Introduction

During the fasting period, the upper gastrointestinal (GI) tract undergoes a temporally coordinated cyclic motor pattern known as the migrating motor complex (MMC) in both humans (Vantrappen et al. 1979) and dogs (Szurszewski 1969). MMC is believed to be physiologically important for the mechanical and chemical cleansing of the empty stomach and preparation for the next meal (Code 1979; Sarna et al. 1983; Sarna 1985; Wingate 1981). It prevents bacterial overgrowth in the upper gut and clears the stomach and intestine of any undigested food that was not removed during normal gastric emptying (Husebye 1999; Nieuwenhuijs et al. 1998; Vantrappen et al. 1977).

Motilin, a 22 amino-acid peptide gastrointestinal hormone, was first isolated by J.C. Brown in 1973 from porcine duodenal mucosa (Brown et al. 1973). It is synthesized by endocrine M-cells of the duodenojejunal mucosa, and is periodically released in the fasted state (Pearse et al. 1974; Poitras 1984). Several physiological factors are needed to sustain motor function in the interdigestive period, but motilin is considered the most important in the regulation of MMC. Some previous studies have revealed that an increase in plasma motilin concentration results in simultaneous contractile activity in the stomach (Itoh et al. 1978; Janssens et al. 1983). Also, exogenous motilin administration induced MMC phase III-like contraction in the stomach (Itoh et al. 1976) and gastric phase III contraction was completely eliminated by neutralizing circulating motilin with motilin antiserum or motilin antagonist (Lee et al. 1983, 1978; Ozaki et al. 2009; Sudo et al. 2008), indicating that endogenous motilin induces gastric phase III in the interdigestive period. However, these rhythmic motor patterns originate from the foregut and propagate downward in the alimentary canal (Kellow et al. 1986; Sanger et al. 2010). Since the plasma motilin peak is associated with the gastric phase III peak, we hypothesized that there could be a specific area in the stomach where motilin binds with its receptor and initiates gastric phase III. There also may be a possibility that different areas of the stomach may respond differently towards motilin.

To test this hypothesis, we used Suncus murinus (Asian house shrew), a small mammal developed as a laboratory animal. Suncus murinus belongs to the order Insectivora, family Soricidae, genus Suncus, and this order of animals has been considered one of the most primitive mammals (Douady and Douzery 2003; Ito et al. 2002; Murphy et al. 2007). Previously, we determined cDNA sequences of S. murinus motilin and ghrelin using PCR cloning (Ishida et al. 2009; Tsutsui et al. 2009). We then studied the contractile properties of the S. murinus stomach, in both conscious free-moving S. murinus and in an organ bath experiment, and found that S. murinus has almost the same GI motility and motilin response as that found in humans and dogs (Sakahara et al. 2010; Tsutsui et al. 2009), indicating that S. murinus can be used for GI motility studies. Moreover, many studies have been performed to identify the rodent motilin gene, no convincing results of sequencing have been reported so far. The anatomical gastric structure of S. murinus is similar to that of humans and dogs. For example, it can be visibly distinguished into the fundus, corpus and antrum, and the connection of gastric mucosa layer and muscle layer is loose (Kuromaru and Mochizuki 1980).

The mechanisms of motilin-induced gastric contractions have been studied, and it has been reported that the effect of motilin is species-specific, since it stimulates GI motility in rabbits (Kitazawa et al. 1994), dogs (Itoh 1997; Szurszewski 1969), and humans (Vantrappen et al. 1979), but has no effect in mice and rats because the genomes of both the mice and the rat only contain remnants of the motilin and motilin receptor genes, which are nonfunctional, thus the mouse and rat are natural motilin and motilin receptor gene knockouts (He et al. 2010; Peeters et al. 2004). From in vivo and in vitro experiments using S. murinus, we demonstrated that motilin-induced gastric contractions are mediated through the myenteric plexus (Mondal et al. 2011; Sakahara et al. 2010). Similar results have been reported in other species (De Smet et al. 2009; Kitazawa et al. 1995; Mizumoto et al. 1993; Ohshiro et al. 2008; Van Assche et al. 1997).

In this study, we investigated the active and most responsive site for motilin-induced gastric contractions in the suncus stomach. Thus, we examined the motilin-induced gastric contractile pattern in different parts of the stomach and its mechanism using an organ bath system. To confirm the functional results, we also measured the mRNA expression of motilin receptor GPR38 in various parts of the S. murinus stomach using quantitative PCR analysis.

Materials and methods

Ethical approval

All procedures were approved and performed in accordance with the Saitama University Committee on Animal Research. All efforts were made to minimize animal suffering and to minimize the number of animals used in the experiment.

Animals

Experiments were conducted with female S. murinus, aged 8 weeks old or more and weighing 50–75 g, of an outbred KAT strain established from a wild population in Kathmandu, Nepal. Animals were housed individually in plastic cages equipped with an empty can as a nest box, and were provided food (trout pellets; Nippon Formula Feed Manufacturing Co., Ltd., Yokohama, Japan) and water ad libitum. The metabolizable energy content of the pellets was 344 kcal 100/g, consisting of 54.1 % protein, 30.1 % carbohydrate, and 15.8 % fat. The animal room was maintained at 21–24 °C and the light and dark cycle was controlled to change every 12 h (lights on from 8.00 to 20.00 h).

Drugs used

The administration volume of each drug was 1 % of the bath volume. Acetylcholine (ACh; Sigma-Aldrich Co. LLC., USA) was dissolved in distilled water (DW) and synthetic S. murinus motilin (Bex, Tokyo, Japan) was dissolved in 0.1 % BSA/PBS. In antagonist or inhibitor experiments, the stomachs were equilibrated before the application of S. murinus motilin with the antagonist or inhibitor: atropine sulfate (10−6 M; Merck, USA) and tetrodotoxin (TTX; 10−6 M; Wako Pure Chemical Industries, Ltd, Osaka, Japan) for 30 min. Concentrations of drugs are expressed as final molar concentrations in the bath solution. Both atropine sulfate and TTX were dissolved in DW before use. All reagents were prepared for each experiment according to the manufacturer’s instructions.

Preparation of S. murinus isolated stomach

After animals were fasted for 8 h, the stomach was dissected out after laparotomy and immediately placed in freshly prepared Krebs’ solution (NaCl 118 mM, KCl 4.75 mM, CaCl2 2.5 mM, MgSO4 1.2 mM, NaH2PO4 1.8 mM, NaHCO3 25 mM, and glucose 11.5 mM; pH 7.2–7.4). The mesentery attachments and fatty tissues were removed, and the inside of the stomach was washed with Krebs’ solution through a small incision in the gastric fundus. The stomachs were sectioned into four parts: the fundus, proximal corpus (includes the cardia), distal corpus, and antrum, as shown in Fig. 1. The cutting sites of the stomach were decided by two notches, the cardiac notch and angular notch. Differences in tissue coloring were also used to distinguish between different parts. The whitish body above the cardiac notch was dissected as fundus. The proximal corpus was cut out along the lower part of the esophagus and the cardiac region was included in the proximal corpus section. The remainder of the cylindrical body was considered distal corpus. The antrum is distinctly visible as a whitish pink body below the angular notch. Tissues with mucosa attached to the muscle layer were used instead of muscle layer strips. The longitudinal and circular muscle layers were not been separated into strips because as previously shown, myenteric plexus is important for motilin-induced Suncus gastric contractions (Mondal et al. 2012), and keeping an intact anatomical structure of the myenteric neuron and smooth muscle is essential for studying motilin-induced contraction. The segmented stomach parts were mounted in 10-mL water-jacketed organ baths along the longitudinal muscle direction with the thread tied on cut edge surfaces. Contractility is hence obtained from longitudinal muscle. Tissues were initially loaded with approximately 0.5 gwt. The temperature of Krebs’ solution was maintained at 37 ± 0.5 °C and the solution was aerated continuously with a mixture of 95 % O2 and 5 % CO2.

Fig. 1
figure 1

Stomach segments used in this study. a Photograph illustrating the different parts of the stomach, which can be divided into the fundus, corpus, and antrum; b for the organ bath experiment, the stomachs were dissected into four parts: (1) fundus, (2) proximal corpus (includes the cardia), (3) distal corpus, and (4) antrum. The arrows indicate cardia (gastroesophageal junction), cardiac notch and angular notch. The arrowheads indicate pylorus

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Gastric contractility study

Contractile activities of the stomach with motilin treatment were monitored using an isometric force transducer (UM-203, Iwashiya Kishimoto Medical Instruments, Kyoto, Japan) and software (PicoLog for Windows, Pico Technology Ltd., St Neots, UK). To normalize contractions in this experiment, each stomach section was first treated with acetylcholine chloride (ACh 10−5 M) twice before the administration of motilin with the absence and presence of an antagonist. At the end of the experiment, ACh (10−5 M) was introduced once again into the organ bath. The percentage of maximal contractions was then calculated by averaging the tonic response induced by these three ACh (10−5 M) administrations. To examine the effect of motilin, each stomach section was treated with the S. murinus motilin (10−10–10−7 M) in the absence or presence of antagonist or inhibitor, and motilin-induced contractions were measured and contraction was measured in g weight tension. Then, the effects of S. murinus motilin in the absence or presence of antagonists or inhibitors were determined. The pH of the bath was checked before drug administration to ensure it was between 7.2 and 7.4.

GPR38 mRNA expression

Stomachs were dissected into four parts: fundus, proximal corpus, distal corpus, and antrum; then, the mucosal layers and the muscle layer were peeled off using a glass slide. Separated tissues were frozen with liquid nitrogen and broken using CRYO PLUS (Microtech Co., Ltd., Chiba, Japan) before being dipped in ISOGEN (Nippon Gene, Tokyo, Japan). The total RNA from the tissues was extracted using ISOGEN (Nippon Gene) according to the manufacturer’s instructions, and then subjected to DNase treatment. cDNA was synthesized from 1 μg total RNA using the High Capacity RNA-to-cDNA kit (Applied Biosystems, USA) according to the manufacturer’s instructions. The oligonucleotide-specific primers for S. murinus β-actin are forward 5′-TGCGTGACATCAGGAGAAG-3′; reverse 5′-TCCAGAGAGGAAGAGGATGC-3′, and those for GPR38 are forward 5′-ACAGGCAGACCATCCGC-3′; reverse 5′-TACATTGTCCGGGTGTCCTT-3′. The quantitative PCR (qPCR) reactions were performed using a Light Cycler (Roche Diagnostics, USA) with SYBR Premix Ex-Taq GC (Takara BIO, Shiga, Japan). The initial template denaturation was programmed for 30 s at 95 °C. PCR was performed with 40 cycles of 10 s at 95 °C, 20 s at 64 °C and 30 s at 72 °C and a final cooling step was performed for 30 s at 40 °C. The S. murinus β-actin mRNA was used as the invariant control. The expression of each mRNA is shown relative to β-actin mRNA expression. All reactions were performed in duplicate, and each transcript was quantitatively measured by establishing a linear amplification curve from serial dilutions of each plasmid containing the amplicon sequence. The amplicon size and specificity were confirmed using a melting curve analysis and 2 % agarose gel electrophoresis.

Statistical analysis

Statistical analysis: The results are expressed as the mean ± SEM. Recording experiments were repeated individually at least three times and similar results were obtained. The number of animals used for statistical analyses are represented in the figure legends. We used GraphPad Prism 5 software (GraphPad Software Inc., CA, USA) to analyze the data. Statistical analyses were performed using one-way ANOVA followed by Tukey’s multiple comparison test. P < 0.05 was considered statistically significant.

Results

Spontaneous contractile pattern in the different segments of isolated stomach

The isolated S. murinus stomach in the organ bath showed spontaneous contraction activity under a basal 0.5-gram weight (gwt) resting tension, and contraction differed among parts of the stomach. In the fundus, the spontaneous contractile activity was unclear and arrhythmic, and the amplitude of spontaneous contraction was about 0.2 gwt (Fig. 2a). The amplitude of the spontaneous contraction of the proximal corpus was approximately 0.4 gwt and the rate was 12–16 cycles per minute (Fig. 2b). In the distal corpus, the amplitude of the spontaneous contraction activity was approximately 0.3 gwt and the rate was approximately 8–12 cycles per minute (Fig. 2c). In the antrum, the amplitude of the spontaneous contraction activity was approximately 0.5 gwt and the rate was approximately 2–6 cycles per minute (Fig. 2d). The maximum tensions produced by ACh (10−5 M) were also different in each stomach segment (Fig. 2a–d). In fundus and proximal corpus, ACh-induced maximum tension was approximately 2.5 gwt, whereas it was 5 gwt in the distal corpus and 4 gwt in the antrum.

Fig. 2
figure 2

Motilin-induced contractile activity in different parts of the isolated stomach. ACh (10−5 M) was used to achieve maximum contraction of the tissue. It induced stomach contractions in all the segmented parts of the stomach, with a maximum tension of approximately 2.5 gwt in the fundus (a left), 2.2 gwt in the proximal corpus (b left), 5.0 gwt in the distal corpus (c left), and 4.1 gwt in the antrum (d left). Treatment with a low dose of motilin (10−10 M) was performed to observe the contraction response in the fundus (a right), the proximal corpus (b right), which responded with 28 % contraction, the distal corpus (c right), and the antrum (d right). Contractile amplitudes were then measured after serial administration with motilin (10−9–10−7 M) in the a fundus, b the proximal corpus, c the distal corpus and d the antrum. Induction of contraction in a concentration-dependent manner started after 10−9 M motilin administration in the fundus (a), the proximal corpus (b), and c the distal corpus whereas d the antrum showed changes in the amplitude of contraction with increasing concentration of motilin. Filled inverted arrow, timing of administration of reagents; the number is drug concentration (−logM)

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Responses to motilin in different segments of stomach

To examine the difference in response to motilin treatment in stomach sites, S. murinus motilin (10−10–10−7 M concentrations) was introduced into the organ bath, and contractile amplitudes and frequencies were measured. Although motilin-induced contraction occurred in a concentration-dependent manner, the responses to motilin varied according to the stomach parts (Figs. 2a–d, 3). In the fundus, motilin-induced contractions in a dose-dependent manner were observed starting from a concentration of 10−9 M. Maximum contractions at the 10−7 M concentration were about 64 % of ACh-induced contractions (Figs. 2a, 3). In the proximal corpus, motilin-induced contractions were observed starting from a concentration of 10−10 M; 50 % response of the ACh was observed in response to this concentration. Maximum contractions (89 %) were observed in response to the 10−9 M concentration, and contractions were the same at higher doses of motilin, 10−8 and 10−7 M (Figs. 2b, 3). In the distal corpus, the 10−10 M motilin concentration did not increase contractile activity. The contraction was induced by motilin concentrations of 10−9 M and higher; contraction responses were 25, 54, and 71 % of the total ACh-induced contractions at 10−9, 10−8, and 10−7 M motilin concentrations, respectively (Figs. 2c, 3). In the antrum, the amplitude of contraction (gwt tension) was increased in a dose-dependent manner starting from the 10−9 M motilin concentration, and showed the maximum contraction response of 28 % at 10−7 M (Fig. 3), although this was not significant. In addition, the frequency of contraction was increased significantly at 10−9, 10−8, and 10−7 M motilin concentrations, and the rate of contractile activity changed to 8–9 cycles per minute (Fig. 2d). The EC50 values of motilin-induced contractions in different regions of the stomach are shown in Table 1. Since 10−11 M motilin concentration did not induce contraction in any stomach part, contractions at this concentration were considered to be zero. It was observed that the proximal corpus had higher potency because it had a low concentration of motilin induced gastric contractions compared to other parts of the stomach. Comparing contraction responses for motilin in each stomach sections, the response in the proximal corpus was significantly higher than that in the other sections to the 10−9, 10−8 and 10−7 M motilin concentrations (Fig. 3). Even at the lower dose (10−10 M), the proximal corpus showed high reactivity.

Fig. 3
figure 3

Motilin-induced contraction responses in segmented sections of stomach. Contraction responses were calculated as a percent maximum contraction of 10−5 M ACh. The concentration response of the proximal corpus was significantly higher than that of other parts at low motilin concentration (10−10 M). Each histogram represents the mean ± SEM, n = 5. Different letters denote significant difference (P < 0.05) among groups

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Table 1 Comparison of contractile activity in different segments of the stomach based on EC50 values of the concentration–response curves to motilin
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The cholinergic pathway of the motilin-induced contraction

Atropine, a muscarinic receptor antagonist, suppressed spontaneous contractile activity in all of the stomach sections (Fig. 4a–d). Under atropine pretreatment, motilin concentrations of 10−10–10−8 M did not induce contraction in any stomach parts (Fig. 4a–e). However, motilin concentration of 10−7 M induced contractions in all parts of the stomach, but they were not significant. (Fig. 4e).

Fig. 4
figure 4

Effects of atropine on the motilin-induced contractions in the different segments of stomach. Atropine administration resulted in immediate suppression of contractile activity in all four parts of the stomach: fundus (a left), proximal corpus (b left), distal corpus (c left), and antrum (d left). Pretreatment with atropine (10−6 M) almost entirely inhibited motilin-induced contraction (10−10–10−7 M) in all parts of the stomach (a–d). These results with motilin treatment under atropine administration are summarized in panel (e). Filled inverted arrow timing of administration of reagents; the number is drug concentration (−logM). Each histogram represents the mean ± SEM, n = 4

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The myenteric plexus pathway of the motilin-induced contraction

We also examined the involvement of TTX, a potent neurotoxin that blocks action potentials in nerves by binding to the voltage-gated Na+ channel, and found that TTX did not affect the spontaneous contraction of the fundus, proximal corpus, and distal corpus (Fig. 5a–c). The spontaneous contraction of the antrum altered after TTX administration, as shown in Fig. 5d, and the amplitude was slightly decreased. Under TTX pretreatment, motilin concentrations of 10−10–10−8 M did not evoke contraction in any stomach parts (Fig. 5a–e) and only the 10−7 M concentration slightly induced contractions, although not significantly, of the fundus, proximal corpus, and distal corpus (Fig. 5e).

Fig. 5
figure 5

Effects of tetrodotoxin (TTX) on contractions induced by motilin in segmented stomach parts. Effect of TTX administration on contractile activity in all segmented parts of the stomach: fundus (a left), proximal corpus (b left), distal corpus (c left), and antrum (d left). Pretreatment with TTX (10−6 M) almost completely inhibited the motilin-induced contraction (10−10–10−7 M) in all of the parts (a–d), with a slight reduction of amplitude in Antrum (d). These results with motilin treatment under TTX administration are summarized in panel (e). Filled inverted arrow timing of administration of reagents; the number is drug concentration (−logM). Each histogram represents the mean ± SEM, n = 4

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GPR38 mRNA expression in the stomach

To analyze the distribution of GPR38 in the stomach, we compared the GPR38 mRNA expression level in the fundus, proximal corpus, distal corpus, and antrum using qPCR. We found that GPR38 mRNA expression was low in the mucosal layer and high in the muscle layer in all areas of the stomach. In the muscle layer, the GPR38 mRNA expression level differed among stomach parts, with the highest expression in the proximal corpus and lowest in the antrum (Fig. 6).

Fig. 6
figure 6

Quantitative PCR analysis of the GPR38 mRNA expression in the stomach. The GPR38 mRNA expression level was low in the mucosal layer and high in the muscle layer. In the muscle layer, the GPR38 mRNA expression level differed among stomach sections, with the highest in the proximal corpus and lowest in the antrum. Each histogram represents the mean ± SEM, n = 6. Different letters denote significant difference (P < 0.05) among groups

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Discussion

In the present study using stomachs of S. murinus, we observed that there are differences in response to motilin in various stomach parts in vitro. The response to motilin in the proximal corpus was higher than that in the other parts of the stomach, especially with the low motilin concentration, i.e., the 10−10 M motilin concentration, which slightly induced contractions and the contraction response at 10−9 M were significantly higher in the proximal corpus than that in the other parts. We previously published reports of concentration-dependent contractile effects of motilin on the isolated S. murinus whole stomach in vitro, and there reported that motilin-induced contraction occurred starting from the 10−9 M concentration (Mondal et al. 2011). Even though the stomach was divided into four sections, each of sections retained the ability to react to motilin, similar to that observed in vitro in the whole stomach. In addition, the speed of onset of contraction and the amplitude of contraction increased in all the parts with increasing dose of motilin. This delay might be caused by the permeation of the motilin into the tissue. Interestingly, the effect of the motilin in the antrum was very different from that in the other sections, a dose-related increase in contractile frequency was observed in the antrum. However, both the fundus and antrum showed dose-dependent increases in contraction and the proximal corpus showed maximum contractile activity at a low dose motilin at 10−9 M concentration. These results suggest that the proximal corpus is the most sensitive to motilin treatment. In humans, different regions of the GI tract responded differently to electrical field stimulation-induced contractions under motilin pretreatment (Broad et al. 2012), suggesting that motilin-induced contraction responses are region-specific.

It has been found that plasma motilin concentration in dogs and humans shows cyclic changes during MMC, and its peak is correlated with phase III contractions. In humans and dogs, the range of plasma motilin concentrations varies widely and it ranges approximately 100–500 pmol/L, corresponding to 10−10–10−8 M (Hall et al. 1983; Itoh and Sekiguchi 1983; Itoh et al. 1978; Janssens et al. 1983; Vantrappen et al. 1979). In the present study, only the proximal corpus responded to motilin at the concentration of 10−10 M, which is close to the physiological dose. Based on this result, the proximal corpus is thought to be the first target site of motilin, and it is probable that contractions begin at the proximal corpus and propagate downwards.

In the present study, we found that motilin-induced contractions in every stomach segment were inhibited by TTX and atropine pretreatment, suggesting that motilin-induced gastric contraction is mediated through the cholinergic neural pathway in the myenteric plexus in all regions of the stomach. In the fundus and corpus, at high concentration, motilin 10−7 M slightly increased contraction even under atropine and TTX treatment. It has been reported that plasma motilin concentration varies approximately between 10−10–10−8 M (Hall et al. 1983; Itoh and Sekiguchi 1983; Janssens et al. 1983; Vantrappen et al. 1979). Contractile action of motilin from 10−10–10−8 M concentration in all the segments of the suncus stomach was significantly eliminated by atropine and tetrodotoxin treatment. In addition, 10−7 M motilin tends to induce contractions in all the parts of the stomach, but the effect is not significant, suggesting that the physiological dose of motilin 10−10–10−8 M as well as a higher dose of 10−7 M motilin stimulated gastric contraction through the neural pathway in all the parts of the Suncus stomach. However, in previous reports, high concentration of motilin that is thought to be pharmacological concentration stimulate gastric contraction by the myogenic effect. For example, motilin induced myogenic contraction in rabbits at 10−6 M (Adachi et al. 1981; Depoortere et al. 1995) and 10−7 M for humans and chickens; (Coulie et al. 1998; Shim et al. 2002; Kitazawa et al. 1995, 1997); however, this was not evident in the Suncus. After the administration of atropine, a competitive antagonist of the muscarinic ACh receptor, the spontaneous contractions were suppressed in all of the stomach parts. In the fundus and proximal corpus, the baseline of the spontaneous contraction was decreased after the administration of atropine, and this action is similar to a relaxation effect. The spontaneous contraction of the distal corpus and antrum were completely suppressed after atropine administration, but the baseline was not changed. On the other hand, TTX did not change the spontaneous contraction of the fundus, proximal corpus, and distal corpus sections; however, the frequency of spontaneous contraction in the antrum was decreased. Van Assche et al. and Venkova et al. found that motilin facilitates antral neurotransmission by acting directly on the cholinergic nerves (Van Assche et al. 1997; Venkova et al. 2009). In the central nervous system, motilin at nanomolar concentrations causes a slow depolarization of neurons (Phillis and Kirkpatrick 1979). Electrophysiological evidence has been provided to support the fact that motilin modulates the excitability of myenteric nerves in the guinea-pig antrum (Tack et al. 1991). These results indicate a difference in the mechanisms of spontaneous contraction in different parts of the stomach i.e., fundus, proximal corpus, distal corpus, and antrum.

We previously identified the S. murinus motilin receptor (GPR38) gene and the fact that its expression was high in the stomach (Suzuki et al. 2012). In the present study, we analyzed this in more detail and showed that the distribution of GPR38 mRNA expression differs in different parts of the stomach. We found that GPR38 mRNA expression was low in the mucosal layer and high in the muscle layer in every part of the stomach. Moreover, in the muscle layer, the GPR38 mRNA expression level differed in each stomach section, with the highest in the proximal corpus and the lowest in the antrum. Therefore, the high reactivity for motilin in the proximal corpus may be caused by the high expression of GPR38, suggesting that the proximal corpus area, including the cardia, may be a site of onset of motilin-induced phase III contraction. In humans, motilin receptor immunoreactivity was identified in the muscle and myenteric plexus in the upper gut. Immunoreactivity studies indicate that the motilin receptor is co-expressed with choline acetyltransferase in neurons (Broad et al. 2012). Together, our results suggest that GPR38 expressed in the neurons of the myenteric plexus is the first target of plasma motilin in MMC

The present study reveals that the proximal corpus and cardia is an important site for motilin-induced contractions, with high GPR38 mRNA expression. Motilin 10−9 M stimulates contraction in all the parts of the stomach, but the lower concentration of motilin 10−10 M stimulates contraction in the proximal corpus, suggesting that the MMC propagates from the proximal corpus to the distal tract and that the proximal corpus is the first site in which contractions are induced by motilin stimulation. Our previous in vivo study showed that ghrelin was important for the initiation of phase III MMC contractions and that co-administration of motilin and ghrelin-induced a synergistic phasic response in the prepared isolated stomachs at concentrations ranging from 10−9 to 10−7 M in a dose-dependent manner (Mondal et al. 2012). Therefore, it would be interesting to conduct further research on the response to motilin and ghrelin co-administration in different parts of the stomach.