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Am J Physiol Gastrointest Liver Physiol 294: G80-G87, 2008. First published October 18, 2007; doi:10.1152/ajpgi.00292.2007
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MUCOSAL BIOLOGY

Gastric mucosal protection against ethanol by EP2 and EP4 signaling through the inhibition of leukotriene C4 production

Youichiro Hattori,2,3 Takashi Ohno,4 Takako Ae,2,3 Takeo Saeki,2 Katsuharu Arai,2 Sumito Mizuguchi,4 Katsunori Saigenji,2 and Masataka Majima1,3

Departments of 1Pharmacology and 2Gastroenterology, Kitasato University School of Medicine; 3Department of Molecular Pharmacology, Kitasato University Graduate School of Medical Sciences, Sagamihara; and 4Department of Internal Medicine, Isehara Kyodo Hospital, Isehara, Kanagawa, Japan

Submitted 27 June 2007 ; accepted in final form 1 October 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prostaglandin (PG)E derivatives are widely used for treating gastric mucosal injury. PGE receptors are classified into four subtypes, EP1, EP2, EP3, and EP4. We have tested which EP receptor subtypes participate in gastric mucosal protection against ethanol-induced gastric mucosal injury and clarified the mechanisms of such protection. The gastric mucosa of anesthetized rats was perfused at 2 ml/min with physiological saline, agonists for EP1, EP2, EP3, and EP4, or 50% ethanol, using a constant-rate pump connected to a cannula placed in the esophagus. The gastric microcirculation of the mucosal base of anesthetized rats was observed by transillumination through a window made by removal of the adventitia and muscularis externa. PGE2 and subtype-specific EP agonists were applied to the muscularis mucosae at the window. Application of 50% ethanol dilated the mucosal arterioles and constricted the collecting venules. Collecting venule constriction by ethanol was completely inhibited by PGE2 and by EP2 and EP4 agonists (100 nM) but not by an EP1 or an EP3 agonist. Ethanol-induced mucosal injury was also inhibited by EP2 and EP4 agonists. When leukotriene (LT)C4 levels in the perfusate of the gastric mucosa were determined by ELISA, intragastric ethanol administration elevated the LTC4 levels sixfold from the basal levels. These elevated levels were significantly (60%) reduced by both EP2 and EP4 agonists but not by other EP agonists. Since LTC4 application at the window constricted collecting venules strongly, and an LTC antagonist reduced ethanol-induced mucosal injury, reductions in LTC4 generation in response to EP2 and EP4 receptor signaling may be relevant to the protective action of PGE2. The present results indicate that EP2 and EP4 receptor signaling inhibits ethanol-induced gastric mucosal injury through cancellation of collecting venule constriction by reducing LTC4 production.

prostaglandin E2; EP receptor; gastric microcirculation; gastric perfusion

THE STOMACHS WERE FREQUENTLY exposed to some substances including drugs, which cause dyspepsia or mucosal injury. Alcohol is the most common chemical that was taken by numerous people. The effects of alcohol on human gastric mucosa were studied extensively in the patients, and high concentrations of ethanol penetrate the deep vascular plexus to impact stasis, disruption of blood vessel walls, deep mucosal hemorrhage, and necrosis (13, 36, 44). E series prostaglandins (PGs) were widely used for mucosal protection against harmful substances and endogenous stimuli such as protons and pepsin (7, 10, 36, 37). E series prostaglandins such as PGE2 increase gastric mucin generation and suppression of motor action, and these activities are quite effective in the prevention of gastric mucosal injury (36). PGE2 is also indispensable to the maintenance of gastric mucosal microcirculatory integrity, which is a major determinant of protective action. We previously reported that gastric mucosal exposure to ethanol caused mucosal damage as a result of congestion of the gastric mucosal microcirculation. Congestion of mucosal blood flow, if persistent, induced intense hypoxia of the gastric mucosa, and as a result, the epithelium of the mucosa was damaged (32, 33). A prostaglandin I2 (PGI2) analog, beraprost sodium, inhibited ethanol-induced gastric mucosal injury through the increased release of a neuropeptide, calcitonin gene-related peptide (CGRP) (39). This was due to the action of CGRP in blocking ethanol-induced constriction of collecting venules and other venules. PGE2 exhibited the same protective action against ethanol, but this PGE2-dependent protection was not mediated by CGRP, since administration of a CGRP antagonist, CGRP-(8–37) did not block PGE2-dependent protection. These observations indicated that PGE2 exhibited protective action independent of CGRP. PGs exert their biological actions by binding to specific receptors that contain seven transmembrane domains. PGE receptors have been defined pharmacologically and cloned, including four subtypes of PGE receptor (EP1, EP2, EP3, and EP4) (30). Genes for each of these receptors have been disrupted, and the corresponding knockout mice have been produced (1, 19, 22, 25, 26, 28, 40, 45). Furthermore, with the use of the cloned receptors, agonists and antagonists highly selective for each of the four EP subtypes have been or are in the process of being developed (41, 47). It is unlikely that any previous pharmacologic tools would neatly prevent clinical gastric mucosal lesions due to ethanol. Thus some effective drugs will be expected. In the present study, we have now identified the EP receptors responsible for the protection of gastric microcirculatory dysfunction with the use of selective compounds for EP receptor subtypes. Responsible EP agonists will be promising agents to prevent ethanol-induced gastric mucosal injury. Our results further clarified that the mechanisms of the protective actions of EP receptor signaling against the ethanol were leukotriene (LT)C4 dependent and suggest that the agents acting on EP2 and EP4 will be therapeutically active against gastric microcircular dysfunction.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Intravital Microscopy

Observation of rat gastric microcirculation. Male Sprague-Dawley rats (specific pathogen free, 250–450 g) obtained from Japan SLC (Hamamatsu, Japan) were housed at constant room temperature (25 ± 1°C) and humidity (60 ± 5%) with a 12-h light-dark cycle. Rats were deprived of food 18–24 h before the start of experimentation but had free access to water. The mucosal microcirculation of rats anesthetized with urethane (Aldrich, Milwaukee, WI) (0.875 g/kg ip) was observed through a modification improving on the method originally reported by Rosenberg and Guth (14, 23, 32, 33, 38).

Surgical preparation for gastric microcirculation experiments. After laparotomy with an electric cautery scalpel (model B-365; Takahashi Shoten, Tokyo, Japan), the greater curvature of the stomach was incised longitudinally leaving the greater omentum attached to the posterior wall while the anterior wall was resected. The dorsal side of the glandular stomach was secured in a plastic perfusion chamber, with the mucosal side facing the interior of the chamber, and was perfused with Tyrode's solution at 37°C (Fig. 1). A small part of the serosa, together with the underlying muscularis externa layers and the submucosa, was carefully dissected away using microsurgical scissors under a stereomicroscope to make an observation window (Fig. 1). This method made possible direct observation, with transillumination through the muscularis mucosae, of the microcirculation of the basal part of the gastric mucosa. Rectal temperature was continuously monitored with an electric thermometer (Thermistor model MGA-3, type 219; Nihon Kohden, Tokyo, Japan) and was maintained at 37–38°C with a heating lamp. Systolic blood pressure was monitored with a pressure transducer (model TP200-T, Nihon Kohden) through a PE-50 tube inserted into the left femoral artery. Rats with systolic blood pressure <90 mmHg or with a body temperature >37°C were discarded. All experimental procedures were approved by the Institutional Animal Care and Use Committee and were performed according to the Guidelines for Animal Experimentation of Kitasato University School of Medicine. The microcirculation of the basal part of mucosa was examined with a light microscope (Optiphot-2; Nikon, Tokyo, Japan) with an objective lens with a long working distance (M plan 10/0.21 SLWD, Nikon). Images of the microcirculation were transmitted through a monochrome TV camera (C-2400 and C2400-7; Hamamatsu Photonics, Hamamatsu, Japan) to a TV monitor screen (model TM 1550; Ikegami Tsushinki, Tokyo Japan) and recorded with a DVD recorder (model DV-HR450; Sharp, Tokyo, Japan). One arteriole, one venule, and two collecting venules were selected in one observation window, and their internal diameters were measured using an adjustable electronic microscaler (Argus-10, Hamamatsu Photonics). Arteriole diameters were expressed as percentages of the original diameter.


Figure 1
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  		Fig. 1. Schema for the experimental setup to observe the microcirculatory dysfunction elicited by ethanol. A small part of the serosa, together with the underlying muscularis externa layers and the submucosa, was dissected away under the stereomicroscope, as described in MATERIALS AND METHODS. A small part of the serosa, together with the underlying muscularis externa layers and the submucosa, was carefully dissected away using microsurgical scissors under a stereomicroscope to make an observation window. Ethanol solution was given to the mucosa, and prostaglandin E (PGE)2 and EP agonists were administered at the window. EtOH, ethanol.

 
Drug application. Ethanol and PGE2 were obtained from Wako Pure Chemicals, Osaka, Japan. EP1 receptor agonist (ONO-DI-004), EP2 receptor agonist (ONO-AE1-259-01), EP3 receptor agonist (ONO-AE-248), and EP4 receptor agonist (ONO-AE1-329) were gifts from ONO Pharmaceutical, Osaka, Japan (47). Ethanol (50%, 1 ml) was placed between the chamber and the gastric mucosa. PGE2, EP1 receptor agonist (ONO-DI-004), EP2 receptor agonist (ONO-AE1-259-01), EP3 receptor agonist (ONO-AE-248), and EP4 receptor agonist (ONO-AE1-329) were applied to the exposed outer surface of the muscularis mucosae at the window (Fig. 1).

Experiments on Perfusion of the Gastric Lumen

Perfusion of rat gastric mucosa. Male Sprague-Dawley strain rats (specific pathogen free, Japan SLC), weighing 250–400 g, were starved for 18–24 h before the experiments began but had free access to water. The experiments were performed on animals anesthetized with urethane (0.875 g/kg, by intraperitoneal injection; Aldrich Chemical).

Surgical preparation for stomach perfusion experiments. After laparotomy of the anesthetized rats, the stomach was doubly cannulated from the esophageal and duodenal ends. The cannulas were secured with thread at the middle of the esophagus and at the pylorus, respectively. Physiological saline (37°C) was perfused at 2 ml/min, using a constant-rate pump (model 11; Harvard Apparatus, Harvard, MA) connected to the esophageal cannula, and was collected from the duodenal cannula. Before the collection of the first sample, to stabilize the stomach, it was perfused with the solution mentioned above for more than 60 min (42). Body temperature was monitored with a thermometer (model CTM-303; Terumo, Tokyo, Japan) and was maintained throughout at 38°C ± 1°C with a desk lamp and a heated table. The passage for air was kept patent by insertion of a cannula (PE-205; Clay Adams, Parsippany, NJ) into the trachea.

Experimental procedure for the perfusion experiments. All solutions for perfusion of the gastric mucosa, comprising solutions of 50% ethanol, PGE2, EP1 receptor agonist (ONO-DI-004), EP2 receptor agonist (ONO-AE1-259-01), EP3 receptor agonist (ONO-AE-248), and EP4 receptor agonist (ONO-AE1-329) were applied to the exposed outer surface of the muscularis mucosae at the window. The following perfusion experiments were performed.

Perfusion of the stomach with 50% ethanol alone. The stomach was perfused at the rate of 2 ml/min with physiological saline. Four milliliters of intragastric perfusate were collected in 2 min and were placed directly into a plastic tube kept on ice. After five consecutive samplings, the perfusion solution was replaced with 50% ethanol (Kanto Chemical, Tokyo, Japan) prepared with distilled water and left for 2 min. We previously reported that 50% ethanol immediately induced mucosal lesions after the exposure of the mucosa (17). Therefore, in the present experiment, we exposed the gastric mucosa to ethanol for 2 min. Then, the perfusate was replaced with physiological saline.

Pre-exposure of the gastric mucosa to physiological saline containing PGE2, EP1, EP2, EP3 or EP4 agonist before perfusion of the mucosa with 50% ethanol. After perfusion for 10 min with physiological saline containing PGE2 (10 µg/ml) or EP receptor agonist (10 µg/ml), 50% ethanol solution was perfused for 2 min. Then the stomach was perfused again with physiological saline. Four milliliters of the perfusate were collected repeatedly at 2-min intervals and were placed directly into a plastic tube kept on ice.

Measurement of intragastric LTC4 levels. The levels of LTC4 in the perfusate from anesthetized rats were measured as described by us previously. Briefly, the perfusate for every 2 min was collected directly in ice-cold absolute ethanol, and after overnight centrifugation at 3,000 g at 4°C, the supernatant was evaporated at reduced pressure. The residue was applied to a Sep-Pack C18, and the resulting fractions for LTC4 were determined by enzyme immunoassay (EIA) (Cayman Chemical, Ann Arbor, MI 48108).

Assessment of gross lesions in the glandular stomach. At the end of each perfusion experiment, the stomach was excised, and the reddened areas were calculated as percentages of the glandular stomach area using Adobe Photo Shop 4.0J. software on a Macintosh computer.

Effects of an LT antagonist on ethanol-induced gastric mucosal injury. An LT antagonist (ONO-1078, 0.03–0.3 mg/kg ip) was given to rats 1 h before ethanol administration, and the area of 50% ethanol-induced gastric mucosal injury was determined as described above.

Immunohistochemistry of 5-Lipoxygenase

The stomachs were immediately fixed with 4% paraformaldehyde in 0.1 M phosphate buffer solution (pH 7.4). After fixation, the stomach tissues were dehydrated with a graded series of ethanol solution, and then embedded in paraffin. The sections (4 µm) from the paraffin-embedded tissues were mounted on glass slides, deparaffinized with xylene, and then placed in cold (4°C) acetone for immunostaining.

The procedure for staining dehydrated sections using a Vectastain ABC Kit (Vector Laboratory, Burlingame, CA) was as follows: 1) incubation with diluted normal horse serum, 2) incubation with diluted (X500) 5-lipoxygenase polyclonal antibody (a gift from Prof. Ueda, Kagawa University, Kagawa, Japan), 3) incubation with biotinylated anti-IgG, 4) incubation with avidin-biotin-peroxidase complex, 5) placement in 0.02% 3,3'-diaminobenzine (DAB) and 0.3% nickel ammonium sulfate in 50 mM Tris·HCl buffer (pH 7.4), 6) color development by immersion in DAB solution containing 0.005% H2O2, and 7) examination and photomicrography with a light microscope.

Statistics

All values in the figures are expressed as means ± SE of n observations. Statistical comparisons for multiple groups were made using one-way ANOVA with a posthoc Scheffé's test. Values of P < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of PGE2 administered at the window on gastric mucosal microcirculation. Figure 2 summarizes the changes in diameter of arterioles 4 min after the administration of PGE2. Application of PGE2 induces concentration-dependent dilatation of arterioles in the basal part of the mucosal microcirculation. PGE2 at concentrations of more than 1 µM/l dilated the arterioles significantly. By contrast, collecting venules and other venules were not dilated but were a little constricted at high concentrations.


Figure 2
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  		Fig. 2. Effects of PGE2 on the diameters of arterioles, venules and collecting (coll.) venules in the basal part of the mucosal microcirculation. PGE2 was administered onto the muscularis mucosae exposed at the window. The values are expressed as means ± SE from 6 rats. ANOVA was used for statistical analysis. *P < 0.05, **P < 0.01; vs. saline.

 
Effects of EP-selective agonists administered at the window on gastric mucosal microcirculation. We examined changes of the diameters of microvessels in the basal part of the mucosal microcirculation when each EP receptor agonist alone was applied to the muscularis mucosae at the window. When EP1 agonist or EP3 agonist was added, the arterioles were not dilated, but EP2 agonist dilated them significantly. EP4 agonist at a high dose (1 mM) dilated arterioles (Fig. 3A). In the collecting venules, no agonist affected the diameters of the microvessels significantly (Fig. 3B). However, EP3 agonist caused slight but significant constriction at a high concentration (100 µM). In the venules, there were few changes in the diameters of microvessels with the application of each EP agonist (Fig. 3C).


Figure 3
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  		Fig. 3. Effects of selective EP agonists on the diameters of arterioles (A), collecting venules (B), and venules (C) in the basal part of the mucosal microcirculation. Selective EP agonists were administered at the window. The values are expressed as means ± SE from 6 rats. ANOVA was used for statistical analysis. *P < 0.05, **P < 0.01; vs. saline.

 
Effect of PGE2 applied at the window on the changes elicited by 50% ethanol in the diameter of gastric microvessels. We examined the actions of PGE2 (100 nM) on the changes elicited during mucosal application of 50% ethanol in the diameter of microvessels. When PGE2 was administered at the window before mucosal exposure to 50% ethanol, ethanol-induced rapid constrictions of the collecting venules and venules were completely inhibited (Figs. 4, B and C). On the other hand, the diameters of arterioles during ethanol exposure were not affected by preadministration of PGE2 (Fig. 4A, see data before ethanol administration). A concentration of 100 nM did not dilate arterioles under normal conditions. Further, even when we applied PGE2 to the muscularis mucosae at the window during ethanol administration, it did not further dilate the arterioles (Fig. 4A).


Figure 4
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  		Fig. 4. Effect of PGE2 on ethanol-induced changes in the diameters of arterioles (A), collecting venules (B), and venules (C). PGE2 was administered at the window prior to the mucosal application of 50% ethanol. Constriction of the collecting venules and venules were inhibited by PGE2. The values are expressed as means ± SE from 5 rats. ANOVA was used for statistical analysis. *P < 0.05, **P < 0.01; vs. 50% EtOH alone.

 
Effects of selective EP agonists administered at the window on changes in diameter of gastric microvessels elicited by 50% ethanol. Further, we tested specification of EP receptor subtypes relevant to gastric mucosal protection to show inhibition of constriction of collecting and other venules (Figs. 5, B and C). We selected a concentration of 100 nM, which did not dilate the arterioles in the normal gastric microcirculation (Fig. 3A), since our preliminary experiments using different concentrations of PGE2 or EP agonists (EP2 and EP4) described below indicated that 100 nM was enough to show the effects. The dilatation of arterioles elicited by 50% ethanol exposure increased the original diameter by about 100%, and this was not affected by prior administration of any EP selective agonist (Fig. 5A). On the other hand, the intense constrictions of the collecting venules and venules were completely blocked by either EP2 or EP4 agonists (Fig. 5, B and C). Administration of an EP1 agonist or an EP3 agonist did not inhibit the ethanol-induced constriction of venules and collecting venules (Fig. 5, B and C).


Figure 5
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  		Fig. 5. Effect of selective EP agonists on ethanol-induced changes in the diameters of arterioles (A), collecting venules (B), and venules (C). Selective EP agonists were administered at the window prior to the mucosal application of 50% ethanol. Constriction of the collecting venules and venules were inhibited by an EP2 agonist and an EP4 agonist. The values are expressed as means ± SE from 5 rats. ANOVA was used for statistical analysis. *P < 0.05, **P < 0.01; vs. 50% EtOH alone.

 
Effects of PGE2, EP1, EP2, EP3, and EP4 agonists on 50% ethanol-induced mucosal injury. In rats, exposure of the gastric mucosa to 50% ethanol resulted in gastric mucosal lesions with a total reddened area covering 22.0 ± 0.645% (n = 6) of the area of the glandular stomach (Fig. 6). Prior intragastric administration of PGE2 (100 nM) significantly reduced the size of the mucosal lesions (7.83 ± 0.743%; n = 6) induced by mucosal exposure of 50% ethanol. Prior administration of EP2 agonist (7.77 ± 0.909%; n = 6) or EP4 agonist (7.62 ± 0.721%; n = 6) inhibited gastric mucosal lesions to the same degree as PGE2 (Fig. 6). However, the administration of EP1 agonist (22.0 ± 1.65%; n = 6) or EP3 agonist (21.0 ± 1.805%; n = 6) did not reduce the mucosal injury (Fig. 6).


Figure 6
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  		Fig. 6. Effects of intragastric perfusion of PGE2 and selective EP agonists on gastric lesions induced by 50% ethanol. The area of reddened lesions after 50% ethanol perfusion was determined during continuous infusion of physiological saline, PGE2, or selective EP agonists. The values are expressed as means ± SE from 6 rats. ANOVA was used for statistical analysis. *P < 0.01; vs. 50% EtOH alone. NS, not significant.

 
Effects of PGE2 and EP agonists on the intragastric levels of LTC4. Intragastric LTC4 levels during perfusion of physiological saline were kept fairly constant; however, the exposure of the gastric mucosa to 50% ethanol after the perfusion of physiological saline immediately increased these levels by six times from the basal levels (Fig. 7A). As Fig. 6B shows, preadministration of PGE2 markedly inhibited the increase in LTC4 levels elicited by 50% ethanol. Similarly, the intragastric pre-exposure to an EP2 agonist and an EP4 agonist reduced the release of LTC4 levels elicited by 50% ethanol. By contrast, an EP1 agonist and an EP3 agonist did not significantly inhibit the release of LTC4.


Figure 7
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  		Fig. 7. Changes in intragastric LTC4 levels during 50% ethanol perfusion (A) and effects of intragastric perfusion of PGE2 and selective EP agonists on gastric LTC4 levels before and after 50% ethanol perfusion (B). After perfusion of PGE2 or selective EP agonists, leukotriene (LT)C released for 6 min after the exposure of 50% ethanol was determined with a specific ELISA. The values are expressed as means ± SE from 6 rats. ANOVA was used for statistical analysis. **P < 0.01; vs. after application of 50% EtOH after physiological saline perfusion.

 
Effects of LTC4 on gastric microcirculation. To evaluate the roles of LTC4 released by 50% ethanol, we tested first the effects of LTC4 applied to the muscularis mucosae at the window on the gastric microcirculation. As shown in Fig. 8, LTC4 constricted collecting venules and venules in a concentration-dependent manner. These changes in gastric microcirculation were abolished with the simultaneous administration of LTC antagonist, ONO1078.


Figure 8
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  		Fig. 8. Effects of LTC4 on the diameter of arterioles, collecting venules, and venules in the basal part of the gastric mucosal microcirculation (A) and effects of simultaneous administration of ONO-1078 (B). LTC4 and ONO-1078 were applied to the exposed muscularis mucosae via the window. Data are expressed as means ± SE from 6 rats. ANOVA was used for the statistical analysis. ***P < 0.001. LTC4, leukotriene C4; ONO, ONO-1078.

 
Immunohistochemical localization of 5-lipoxygenase in rat gastric mucosa. Immunohistochemical studies reveal that immunoreactive 5-lipoxygenase positive cells were localized in the interstitial spaces of the gastric mucosa (Fig. 8, A and B). These positive cells were strongly stained with toluidine blue (data not shown), suggesting that these cells may be mast cells.

Effects of LT antagonist and 5-lipoxygenase inhibitor on ethanol-induced gastric mucosal injury. To observe the function of LTC4 released by ethanol, we administered an LT antagonist, ONO1078, during ethanol administration. ONO1078 inhibited ethanol-induced gastric mucosal injury significantly in a dose-dependent manner (Fig. 8C). When a sufficient dose of the 5-lipoxygenase inhibitor AA861 was given, the same magnitude of inhibition was observed as that seen with the highest dose of ONO1078 (Fig. 8C).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Disturbances in the gastric mucosal microcirculation are thought to be an important cause of injury, and observation of the microcirculation is therefore important for revealing the pathophysiology of mucosal injury (32, 33). As described above, 50% ethanol applied to the gastric mucosa induces mucosal congestion as a result of constriction of the collecting venules and other venules. We have previously reported that capsaicin applied to the mucosa prevented gastric lesions (2, 8). The importance of the cancellation of constriction of the collecting and other venules by capsaicin was confirmed by the action of CGRP, a major neuropeptide released by capsaicin. PGE2, when administered exogenously, inhibits gastric mucosal injury, but this action was not inhibited by a CGRP antagonist, although the inhibition due to a PGI2 analog was inhibited by CGRP antagonist (2, 8).

EP1–4 receptor signaling may be involved in the protective actions of PGE2. As shown here, EP2 agonist, together with EP4 agonist, neatly suppressed the constriction of collecting and other venules resulting in a reduced area of injury in the gastric mucosa. The activity of EP2/4 agonists may be not CGRP-dependent, as mentioned above. Arteriole dilatation was induced with the use of a high dose of PGE2 or an EP2 agonist. In terms of arteriole-dilating activity, an EP2 agonist was potent, although EP4 signaling did not dilate them, suggesting that the dilatation of arterioles was not a critical determinant for the protective action of EP2/4 signaling. EP4 signaling that did not dilate arterioles may be attributable to other processes that affect nonvascular tissues. It is also important for the protective action of EP2 signaling to take place at concentrations that do not affect vascular components.

EP receptors involved in the indirect protection of the gastric mucosa by PGE2 have been revealed to have the following actions: inhibition of gastric motility and stimulation of bicarbonate secretion by PGE2 are mediated by the EP1/EP3 receptors (43, 46); the stimulation of mucin production by PGE2 is mediated by EP4 receptors (15); the increase in gastric cytoprotection by PGE2 is mediated by EP3 receptors (3); and inhibition of acid secretion by PGE2 is mediated by EP2/EP3 receptors (29, 48). However, at the present we have had no concrete evidence as to the role of EP receptors in terms of the prevention of congestion of the gastric mucosa by PGE2. The present results clarified the novel mechanisms of the preventive actions of PGE2 on the microcircular dysfunction in ethanol-induced gastric mucosal injury.

There is not much evidence that an EP2/4 expressing cellular component was present in the gastric mucosa. The expression of EP receptors in the gastrointestinal tract of the mouse has been examined by in situ hybridization studies where it was shown that the expression of EP4 mRNA could be detected in gastric mucosal cells; however, expression of EP2 mRNA was not detected in any types of cells from the stomach by this method (27), although it was recently reported (21) that EP2 is expressed in the stomach of the mouse, using RNase protection assay. Direct protective actions, such as inhibition of apoptosis of PGE2 have been reported (20), but, as shown here, the indirect actions of PGE2 were protective of nonvasculature elements.

It was previously reported that LTC4 was a major mediator of ethanol-induced mucosal injury (6, 18, 23, 34, 35, 49). The activity of LTC4 in inducing CV constriction was confirmed in the present experiment, suggesting that LTC4 was a critical factor for ethanol-induced mucosal injury. In fact, an antagonist for LTC inhibited the injury elicited by ethanol in a dose-dependent manner (Fig. 8). The source of LTC4 was reported to be the mast cells (12, 24). It was frequently mentioned that there is a heterogeneity among mast cells (4). In terms of generation of the arachidonate metabolites, the mucosal type mast cells generate LTC4, whereas those from connective tissues do PGD2 (11). The gastric mucosal mast cells may be the source of LTC4 that constricts mucosal collecting and other venules. The mucosal localization of the mast cells expressing 5-lipoxygenase corresponded well with the locations of microcirculation, which can be protected with an antagonist for LT, judging from the results of the present immunohistochemical study (Fig. 9). Further, we clarified here that the mast cells may be regulated by EP2/4 signaling (Fig. 7), suggesting that the site of action of EP2/4 signaling is the mucosal mast cells that express 5-lipoxygenase. The presence of EP2/4 receptors on the mucosal mast cells was reported previously (31). EP2/4 signaling links to the adenylate cyclase activation (9, 30), and it was reported that the elevation in cAMP levels stabilizes the mast cell activation (16). The released LTC4 was active in induction of gastric mucosal injury, since ONO-1078, which antagonized LTC4, inhibited ethanol-induced gastric mucosal injury. The same was true of the 5-lipoxygenase inhibitor AA861.


Figure 9
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  		Fig. 9. Immunohistochemical (IH) staining of 5-lipoxygenase in the gastric mucosa (A and B) and effects of ONO-1078 and AA-861 on ethanol-induced gastric mucosal lesions (C). Data are expressed as means ± SE from 6 rats. ANOVA was used for the statistical analysis. *P < 0.05; ***P < 0.001.

 
In summary, the administration of an EP2 agonist and an EP4 agonist completely inhibited ethanol-induced constriction of collecting venules and other venules in our microcirculation experiments. Arterioles were not markedly dilated by these agonists at 100 nM, a concentration at which the agonists can protect the gastric mucosa from 50% ethanol. The elevated LTC4 levels elicited by ethanol were significantly reduced with an EP2 agonist or an EP4 agonist, whereas neither an EP1 agonist nor an EP3 agonist reduced the LTC4 levels. Also, LTC4 released by ethanol has a significant role in mucosal injury. These results suggest that EP2 and EP4 agonists inhibit ethanol-induced gastric mucosal injury through the inhibition of gastric generation of LTC4, and that an EP2 agonist and an EP4 agonist are promising agents for treating gastric mucosal injury.


   ACKNOWLEDGMENTS
 
We thank Michiko Ogino, Keiko Nakamigawa, and Osamu Katsumata for their technical assistance and C.W.P Reynolds for correcting the English manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Masataka Majima, Dept. of Pharmacology, Kitasato Univ. School of Medicine, Sagamihara, Kanagawa 228-8555, Japan (e-mail: mmajima{at}med.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. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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