric
lesions in both wild-type and IP receptor knockout mice.
PGE
2
, given i.v. at 0.1 to 1 mg/kg 5 min before reperfusion,
dose-dependently reduced the severity of I/R-induced gastric
lesions in wild-type mice, and a significant effect was ob-
tained only at 1 mg/kg, the degree of protection being 39.1%,
which is less pronounced compared with that of iloprost at 3
g/kg (67.3%; Fig. 8A). The protective effect of PGE
2
was
observed even in IP receptor knockout mice, although the
degree of protection was 25.8%, slightly less than that in
wild-type mice (Fig. 8B).
Discussion
Ischemia followed by reperfusion leads to tissue injury
(Farber et al., 1981; Cheung et al., 1986; Piper et al., 2003).
Whereas there is a substantial body of experimental data
characterizing the factors that promote gastric lesions under
I/R-induced conditions, tissue defense reactions that counter-
balance the noxious effects of I/R remain less understood.
The present study clearly demonstrated that endogenous
PGs derived from COX-2 play a crucial role in the mucosal
defense of the stomach under I/R-induced conditions, and
this action is mainly mediated by PGI
2
through activation of
IP receptors.
It has been thought that COX-1 functions as a housekeep-
ing enzyme, catalyzing the formation of PGs that contribute
to the maintenance of the mucosal integrity of the stomach
through the modulation of various functions (Soll et al., 1991;
Vane and Botting, 1995), whereas COX-2, the inducible en-
zyme up-regulated by proinflammatory cytokines and growth
factors, mediates pathological reactions such as inflamma-
tion and tumor growth (Davies et al., 1997; Koga et al., 2004).
Recent studies, however, showed that COX-2 is also involved
in mucosal defense under certain conditions (Muscara et al.,
2000; Tanaka et al., 2002) and plays an important role in the
healing of gastric ulcers (Mizuno et al., 1997). In the present
study, we found that the selective COX-2 inhibitor rofecoxib
significantly aggravated the development of gastric lesions in
response to I/R, similar to indomethacin, confirming the in-
Fig. 7. Effect of COX inhibitors on mucosal levels of 6-keto-PGF
1
␣
, the
stable metabolite of PGI
2
(A), and PGE
2
(B) in the stomach of wild-type
mice subjected to I/R treatment. Indomethacin (5 mg/kg), SC-560 (5
mg/kg), or rofecoxib (5 mg/kg) was given p.o. 60 min before ischemia. Data
are presented as the mean ⫾ S.E.M. from five mice. Significant difference
at p ⬍ 0.05: ⴱ, from sham; #, from control.
TABLE 2
Effect of various COX inhibitors and iloprost on gastric secretion in
wild-type mice
Acid secretion was measured in wild-type mice provided with an acute gastric fistula
under urethane anesthesia. The stomach was filled with 0.4 ml of saline through the
fistula, the solution was changed after 20 min, and the collected samples were used
to measure acid output. Indomethacin (5 mg/kg), SC-560 (5 mg/kg), or rofecoxib (5
mg/kg) was given i.d. 1 h before the instillation of saline in the stomach, whereas
iloprost (0.1⬃3
g/kg) was given i.v. 5 min before. Data are presented as the mean ⫾
S.E.M. from four to five mice.
Group Dose No. of Mice Total Acid Output
Eq/2 h
Saline 4 3.02 ⫾ 0.36
Indomethacin 5 mg/kg 5 2.67 ⫾ 0.39
SC-560 5 mg/kg 5 2.86 ⫾ 0.29
Rofecoxib 5 mg/kg 5 3.21 ⫾ 0.17
Iloprost 0.3
g/kg 5 3.01 ⫾ 0.40
1
g/kg 5 3.18 ⫾ 0.10
3
g/kg 5 2.64 ⫾ 0.55
TABLE 3
Basal acid secretion in wild-type and IP receptor knockout mice
Acid secretion was measured in wild-type and IP receptor knockout mice provided
with an acute gastric fistula under urethane anesthesia. The stomach was filled with
0.4 ml of saline through the fistula, the solution was changed after 20 min, and the
collected samples were used to measure acid output. Data are presented as the
mean ⫾ S.E.M. from four mice.
Group No. of Mice Total Acid Output
Eq/2 h
Wild-type mice 4 3.01 ⫾ 0.40
IP knockout mice 4 3.51 ⫾ 0.43
552 Kotani et al.
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volvement of COX-2/PGs in mucosal defense during I/R.
These results are consistent with the findings of Maricic et al.
(1999), who showed that I/R-induced gastric damage was
aggravated by administration of selective COX-2 inhibitors
such as NS-398 and DFU as assessed both macroscopically
and histologically. They also showed that I/R increased
COX-2 mRNA levels in the gastric mucosa and that dexa-
methasone aggravated the I/R-induced gastric lesions at a
dose that inhibited COX-2 expression following I/R (Maricic
et al., 1999). We also found in the present study the up-
regulation of COX-2 expression in the stomach following I/R.
These findings suggest that the gastric mucosa has the ca-
pacity to rapidly up-regulate COX-2 expression in response
to various stimuli. It should be noted that ischemia alone
neither caused any macroscopic damage nor up-regulated
COX-2 expression in the gastric mucosa. Schmedtje et al.
(1997) reported that hypoxia increased the expression of the
COX-2 gene in human vascular endothelial cells in vitro. It is
possible that the expression is up-regulated by ischemia
alone when examined at a later period. Notwithstanding, it is
assumed that COX-2 expression is up-regulated as one of the
protective mechanisms when the stomach is exposed to ul-
cerogenic stimuli.
On the other hand, we observed in this study that the
selective COX-1 inhibitor SC-560 did not significantly affect
the severity of I/R-induced gastric lesions, suggesting no role
for COX-1 in mucosal defense during I/R. However, Hirat-
suka et al. (2004) reported that SC-560 reduced the severity
of I/R-induced gastric lesions in mice, probably due to down-
regulation of free radical production during reperfusion by
decreasing blood flow. The discrepancy in these results re-
mains unexplained at present, yet it may be due to different
experimental conditions such as the dose of SC-560; they
used 40 mg/kg of this agent, 8 times higher than that (5
mg/kg) in the present study.
The most important finding of the present study is that the
severity of I/R-induced gastric lesions was markedly in-
creased in IP receptor knockout mice but not in the animals
lacking EP1 or EP3 receptors. These results suggest that
although I/R stimulated the generation of PGs to increase the
mucosal levels of both PGE
2
and 6-keto-PGF
1
␣
, the stable
metabolite of PGI
2
, in the stomach, the type of prostanoid
responsible for mucosal defense during I/R is PGI
2
not PGE
2
.
These results are understandable, because the expression of
COX-2 in the gastric mucosa following I/R was observed
mainly in the endothelial cells (Hiratsuka et al., 2004) and
because PGI
2
is a major prostanoid produced in the endothe
-
lial cells (Konturek and Robert, 1982). We observed in this
study that iloprost, a stable analog of PGI
2
, significantly
prevented the I/R-induced gastric lesions in the absence or
presence of COX inhibitors, supporting the involvement of
endogenous PGI
2
in mucosal defense during I/R. This PGI
2
analog has an affinity for not only IP receptors but also EP
receptors as well (Narumiya and FitzGerald, 2001). In the
present study, however, iloprost had no effect on the devel-
opment of I/R-induced gastric lesions in IP receptor knockout
mice, excluding the involvement of EP receptors in the pro-
tective action of this agent. Harada et al. (1999, 2000) re-
ported that iloprost prevented stress-induced gastric lesions,
primarily by inhibiting leukocytes from accumulating. Since
I/R injury is a neutrophil-dependent response (Zimmerman
et al., 1990), it is assumed that selective COX-2 inhibitors
promote the adherence of leukocyte to the vascular endothe-
lium during I/R, thereby resulting in aggravation of the le-
sions in the stomach (Muscara et al., 2000). In the present
study, we observed a marked increase in MPO activity in the
gastric mucosa following I/R in wild-type mice, and this re-
sponse was significantly reduced by iloprost and further en-
hanced in IP receptor knockout animals, confirming the in-
hibitory role for PGI
2
/IP receptors in the neutrophil-related
process of I/R-induced gastric injury. These results all
strongly suggest that endogenous PGI
2
produced by COX-2
plays a role in mucosal defense during I/R through the acti-
vation of IP receptors.
We previously examined, using various subtype-specific
EP receptor agonists and antagonists, the relationship be-
tween EP receptor subtypes and PGE
2
-induced gastric cyto
-
protection and found that PGE
2
exhibits a protective action
against a variety of gastric lesions mediated by the activation
of EP1 receptors (Araki et al., 2000; Suzuki et al., 2001;
Takeuchi et al., 2002a). The present results, however, sug-
gest that neither EP1 nor EP3 receptors participate in mu-
cosal defense during I/R in the stomach, although the muco-
Fig. 8. A, effect of PGE
2
on I/R-induced gastric lesions in wild-type mice.
PGE
2
(0.1⬃1 mg/kg) was given i.v. 5 min before reperfusion. Data are
presented as the mean ⫾ S.E.M. from five to six mice. ⴱ, significant
difference from control at p ⬍ 0.05. B, effect of PGE
2
on I/R-induced
gastric lesions in IP receptor knockout mice. PGE
2
(1 mg/kg) was given
i.v. 5 min before reperfusion. Data are presented as mean ⫾ S.E.M. from
four to six mice.
PGI
2
and Ischemia/Reperfusion-Induced Gastric Injury 553
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sal PGE
2
content was significantly elevated following I/R. Of
course, the present data do not totally exclude the involve-
ment of PGE
2
in mucosal defense during I/R. It has been
reported that PGE
2
protects against ischemia- or I/R-induced
injury in brain, liver, and heart through EP2 or EP4 recep-
tors (McCullough et al., 2004; Xiao et al., 2004; Kuzumoto et
al., 2005). We also found in the present study that PGE
2
significantly reduced the severity of these lesions in both
wild-type and IP receptor knockout mice, yet the effective
dose was much higher and the effect was much less pro-
nounced compared with iloprost. In a preliminary study, we
also observed that the effect of PGE
2
was significantly af
-
fected by neither EP1, EP3, nor EP4 antagonists (data not
shown). At present, it remains unknown whether or not this
effect of PGE
2
is physiological action, yet there is a possibility
that PGE
2
exhibits a protective effect against the I/R-induced
gastric lesions, probably through EP2 receptors. Hoshino et
al. (2003) reported that PGE
2
inhibited the irritant-induced
apoptosis via EP2/EP4 receptors and speculated that this
action is involved in the gastroprotective action of PGE
2
in
vivo conditions. However, we previously reported that nei-
ther specific EP2 nor EP4 agonists protected the stomach
against acidified ethanol or indomethacin in rats (Araki et
al., 2000; Suzuki et al., 2001). Furthermore, no evidence has
been reported the involvement of apoptotic changes in the
I/R-induced gastric lesions. On the other hand, it is known
that PGE
2
inhibits the neutrophil migration in the gastric
mucosa via EP2/EP4 receptors (Suzuki et al., 2001). Thus, it
is assumed that PGE
2
prevents I/R-induced gastric lesions
via inhibition of the neutrophil-related process but not apo-
ptosis, similar to PGI
2
.
It has been reported that gastric acid secretion is substan-
tially decreased after ischemia and remained reduced for
several hours even after reperfusion (Takeuchi et al., 1986;
Nakamoto et al., 1998). However, Kitano et al. (1997) showed
that cimetidine, the histamine H
2
receptor antagonist, had a
protective effect on I/R-induced gastric lesions through the
suppression of acid secretion. In a preliminary study, we
observed that lansoprazole, a proton pump inhibitor, also
significantly reduced the severity of I/R-induced gastric le-
sions in wild-type mice (data not shown), suggesting the
participation of gastric acid in the pathogenesis of these
lesions. Since iloprost at the highest dose (3
g/kg) caused a
slight decrease in acid secretion, consistent with the finding
by Seidler et al. (1989), it is possible that the protective effect
of this prostanoid, especially at high doses, on I/R-induced
gastric lesions is partly accounted for by its antisecretory
action.
Based on all the results of the present study, we confirmed
that I/R induced gastric lesions with the up-regulation of
COX-2 expression and that the damage was significantly
aggravated by indomethacin as well as the selective COX-2
inhibitor rofecoxib but not the selective COX-1 inhibitor SC-
560. We further showed for the first time that the I/R-induced
gastric lesions were significantly worsened in IP receptor
knockout mice but not in the animals lacking EP1 or EP3
receptors. Thus, it is assumed that endogenous PGs derived
from COX-2 play a crucial role in gastric mucosal defense
during I/R, and this action is mainly mediated by PGI
2
through the activation of IP receptors.
Acknowledgments
We thank Professor Shu Narumiya, Kyoto University Faculty
(Kyoto, Japan) of medicine for kindly supplying EP1, EP3, and IP
receptor knockout mice.
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2
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