It is widely accepted that the inhibition of gastric motor activity as well as the maintenance of gastric mucosal blood flow and mucous secretion are important for the homeostasis of the gastric mucosa. The present study was performed to ascertain whether or not endogenous PGs, which can protect the stomach from noxious stimuli, affect gastric motor activity and emptying. The myoelectrical activity of rat gastric smooth muscle was increased at intragastric pressures of over 2 cmH2O. Replacement of intragastric physiological saline with 1 M NaCl solution significantly increased PGI2 and PGE2 in stomach and suppressed the myoelectrical activity under a pressure of 2 cmH2O by 70%. Indomethacin inhibited the suppression of myoelectrical activity by 1 M NaCl. The myoelectrical activity under a pressure of 2 cmH2O was suppressed by continuous infusion of a selective EP1 agonist (ONO-DI-004, 3–100 nmol·kg−1·min−1) into the gastric artery in a dose-dependent manner, but not by that of the PGI receptor agonist beraprost sodium (100 nmol·kg−1·min−1). Suppression of myoelectrical activity with 1 M NaCl was inhibited by continuous infusion of a selective EP1 antagonist (ONO-8711, 100 nmol·kg−1·min−1) into the gastric artery. Furthermore, gastric emptying was tested in EP1 knockout mice and their wild-type counterparts. Gastric emptying was strongly suppressed with intragastric 1 M NaCl in wild-type mice, but this 1 M NaCl-induced suppression was not seen in EP1 knockout mice. These results suggest that PGE2-EP1 signaling has crucial roles in suppression of myoelectrical activity of gastric smooth muscles and inhibition of gastric emptying and that EP1 is an obvious target for drugs that control gastric emptying.
- myoelectrical activity
- gastric emptying
during digestion, stomach is continuously exposed to various harmful substances such as gastric acid and pepsin. Despite exposure of such harmful substances, stomach is certainly protected by maintaining gastric mucosal blood flow and/or increasing the generation of mucin (15). Endogenous PGs are widely known to exhibit gastric protective actions against harmful substances (9). PGs including PGE2 and PGI2 constitute a group of oxygenated metabolites of arachidonic acid that are produced by the sequential actions of phospholipase A2, cyclooxygenase (COX), and specific PG synthases. PGE2 and PGI2 are the major PGs generated in the stomach (4). We had reported previously that PGs could protect gastric mucosa from ethanol through the inhibition of microcirculatory dysfunctions (37). When gastric mucosa was exposed to capsaicin or high-osmolarity solution, such as 1 M NaCl, both of which can increase PGE2 and PGI2 in the stomach, PGI2-PGI receptor (IP) signaling exhibits protective actions through the release of a protective neuropeptide, calcitonin gene-related peptide (CGRP) (4, 2). However, the protective action of PGE2 was CGRP-independent (4, 37).
It is widely accepted that the inhibition of gastric motor activity, as well as the maintenance of gastric mucosal blood flow, is important for the homeostasis of the gastric mucosa. Furthermore, the importance of the regulation of gastric motor activity in physiological conditions has long been recognized. Increasing concentrations of nutrients, salts, or acidity in liquid meals generally prolong the emptying time of the stomach (23). The half-time for stomach emptying can be also prolonged by increasing the osmolality of such liquid meals (34). The mechanism of this prolongation of emptying has not been fully clarified. However, we had previously reported that endogenous PGE2, generated by mild hyperosmolality, suppressed the myoelectrical activity of the gastric smooth muscle as a parameter of gastric motor activity, suggesting that PGs may be a regulator of gastric motor function (29).
PGs exert their biological actions by binding to specific receptors that contain seven transmembrane domains. Eight different PG receptors have been defined pharmacologically and cloned, including the PGD receptor, four subtypes of PGE receptor (EP1, EP2, EP3, EP4), the PGF receptor, IP, and the thromboxane receptor (28). Genes for each of these receptors have been disrupted, and the corresponding knockout mice have been produced (1, 13, 17, 20, 22, 27, 42, 46). Furthermore, with the use of the cloned receptors, agonists and antagonists highly selective for each of the four prostaglandin E receptor (EP) subtypes have been or are in the process of being developed (13, 43, 48).
In the present study, we have now identified the PG species responsible for high osmolarity-induced suppression of the myoelectrical activity of the gastric smooth muscle in the antrum as a parameter of gastric motor activity with the use of these selective compounds and PG receptor knockout mice. Our results indicate that gastric PGE2-EP1 signaling appears critical for controlling gastric motor activity and suggest that agents acting on EP1 signaling will become novel therapeutic tools for gastric function.
MATERIALS AND METHODS
Measurement of myoelectrical activity.
Male Sprague-Dawley strain rats (specific pathogen free; Japan SLC, Hamamatsu, Japan) weighing 400–500 g were starved of food for 24 h before the experiments but had free access to water. The studies were carried out in accordance with the Guidelines for the Treatment of Experimental Animals of Kitasato University School of Medicine. The study protocol was approved by the Animal Care and Use Committee at the Kitasato University School of Medicine (2078, 2009-125).
A laparotomy was performed under urethane anesthesia (0.875 g/kg ip), and the stomach was cannulated with two catheters, one from each end (esophageal and duodenal; polyethylene tubes of 2 and 4 mm in external diameter, respectively), as described in a previous report (44). The intragastric pressure was monitored from the side arm of the cannula inserted into the duodenum, and physiological saline (300 mosmol/kg) was injected into the stomach through the esophageal catheter to maintain the intragastric pressure at a fixed value. A fixed intragastric pressure was maintained by closing both catheters for 10 min. Body temperature was maintained at 36.5–37.5°C by means of a heating lamp and a warm plate during the experiments.A bipolar electrode (TF201-076, Unique Medical, Tokyo, Japan), newly made as an improvement on the original bipolar electrode (24, 25
Sixty minutes were allowed before the start of the experiments to avoid the influence of any artificial changes caused by touching the gastric wall (44). After the collection of physiological saline, 1 M NaCl solution (2,000 mosmol/kg) was placed in the stomach for 10 min, and the intragastric solutions were then replaced with physiological saline. This procedure can mimic the increased osmolarity during intake of nutrients in daily meals.
Measurement of intragastric PGE2 and 6-keto-PGF1α levels.
The levels of PGE2 and 6-keto-PGF1α in the perfusate from anesthetized mice and rats were measured as reported previously (4). Briefly, the perfusate for every 4 min was collected directly in ice-cold absolute ethanol, and after centrifugation at 6,000 g for 15 min at 4°C the supernatant was evaporated at a reduced pressure. The residue was applied to a Sep-Pack C18 and HPLC, and the resulting fractions for PGE2 and 6-keto-PGF1α were determined by specific ELISAs (Cayman Chemical, Ann Arbor, MI), as reported previously (2, 4).
Continuous infusion of PGE2, PGI2, and EP-selective agonists to rats.
A polyethylene cannula (PE10) was inserted into the splenic artery of an anesthetized rat (25), and physiological saline containing PGE2 and a PGI2 analog, beraprost sodium (a gift from Toray, Tokyo, Japan), was retrogradely infused (∼1–100 nmol·kg−1·min−1) into the gastric artery 10 min before the first sample (physiological saline) collection. During the experimental period, the stomach was kept full of physiological saline, and the intragastric pressure was maintained at 2 cmH2O. The myoelectrical activity of the gastric smooth muscle was recorded during every 10 min.
In a separate experiment, physiological saline containing EP receptor-selective agonists, ONO-DI-004, ONO-AEI-257, ONO-AE-248, and ONO-AEI-329, which were developed by us previously (48) and were agonists for EP1, EP2, EP3, and EP4, respectively, were retrogradely infused (∼1–1,000 nmol·kg−1·min−1) into the gastric artery. The myoelectrical activity of the gastric smooth muscle under 2 cmH2O of the intragastric pressure was determined over periods of 10 min. The first 10-min results of the myoelectrical activity were recorded to assess the effects of EP agonists.
To test the effect of blockade of EP1 signaling on the gastric myoelectrical activity during 1 M NaCl intragastric administration, an EP1 antagonist, ONO-8711 (33), was retrogradely infused (100 nmol·kg−1·min−1) into the gastric artery from 10 min before the intragastric application of 1 M NaCl throughout the experimental period.
Transcripts encoding COX-1, COX-2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined by RT-PCR analysis, as described previously (17). The amplification protocol comprised 28 cycles (COX-1, COX-2) and 23 cycles (GAPDH) of 45 s at 94°C, 60 s at 55°C, and 60 s at 72°C. The reaction mixtures were subsequently applied to a 2% agarose gel and the amplified products were stained with ethidium bromide. Primers used were as follows: 5′-TCCCTGAGATCTGGACCTGGC-3′ (sense) and 5′-TGAGTACTTCTCGGATGAAGG-3′ (antisense) for COX-1, 5′-TGGGTGTGAAGGGAAATAAGG-3′ (sense) and 5′-CATCATATTTGAGCCTTGGGG-3′ (antisense) for COX-2, and 5′-CCCTTCATTGACCTCAACTACAATGGT-3′ (sense) and 5′-GAGGGGCCATCCACAGTCTTCTG-3′ (antisense) for GAPDH. The glandular stomachs were isolated from nontreated wild-type (WT) mice and EP1−/− mice and were rapidly frozen in liquid nitrogen. The frozen tissues were pulverized, and total RNA was extracted from the pulverized tissue with Isogen. The glandular stomachs were also isolated from WT mice and EP1−/− mice 20 min after the ingestion of 1 M NaCl by gavages.
Determination of gastric emptying.
Male C57BL/6 WT mice and EP1 knockout (EP1KO) mice weighing 20–30 g were starved of food for 12 h before the experiments but had free access to water. The experiments were performed on the animals after they had been anesthetized with urethane by intraperitoneal injection (1.225 g/kg, Aldrich Chemical, Milwaukee, WI) (4). The studies were carried out in accordance with the Guidelines for the Treatment of Experimental Animals of Kitasato University School of Medicine.
After laparotomy of the anesthetized mice, the stomach was doubly cannulated from the esophageal and duodenal ends. The cannulae were secured with thread at the middle of the esophagus and at the pylorus, respectively. Physiological saline or 1 M NaCl solution (37°C) containing 50 mg/ml BSA prelabeled with 99mTc was applied to the stomach through the esophageal cannula, and the movement of 99mTc-labeled BSA from stomach to duodenum was determined with a small semiconductor gamma camera (Acrorad, Tokyo, Japan) (45). The radioactivity of the duodenum 15 min after application was expressed as a percentage of the initial radioactivity applied to the stomach.
Male Sprague-Dawley strain rats (specific pathogen free; Japan SLC) weighing 200 g were starved of food for 24 h before the experiments but had free access to water. Gastric emptying was determined in unanesthetized rats with use of phenol red, a nonabsorbable marker. Phenol red was dissolved in the physiological saline or 1 M NaCl solution at the concentration of 60 μg/ml. These solutions (3 ml/rat) were given to the unanesthetized rats by gavage, and the residual dye amounts in the stomach was determined from volume and dye concentration. Phenol red concentration was determined by spectrophotometrically by absorbances read at 550 nm. To test the effect of blockade of EP1 signaling on the gastric emptying, ONO-8711 was administered 30 min before the ingestion of test solutions (10 mg/kg ip). Indomethacin was also given to rats 30 min before the ingestion of test solutions (10 mg/kg ip).
In situ hybridization of EP1.
In situ hybridization was performed as described previously (26). Stomach sections (10 μm in thickness) were cut on a Jung Frigocut 3000E cryostat (Leica Instruments, Nussloch, Germany) and thaw-mounted onto poly-l-lysine-coated glass slides. The sections were fixed in 4% formalin in PBS for 10 min, rinsed in PBS, and acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine-0.9% NaCl for 10 min at room temperature. After dehydration sequentially in 70, 95, and 100% ethanol, the sections were air dried and stored at −80°C until use. An antisense cRNA probe for mouse EP1 was prepared as follows; a 1,312-base pair EcoRI fragment of MK643 (47) was subcloned into pBluescript II (Stratagene, La Jolla, CA) for synthesis of antisense and sense complementary RNA probes. Riboprobes were synthesized by transcription with T3 or T7 RNA polymerase (Stratagene, La Jolla, CA) in the presence of [α-35S] CTP. The probes had specific activities of ∼2 × 109 dpm/μg. Cold antisense riboprobes were also synthesized by the same procedure with unlabeled nucleotides. The specificity of the signal with each antisense probe was verified both by its disappearance with the addition of an excess of unlabeled probe and by the absence of specific hybridization with the sense probe (data not shown). Hybridization sections were dipped in nuclear track emulsion. After exposure for 5 wk at 4°C, the dipped slides were developed, fixed, and counterstained with hematoxylin and eosin.
Data are expressed as means ± SE. Comparisons among multiple groups were performed by factorial ANOVA followed by Scheffé's test. Comparisons between two groups were performed with Student's t-test. A P value of <0.05 was considered statistically significant.
Detection of myoelectrical activity and regulation by endogenous prostaglandins.
First, we tested the effects of intragastric pressure on myoelectrical activity. Myoelectrical activity of the gastric smooth muscle in the antrum was recorded both when the intragastric pressure was raised over 0.5 cmH2O and when it was maintained at 2 cmH2O (Fig. 1A). This myoelectrical activity was composed of a group of spikes of different amplitudes, the frequency of the burst discharge being five to eight times per minute. The voltage of the gastric myoelectrical activity was ∼200–2,000 μV. However, even if the pressure was increased to 4 or 6 cmH2O, the myoelectrical activity did not further increase (Fig. 1A). Thus, in the present study, we adopted the pressure of 2 cmH2O, which was a minimum pressure to induce the substantial electric activities, to see the effect of endogenous PGs. At the pressure of 2 cmH2O, we could constantly record myoelectrical activity for at least 2 h.
At the intragastric pressure of 2 cmH2O, when intragastric fluid was changed to 1 M NaCl solution, within 1 min, myoelectrical activity was markedly suppressed (Fig. 1B). As shown in Fig. 1C, when the first 10-min results were summarized and NaCl solutions of different concentrations were applied, the suppressive NaCl effects were concentration dependent. Application of 1 M NaCl suppressed myoelectrical activity by 70%, and the effect was significant over 0.5 M. Furthermore, prior systemic administration of indomethacin (10 mg/kg ip, 30 min before NaCl application) completely blocked NaCl effects (Fig. 1C). These results indicate that endogenous PGs can suppress myoelectrical activity elicited by mild distension.
Increase in endogenous prostaglandins by 1 M NaCl.
Next, we examined which kinds of endogenous PGs were involved in the suppression of myoelectrical activity. When we determined the released amounts of PGE2 (Fig. 2A), and PGI2 (Fig. 2B) from rat stomach, using ELISA, application of 1 M NaCl into the gastric cavity certainly increased both levels significantly. The levels in Fig. 2 were those determined at the first 10-min period after 1 M NaCl administration, suggesting that 1 M NaCl rapidly increased the levels of endogenous PGE2 and PGI2. Both PGs may be candidates to elicit the effect of 1 M NaCl.
The levels from WT C57/Bl6 mice before 1 M NaCl were 21.3 ± 1.9 pg·stomach−1·10 min−1 for PGI2 (6-keto-PGF1α) and 15.7 ± 2.3 pg·stomach−1·10 min−1 for PGE2. These were increased with 1 M NaCl to 94.5 ± 9.0 pg·stomach−1·10 min−1 for PGI2 (6-keto-PGF1α) (P < 0.05) and 40.9 ± 3.1 pg·stomach−1·10 min−1 for PGE2 (P < 0.05).
Effects of agonists for EP receptors and IP receptor on myoelectrical activity.
Next, we tested the effects of agonists for EP receptors and IP receptor continuously infused into the gastric artery on myoelectrical activity elicited by mild distension (Fig. 3A). Even though we administered quite a high dose of the PGI2 analog beraprost sodium, this compound did not suppress myoelectrical activity. By contrast, PGE2 suppressed it in a dose-dependent manner. These results suggest that PGE2 may be responsible for the suppression of myoelectrical activity.
Since there are four subtypes in EP receptors, to identify responsible receptor subtypes the four kinds of EP subtype-specific agonists were infused to the gastric artery (Fig. 3B). An EP2 agonist and an EP3 agonist infused into the gastric artery did not show any inhibitory effects. Although infusion of an EP4 agonist slightly suppressed myoelectrical activity, that of an EP1 agonist strongly inhibited it (Fig. 3B).
Effects of an EP1 antagonist on the suppression of myoelectrical activity by 1 M NaCl.
We further tested whether endogenous PGE2 increased by 1 M NaCl can inhibit myoelectric activity through the stimulation of EP1 receptor (Fig. 4). As shown in Fig. 4A, intragastric administration of 1 M NaCl certainly suppressed myoelectrical activity. After returning to physiological saline, suppression was restored gradually. The suppressive effect of 1 M NaCl was completely inhibited by continuous infusion of EP1 antagonist into the gastric artery (Fig. 4B). From these results, we can conclude that endogenous PGE2 generated by 1 M NaCl can suppress motor activity by stimulating an EP receptor, EP1.
Lack of 1 M NaCl-induced suppression of gastric emptying in EP1KO mice.
We further evaluated more physiological roles of PGE2-EP1 receptor signaling. Gastric emptying was examined in EP1KO mice and their WT counterparts receiving 1 M NaCl solution (Fig. 5). Anesthetized EP1KO mice and WT mice were cannulated with a catheter from esophagus, and 99mTc-labeled albumin solution was administered (Fig. 5A). We determined the movement of 99mTc-labeled albumin solution from stomach to duodenum using a small semiconductor gamma camera (Fig. 5B). Figure 5B depicts a typical result of gamma camera in WT mice. When we put physiological saline containing Tc-labeled albumin in the stomach, within 15 min nearly 50% of isotope was moved to the duodenum in WT mice. By contrast, the movement of 99mTc-labeled albumin resolved in 1 M NaCl solution from stomach to duodenum was markedly delayed in WT mice.
We performed the same experiments in EP1KO mice and compared with those in WT mice (Fig. 5C). When physiological saline was administered to EP1KO mice, the movement was rapid as in WT mice. There is no significant difference between the movements of 99mTc-labeled albumin resolved in physiological saline in WT mice and EP1KO mice (Fig. 5C). By contrast, the movement of 1 M NaCl solution, which was markedly suppressed in WT mice, was not suppressed in EP1KO mice (Fig. 5C). These results suggested that endogenous PGE2 generated by 1 M NaCl suppressed gastric emptying through the stimulation of EP1 and that EP1 signaling may provide sufficient time for food digestion.
Expressions of COX-1 and COX-2 in the stomachs in EP1−/− mice and WT mice.
When the expressions of COX-1 and COX-2 in the naive glandular stomachs were tested with RT-PCR, COX-1 was detected in both WT mice and EP1−/− mice, whereas COX-2 was not detected (Fig. 6). Even after the ingestions of 1 M NaCl, the same was true in both WT mice and EP1−/− mice.
Localization of EP1 receptors in mouse stomach.
Figure 7 depicts a typical result of EP1 in situ hybridization in glandular stomach in WT mice. Strong hybridization signals for EP1 mRNA were detected in a neuron located in the submucosal region (arrow) in addition to cells in the muscularis mucosae layer (arrowhead).
Effect of an EP1 receptor antagonist on gastric emptying in unanesthetized rats.
We finally tested effects of EP1 antagonist on 1 M NaCl-induced delay in gastric emptying in unanesthetized rats. As shown in Fig. 8, phenol red dissolved in 1 M NaCl solution delayed significantly, compared with that in physiological saline. Prior administration of an EP1 antagonist, ONO-8711, markedly inhibited 1 M NaCl-induced delay in emptying, and effect of ONO-8711 was essentially the same as indomethacin.
Increasing concentrations of nutrients are known to prolong the emptying time of the stomach (23, 34). Under fasting conditions, the pattern of gastric motility is reported to be characterized by the recurrence of the migrating motor complex (MMC) (7); when we ingest a meal this MMC pattern is interrupted and is replaced with irregular contractile activity (8, 35). Gastric distention markedly alters the motor activity of the gut, even though gastric accommodation prevents a major increase in intragastric pressure (3). In the present experiments, the myoelectrical activities of the gastric antral smooth muscle, however, were barely recorded in the resting state or during instillation of small amounts of intragastric solutions, but the activity could be successfully recorded when the stomach was distended at 2 cmH2O with intragastric application of physiological saline. The myoelectrical activity was the same as that reported by Mersereau and Hinchey (24), using a miniature balloon and a pair of silver bipolar electrodes.
Although the source of this myoelectrical activity was not clarified in the present experiments, the suppressed myoelectrical activity by 0.3–1.0 M NaCl solutions was disappeared after indomethacin treatment. These findings imply that cyclooxygenase products, generated by instillation of NaCl solution, suppressed the myoelectrical activity. We had previously observed that the mechanical stimulation such as touching to the stomach and the cutting of the gastric walls with scissors resulted in the generation of PGE2 more than that seen in the present study. This indicated that the activation of phospholipase A2 and the release of arachidonic acids from the phospholipids can be induced under such stimulation. We had clarified previously that 1 M NaCl ingestion also released the arachidonic acid and generated PGs in the gastric walls. We can observe this concentration of NaCl when we take a salty food or salty fluids during daily life. In terms of osmolarity in the gastric contents, 1 M NaCl is not so high. Although the precise mechanisms of 1 M NaCl solutions to activate PLA2 activity and to enhance PG generation are not fully elucidated, high concentration of mannitol also increased PG levels in the gastric walls. Relevant COX molecule to 1 M NaCl-induced PG generations may be COX-1, as shown in Fig. 6. In our preliminary experiment, the COX-1 immunoreactivity was localized throughout the mucosal epithelial layers. This was consistent with the previous report by others (12).
EP1 signaling is known to induce the constriction of arterioles in some organs (14, 30, 46a); however, we had previously reported (11) that even a high dose of an EP1 agonist did not induce the strong constrictions of the arterioles in the gastric mucosa. The changes in the gastric hemodynamics may not affect the suppressive activity of EP1 signaling in stretch-induced myoelectric activity.
Regional and cellular distribution of mRNAs for PGE receptor subtypes was reported in the mouse gastrointestinal tract by in situ hybridization (39). Intense signals for EP3 transcripts were detected in neurons of the myenteric ganglia throughout the gastrointestinal tract. Moderate EP3 mRNA expression was also observed in fundic gland epithelial cells, except for surface mucous cells in the stomach. Expression of EP4 mRNA was moderate in surface epithelial cells of the corpus and in glands from the surface to the base of the antrum. However, no or weak signals for EP2 transcripts were detected. By contrast, strong signals for EP1 transcripts were detected in cells of the muscularis mucosae layer, especially in the body of the stomach. Furthermore, it was reported that EP1 and EP3 receptor mRNA were detected in the smooth muscle and enteric ganglia of rat stomach (31). Localization of signals for EP1 transcripts suggested that the site of actions of PGE2 and/or EP1 agonist may be the muscularis mucosa layer and/or enteric ganglia. In our separate experiments, the enhancement of the myoelectrical activity introduced by a cholinergic agent, bethanechol was suppressed after the intragastric administration of a 1.0 M NaCl solution (29), suggesting that one of the sites of action of endogenous PGE2 may be the muscularis mucosa layer and/or enteric ganglia that may regulate the smooth muscle contractivity. We had previously tested that the PGE2 constricted the longitudinal smooth muscles of gastric walls (data not shown). Because EP1 receptor signaling was reported to constrict the smooth muscles of gastric walls (38), endogenous PGE2 generated in response to 1 M NaCl may stimulate the EP1 receptors possibly present on the inhibitory enteric neurons. However, further careful examination should be necessary to show physiological roles of EP1 receptor relevant to the suppression of myoelectrical activity in the intrinsic nerves or in the extrinsic nerves. We recently reported that CGRP released from sensory nerves with the actions of capsaicin suppressed the myoelectrical activities (25). The inhibitory effect of 1 M NaCl was not blocked by arterial infusion of a CGRP antagonist, CGRP-(8–37) (25). Thus EP1-mediated suppression may be independent of CGRP.
It was reported that increased osmolality prolongs gastric emptying (20, 30) and that PGs may be determinant of gastric emptying (5, 6, 10, 21, 32, 40, 41). Despite these earlier findings, the mechanism of the prolongation of emptying by hyperosmotic solutions had not been clarified. The osmolality of the 1.0 M NaCl solution used in the present experiments was 2,000 mosmol/kg and may not exceed the range found in normal meals (up to 2,000 mosmol/kg) (19). These suggested that hyperosmolarity, not hypoosmolarity, may play a key role in gastric function. The observation of the present experiment that inhibition of the myoelectrical activity was attributable to PGE2 generated in the gastric wall may be extended to the prolongation of emptying by slightly hyperosmotic solutions in the gastric lumen, when the myoelectrical activity is taken as parameter of gastric motility. To reveal the precise mechanism of PG-mediated prolongation of gastric emptying by hyperosmotic solutions, we used EP1KO mice together with their WT counterparts in the present experiments. This is the first report that endogenous PGE2 suppresses the gastric emptying predominantly through the activation of EP1 receptor when the gastric mucosa is exposed to the hyperosmotic solution that mimics food intake. The mechanistic avenue of this suppression was not clarified precisely in the present study. However, the gastric EP1 receptor present in the muscularis mucosae layer and/or enteric ganglia may be functionally active in gastric emptying and could be a good target to control gastric motility and the mucosal protection system. It was reported that surgical procedures to annulate the stomach and duodenum increased endogenous PGs. The results in the knockout mice may be affected by such PGs generated by the surgical approaches. However, when we tested classical method using dye without anesthesia and surgical approaches, the delay by the hyperosmotic solution was blocked significantly by an EP1 antagonist (Fig. 8). This also supported the results from knockout mice (Fig. 5).
The source of this myoelectrical activity was not clarified in the present experiments, but the myoelectrical activity may originate from circular smooth muscle of the stomach, since the myoelectrical activity was inhibited by 1.0 M NaCl and the antral circular smooth muscle of isolated canine stomach is relaxed by endogenous PGI2 or PGE2 (39). In a separate experiment, we confirmed that the myoelectrical activities also may be caused by a cholinergic never-related reflex, which was induced both by distension of the gastric wall and by the enhanced activity of cholinergic, muscarinic fibers, since the activity was induced by large volumes intragastric saline and inhibited by atropine (29). Changes in motor activity are a basic response to filling of smooth muscle organs, and responses to gastric filling, for example, are thought to be regulated by neural reflexes. It was demonstrated previously that stretch-dependent responses in visceral smooth muscles is mediated by mechanosensitive interstitial cells of Cajal. Responses to stretch were inhibited by indomethacin (1 μM), suggesting that cyclooxygenase-derived eicosanoids may mediate these responses (48a). Dual microelectrode impalements of muscle cells within the corpus and antrum showed that stretch-induced changes in slow-wave frequency uncoupled proximal-to-distal propagation of slow waves. This uncoupling could interfere with gastric peristalsis and impede gastric emptying. Stretch of antral muscles of W/WV mice, which lack intramuscular interstitial cells of Cajal, did not affect membrane depolarization or slow-wave frequency. These findings suggested that stretch reflex in gastric muscles is related to a mechanosensitive role for interstitial cells of Cajal in smooth muscle tissues.
In the present study, we have now identified the PG species and receptor signaling responsible for intragastric osmolarity-induced suppression of the myoelectrical activity of the gastric smooth muscle as a parameter of gastric motor activity with the use of these selective compounds and PG receptor knockout mice. The mechanism of gastric muscular relaxation caused by hyperosmotic nutrients in the gastric lumen may be quite “physiological” and reasonable for providing sufficient time for digestion. Our results indicate that gastric PGE2-EP1 signaling appears critical for controlling gastric motor activity and the gastric emptying. These results suggest that agents acting on EP1 signaling will become novel therapeutic tools for gastric function.
This work was supported by a grant from the Integrative Research Program of the Graduate School of Medical Science, Kitasato University, and by a Grant from the Parents' Association of Kitasato University School of Medicine. It was supported also by a research grant (nos. 15390084 and 16022256), a “High-tech Research Center” grant, and a grant from The 21st Century COE Program, from the Ministry of Education, Culture, Sports, Science and Technology (MEXT).
No conflicts of interest, financial or otherwise, are declared by the author(s).
We express our thanks to C. W. P. Reynolds for correcting the English of this manuscript and to Katsumi Aoki for technical assistance.
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