Figures
Abstract
Here, we have reported that motilin can induce contractions in a dose-dependent manner in isolated Suncus murinus (house musk shrew) stomach. We have also shown that after pretreatment with a low dose of motilin (10−10 M), ghrelin also induces gastric contractions at levels of 10−10 M to 10−7 M. However, the neural mechanism of ghrelin action in the stomach has not been fully revealed. In the present study, we studied the mechanism of ghrelin-induced contraction in vitro using a pharmacological method. The responses to ghrelin in the stomach were almost completely abolished by hexamethonium and were significantly suppressed by the administration of phentolamine, prazosin, ondansetron, and naloxone. Additionally, N-nitro-l-arginine methylester significantly potentiated the contractions. Importantly, the mucosa is essential for ghrelin-induced, but not motilin-induced, gastric contractions. To evaluate the involvement of intrinsic primary afferent neurons (IPANs), which are multiaxonal neurons that pass signals from the mucosa to the myenteric plexus, we examined the effect of the IPAN-related pathway on ghrelin-induced contractions and found that pretreatment with adenosine and tachykinergic receptor 3 antagonists (SR142801) significantly eliminated the contractions and GR113808 (5-hydroxytryptamine receptor 4 antagonist) almost completely eliminated it. The results indicate that ghrelin stimulates and modulates suncus gastric contractions through cholinergic, adrenergic, serotonergic, opioidergic neurons and nitric oxide synthases in the myenteric plexus. The mucosa is also important for ghrelin-induced gastric contractions, and IPANs may be the important interneurons that pass the signal from the mucosa to the myenteric plexus.
Citation: Mondal A, Aizawa S, Sakata I, Goswami C, Oda S-i, Sakai T (2013) Mechanism of Ghrelin-Induced Gastric Contractions in Suncus murinus (House Musk Shrew): Involvement of Intrinsic Primary Afferent Neurons. PLoS ONE 8(4): e60365. https://doi.org/10.1371/journal.pone.0060365
Editor: Wolfgang Blenau, Goethe University Frankfurt, Germany
Received: December 28, 2012; Accepted: February 26, 2013; Published: April 2, 2013
Copyright: © 2013 Mondal et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a Grant-in-Aid for the Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) KAKENHI (grant number 21590785). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Ghrelin is an orexigenic peptide isolated from the stomach as an endogenous ligand for growth hormone secretagogue receptors (GHS-Rs) [1]. Ghrelin is mainly involved in the stimulation of growth hormone secretion [1] and food intake [2] and forms a peptide family with motilin owing to their similarities in not only peptides but also receptors [3]. Moreover, endogenous ghrelin is generally considered to regulate gastric contractions in the interdigestive state [4]; administration of ghrelin stimulates gastric contractions in rats [5], mice [6], and humans [7] and may be an alternative to motilin for gastrointestinal (GI) motility in motilin-lacking rodents [3]. However, the mechanism underlying the role played by ghrelin in the regulation of fasted motor activities in the GI tract is not fully understood.
Functional analyses of ghrelin-induced GI motor-stimulating actions both in vivo and in vitro have suggested 2 main mechanisms for these responses. Ghrelin stimulates fasted intestinal motor activity in rats through ghrelin receptors on vagal afferent nerves [8]. Moreover, the gastric motor-stimulating action of ghrelin in rats shows vagovagal sensitivity [5], [9]. The expression of ghrelin receptors in the nodose ganglion [10], [11] and the capability of ghrelin to modify the discharges of afferent vagal neurons [12] also support the essential role of a vagovagal reflex pathway in ghrelin-induced responses. In addition to this reflex pathway is a mechanism via direct activation of the enteric nervous system in ghrelin-stimulated contraction. In rats and mice, the gastroprokinetic activity of ghrelin is observed in vitro as an increase in neuronally mediated contractions evoked by electrical field stimulation (EFS) [9], [13], [14], [15], [16], [17], and a ghrelin-induced fasted motor pattern has also been observed in vagotomized rats [8]. Together, these results suggest that at least one of the target sites of ghrelin in rodents is the enteric nervous system. However, the phenotypes of ghrelin-sensitive enteric nerves have not been clearly described to date.
One explanation for the gap in information is that the effects of ghrelin activity have thus far been investigated using EFS systems in the case of smooth muscle preparations [9], [13], [14], [15], [16], [17]. Therefore, the presence of a complete neural package in the stomach has not been studied. Moreover, the actions of ghrelin are species dependent, similar to those of the ghrelin-related peptide motilin. For example, ghrelin does not stimulate canine and rabbit GI motility [7], [18] but induces gastric contractions in rats, mice, and humans, and although motilin stimulates GI motility in rabbits [19], dogs [20], and humans [21], it has no effect in mice and rats. To address these dissimilarities, we used Suncus murinus (house musk shrew) in an organ bath study. S. murinus belongs to the order Insectivora, family Soricidae, and this order of animals is considered one of the key groups for understanding the origin of mammals [22], [23]. We have already identified the complementary DNA sequences of suncus motilin and ghrelin in S. murinus using polymerase chain reaction cloning [24], [25]. We have also identified GHS-R and G protein-coupled receptor 38 genes in S. murinus [26]. Moreover, we have studied the contractile properties of the stomach in conscious, free-moving S. murinus as well as in organ-bath experiments and found that S. murinus has GI motility that is almost identical to that in humans and dogs [24], [27]. We have also published the mechanism of motilin-induced gastric contractions in the S. murinus stomach [28].
Recently, we demonstrated that ghrelin can induce gastric contractions after pretreatment with a low dose of motilin, and this coordination of motilin and ghrelin may be necessary for the initiation of phase III contractions [29]. However, the mechanism and neural pathway of that synergistic effect in the enteric nervous system is unknown. To clarify this point, we investigated the mechanism of ghrelin-induced contractions in vitro using the whole stomach of S. murinus.
Materials and Methods
Animals
The experiments were conducted using female suncuses (weight, 45–75 g) older than 5 weeks, from an outbred KAT strain established from a wild population in Kathmandu, Nepal [30]. Animals were housed individually in plastic cages equipped with empty cans for nest boxes. The animals were given food (trout pellets; Nippon Formula Feed Manufacturing Co. Ltd., Yokohama, Japan) and water ad libitum. The animal room was maintained at 21°C to 24°C and the light-dark cycle was controlled to change every 12 h (lights on from 0800 to 2000 h). All procedures were approved by and performed in accordance with the guidelines of the Saitama University Committee on Animal Research. All efforts were made to minimize animal suffering and reduce the number of animals used in the experiment.
Preparation of Suncus Isolated Stomach
After being deeply anesthetized with diethyl ether, the animals were killed via decapitation, and their stomachs were immediately placed into freshly prepared Krebs solution (composition in mM: NaCl, 118; KCl, 4.75; CaCl2, 2.5; MgSO4, 1.2; NaH2PO4, 1.8; NaHCO3, 25; and glucose, 11.5; pH 7.2) after laparotomy. The mesentery attachments and fatty tissues were removed, and the inside of each stomach was washed with Krebs solution through a small incision in the gastric fundus. The stomachs were then mounted in 10-mL water-jacketed organ baths and initially loaded with weight totaling approximately 1.0 g. The temperature of the Krebs solution was maintained at 37°C ±0.5°C, and the solution was aerated continuously with a mixture of 95% O2 and 5% CO2.
Gastric Contractility Study
The contractile activities of the stomach in response to ghrelin 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 the contractions in this experiment, we added acetylcholine (ACh; 10−5 M) to the organ bath twice before the cumulative administration of ghrelin (after pretreatment with 10−10 M motilin) in the absence and presence of an antagonist. At the end of the experiment, ACh (10−5 M) was added once again to the organ bath, and the percentage of maximal contractions was calculated by averaging the tonic response induced by these 3 administrations. Note that in each case, the ACh administration evoked almost the same tonic gastric contractions. Then the effects of acyl ghrelin in the absence or presence of antagonists were expressed as a percentage of the control contractions. Concentration-response curves were obtained through cumulative addition of acyl ghrelin with or without antagonists or an inhibitor at appropriate intervals to the organ bath.
Damaging the Mucosa
The mucosa was damaged mechanically. In brief, the ghrelin-induced (with low-dose motilin pretreatment) and motilin-induced gastric contractions were recorded as a control, the stomach was removed from the organ bath, and the mucosa was damaged through gentle pressing with cover-glass forceps. These damaged stomachs were loaded into the organ bath and washed several times with Krebs buffer. The stomachs were again mounted in 10-mL water-jacketed organ baths, and the contractile properties were measured using ghrelin and motilin.
Drugs Used
Acetylcholine chloride (Sigma, USA) was dissolved in distilled water, and synthetic suncus motilin (Bex, Tokyo, Japan) was dissolved in 0.1% bovine serum albumin/phosphate-buffered saline. In antagonist or inhibitor experiments, the stomachs were equilibrated before the application of acyl ghrelin (pretreated with a low dose of motilin) with the following antagonists: hexamethonium bromide (10−4 M; Wako, Osaka, Japan) [31], prazosin hydrochloride (10−6 M; Wako) [32], timolol maleate (10−6 M; Wako) [33], naloxone (10−6 M; Wako) [34], FK888 (10−6 M; Tocris Bioscience, Ellisville, USA) [35], ondansetron (10−5 M; Hikari Pharmaceutical, Imado, Japan) [36], and phentolamine mesylate (10−5 M; MP Biomedicals, France) [32] for 30 min; yohimbine hydrochloride (10−6 M; Tocris Bioscience) [33] for 25 min; ritanserin (10−7 M; Tocris Bioscience) [37] for 1 h, or N-nitro-l-arginine methylester (L-NAME; 10−4 M; Sigma) [38], adenosine (10−8.5 M; Sigma) [39], and GR113808 (10−7 M; Tocris Bioscience) [40] for 15 min; and SR142801 (10−7 M; Axon Medchem, Groningen, The Netherlands) [41] for 20 min. Concentrations of drugs were expressed as final molar concentrations in the bath solution. Ritanserin and FK888 were dissolved in ethanol, and the other compounds were dissolved in distilled water before use. All reagents were prepared for each experiment according to manufacturer instructions.
Statistical Analysis
The results of experiments have been expressed as mean ± standard error of the mean values of more than 4 separate experiments using whole stomachs. One-way analysis of variance followed by the Student’s t-test was used for the statistical analysis of data. P<0.05 and P<0.01 were considered significant.
Results
Cholinergic Pathway
We have previously shown that ghrelin induces gastric contractions after pretreatment with a low dose of motilin (10−10 M) [42]. We have also reported that atropine, a muscarinic receptor antagonist, completely abolishes the response to ghrelin in the stomach and suppresses spontaneous contractile activity [42]. Similarly, hexamethonium, a nicotinic receptor antagonist, almost completely eliminates contraction induced by ghrelin (10−10–10−9 M; Figure 1A–C) and hexamethonium reduced the effect of 10−7 M dose of ghrelin by 85.1±3.2%.
(A) After pretreatment with motilin (10−10 M), ghrelin stimulated gastric contractions from 10−10 M. (B) Trace showing that ghrelin-induced gastric contractions was considerably attenuated by pretreatment with hexamethonium (10−4 M). (C) Concentration-response curve showing the almost complete inhibitory effect of hexamethonium on ghrelin-induced gastric contractions. ▾, timing of the administration of reagents; the number is the concentration of ghrelin (-Log[ghrelin]M). Each value is the mean ± standard error of the mean (SEM; n = 6). •: Control; ▴: antagonist treatment; **P<0.01. ACh, acetylcholine.
Adrenergic Pathway
We also examined the involvement of adrenergic neurons. Phentolamine, an α receptor antagonist, markedly inhibited ghrelin-induced contraction (Figure 2A) and decreased spontaneous contraction (data not shown). Ghrelin-induced tonic contraction was also significantly suppressed by pretreatment with prazosin, an α1 receptor antagonist (Figure 2B). Phentolamine and prazosin reduced the stimulatory effect of 10−7 M dose of ghrelin by 74.1±6.3% and 55.6±6.5%, respectively. Conversely, yohimbine, an α2 receptor antagonist, and timolol, a β receptor antagonist, did not affect ghrelin stimulatory contraction (Figure 2C, D).
(A) Phentolamine (α adrenergic receptor antagonist; 10−5 M) significantly decreased contractions. (B) Prazosin (α1 adrenergic receptor antagonist; 10−6 M) also significantly suppressed the control contractions. (C) Yohimbine (α2 adrenergic receptor antagonist; 10−6 M) had no significant effect. (D) Timolol (β adrenergic receptor antagonist; 10−6 M) had no effect. Each value is the mean ± SEM (n = 6). •: Control; ▴: antagonist treatment; **P<0.01. ACh, acetylcholine.
Serotonergic Pathway
In the present study, ritanserin, a 5-hydroxytryptamine (HT) 2 receptor antagonist, did not decrease ghrelin-induced contractions at any concentration (Figure 3A) but markedly suppressed spontaneous contractile activity (data not shown). In contrast to ritanserin, ondansetron, a 5-HT3 receptor antagonist, significantly suppressed the contractions induced by ghrelin (Figure 3B) and the rate (%) of reduction by the ondansetron on the stimulatory effects of 10−7 M dose of ghrelin was 58.1±3.2%.
(A) Ritanserin (10−7 M) showed no antagonistic effect on ghrelin-induced contractions. (B) Ondansetron treatment (10−5 M) significantly reduced ghrelin-induced contractions. (C) Naloxone (10−6 M) significantly inhibited ghrelin-induced contractions. (D) FK888 had no significant effect on ghrelin-induced contractions. (E) L-NAME significantly potentiated the contractions at doses of 10−10 and 10−9 M ghrelin. Each value is the mean ± SEM value (n = 6). •: Control; ▴: antagonists or inhibitor treatment; *P<0.05, **P<0.01. ACh, acetylcholine.
Other Receptor Antagonists and Nitric Oxide (NO) Synthase Inhibitor
The effects of other receptor antagonists and NO synthase inhibitors were also investigated for further characterization of the ghrelin response. Naloxone, an opiate receptor antagonist, potentially suppressed the contractions induced by ghrelin (Figure 3C) but did not affect spontaneously occurring phasic contractions (data not shown) and the inhibitory rate (%) of the naloxone on the effect of 10−7 M ghrelin was 62.9±6.1%. Neither spontaneously occurring contractions nor motilin-induced contractions were decreased by FK888, a neurokinin (NK) 1 receptor antagonist (Figure 3D). N-nitro-l-arginine methylester, an inhibitor of NO synthase, potentiated the contractions induced by 10−10 and 10−9 M ghrelin but did not significantly change contractions induced by other ghrelin concentrations (Figure 3E).
Importance of Mucosa in Ghrelin-induced Contraction
Because ghrelin-producing cells have been observed in the gastric mucosa of S. murinus [25], we predicted that the mucosa plays an important role in the ghrelin stimulatory pathway. Figure 4A depicts the synergistic effect of motilin and ghrelin with and without damage to the gastric mucosa. Interestingly, after the breakdown in mucosa, ghrelin-induced gastric contraction was completely abolished (see Figure 4A, B), whereas motilin-induced contraction was preserved (see Figure 4A, C), suggesting that mucosa has a central role in triggering ghrelin-induced contractions.
(A) Chart showing the increasing pattern of ghrelin-induced (after pretreatment with a low dose of motilin) and motilin-induced gastric contractions before and after damage to the mucosa. (B) Magnification of part A showing that ghrelin can stimulate gastric contractions with the mucosa intact, but gastric contraction was absent after damage to the mucosal layers. (C) Motilin evoked the same gastric contraction even after mucosal damage. ▾, timing of the administration of reagents; the number is the concentration of ghrelin (-Log[ghrelin]M; n = 4). ACh, acetylcholine.
Effects of Adenosine, SR142801, and GR113808 on Ghrelin-induced Contraction
Intrinsic primary afferent neurons (IPANs) reportedly play a pivotal role in controlling the motility in the GI tract through synaptic connections with other neurons in the myenteric, submucosal, and mucosal ganglia [43], [44]. Therefore, we predicted that IPANs play a significant role in the ghrelin regulatory pathway. IPAN is the primary transmitter of ACh and tachykinin [39], [44]. The 5HT4 receptor is also expressed on IPANs [45]. To confirm the involvement of these factors, we used pretreatment with GR113808 (5HT4 receptor antagonist), adenosine, an agonist of adenosine receptors (to inhibit the release of ACh from IPANs through A1 receptors), and SR142801 (NK3 receptor antagonist), and found that GR113808 completely eliminated ghrelin-induced contractions (Figure 5A, B). The concentration-response curve showed that adenosine pretreatment significantly abolished ghrelin-induced gastric contraction (Figure 5C). Pretreatment with SR142801 almost completely inhibited ghrelin-induced contractions at ghrelin doses of 10−10 and 10−9 M and significantly suppressed it at a concentration of 10−8 M ghrelin (Figure 5D). GR113808 and adenosine reduced the effects of 10−7 M dose of ghrelin by 92.6±2.5% and 39.9±18.7%, respectively, and SR142801 reduced the effects of 10−9 M ghrelin by 82.1±7.3%.
(A) Traces of ghrelin-induced contractions with and without pretreatment with GR113808 (10−7 M). (B) Concentration-response curve showing that GR113808 completely abolished ghrelin-induced contractions. (C) Concentration-response curve showing that adenosine significantly attenuated ghrelin-induced contractions. (D) SR142801 almost completely inhibited ghrelin-induced gastric contraction at doses of 10−10 and 10−9 M ghrelin. ▾, timing of the administration of reagents; the number is the concentration of ghrelin (-Log[ghrelin]M). Each value is the mean ± SEM (n = 4). •: Control; ▴: antagonist treatment; *P<0.05; **P<0.01. ACh, acetylcholine.
Discussion
Since the discovery of ghrelin, the mechanisms of ghrelin activity with respect to GI motility have been increasingly investigated; however, clear insights into these mechanisms have been elusive thus far. In the present study, we used the stomach of S. murinus, a new model animal for the study of motilin/ghrelin, and revealed mechanisms of ghrelin-induced contractions with a pharmacological in vitro method. To investigate the response of the neural network to ghrelin in the enteric nervous system, we examined the effects of various receptor antagonists and a NO synthase inhibitor on ghrelin-induced contractions and characterized the pharmacological properties in the suncus stomach in vitro.
We have already reported the novel observation that ghrelin (10−10 to 10−7 M) can induce contraction in isolated S. murinus stomach in a dose-dependent manner when pretreated with a low concentration of motilin (10−10 M) [29]. Ghrelin-induced S. murinus gastric contractions have also been confirmed to operate in a vagus-independent manner [29]. In the present study, hexamethonium, a ganglion-blocking agent, almost completely suppressed the action of ghrelin and, as we have reported in a previous study, atropine also completely inhibits ghrelin-induced gastric contractions [29]. Several functional in vivo and in vitro studies have reported that the cholinergic system may be the dominant motor pathway in ghrelin-induced contractions [5], [17]. These results together indicate that myenteric preganglionic cholinergic neurons and postganglionic cholinergic neurons are equally important for ghrelin-induced gastric contractions. Moreover, given the inhibitory potency of hexamethonium (Table S1), presynaptic cholinergic activation plays a much more prominent role than that of motilin in ghrelin-induced gastric contraction [28].
The notable inhibitory effects of phentolamine and prazosin indicate that α receptors, specifically α1 receptors, but not α2 and β receptors, are also involved in ghrelin-induced contraction. Conversely, our previous study clearly showed that α2 receptors are important for motilin-induced gastric contraction [28]. Taken together, these results suggest that different neural pathways exist between ghrelin- and motilin-induced gastric contractions.
The significance of 5HT neurons has been observed in intestinal motility with vagus dependency [46], [47]. By contrast, we showed that 5HT3 but not 5HT2 neurons are involved in ghrelin-induced contractions with vagus independence. Similarly, for the first time, we observed that the opioidergic pathway has a vital role in ghrelin-stimulated contraction. Moreover, compared with motilin-induced gastric contraction in S. murinus [28], ghrelin-induced gastric contractions is strongly suppressed by ondansetron and naloxone pretreatment (see Table S1). Bassil et al. [16] have reported an EFS study in which ghrelin-induced contractions was evoked with multiple neurotransmitters. Ghrelin increases EFS-evoked contraction mediated by a combination of cholinergic and non-cholinergic excitatory activity in rats [15]. These results suggest that not only pre-ganglionic cholinergic neurons but also the involvement of 5HT3 and opioidergic neurons are likely of similar importance for ghrelin-induced contraction in S. murinus stomach.
Another significant observation is that motilin is essential for ghrelin-induced gastric contractions [42]. Ghrelin administration alone, despite the administration of high doses (10−7 M), failed to stimulate any gastric contractions; however, pretreatment with a low dose of motilin restored sensitivity to ghrelin, implying that motilin opens the gate of the ghrelin pathway. Two possibilities explain this gate-opening property: (1) motilin may synergistically stimulate the ghrelin pathway, or (2) motilin may produce inhibitory effects on some strong suppression mechanism. Considering the differing properties between the independent administration of ghrelin (1000 times higher doses) and motilin-pretreated ghrelin, which can induce gastric contractions (i.e., 10−7 M versus 10−10 M ghrelin), it is reasonable to predict that in restoring ghrelin activity, a low dose of motilin may affect the inhibitory neural network in the myenteric plexus in a flip-flop manner. However, we cannot draw any conclusions at this time, and therefore, this area remains an important one for further investigation.
Apart from this, the most interesting finding in this study is the significance of the mucosa for ghrelin-induced, but not motilin-induced, gastric contractions. We have reported that ghrelin-producing cells are found in suncus gastric mucosa [25] and that GHS-R messenger RNA expression is found in both the mucosa and the muscle layers of S. murinus stomach [26]. Therefore, some specialized neurons may responsible for the transfer of signals from the mucosa to the myenteric plexus in ghrelin-induced gastric contraction. Mucosal stimulation is thought to activate IPANs in both the submucosal [48], [49], [50], [51] and the myenteric plexuses [39], [52], [53], and this distinctive shape is known as Dogiel type II morphology [44]. Therefore, we assumed that the IPANs may act as interneurons in ghrelin stimulatory action. The primary transmit component of IPANs are ACh and tachykinergic neurons [44]. Moreover, adenosine activates adenosine receptors, and most afterhyperpolarizing/Dogiel type II neurons (about 85%) are hyperpolarized by adenosine treatment–largely through A1 receptors [54] via inhibition of ACh release in the myenteric plexus [55]–and many studies have shown that the cell bodies of myenteric IPANs bear NK3 receptors [56], [57], [58], [59]. In the present study, we found that pretreatment with adenosine significantly suppressed ghrelin-induced gastric contractions and that an NK3 receptor antagonist (SR142801) almost completely abolished the contraction at doses of 10−10 and 10−9 M. Conversely, we also observed that both adenosine and SR142801 had no effect on motilin-induced gastric contractions (Figures S1A, B). Although GR113808 (5HT4 receptor antagonist) treatment completely inhibited ghrelin-induced contraction, motilin-induced contraction was partially suppressed by GR113808 treatment (Figure S2). 5HT4 receptors are reportedly present in IPANs and in some other neuron types in the myenteric plexus [45]. Recently, Takahashi et al. [60] observed that 5HT4 receptors are important in regulating migrating motor complexes through IPANs. Taken together, these data indicate that a different stimulatory pathway exists between the motilin and the ghrelin effects and that IPANs may be implicated in the effects of ghrelin on GI motility.
IPANs with cell bodies in myenteric ganglia have been shown to transmit via slow excitatory postsynaptic potentials (EPSPs) to interneurons and motor neurons, and indirect evidence suggests that they also transmit via fast EPSPs [39]. Slow EPSPs in IPANs are mimicked by the NK3 receptor agonist senktide [61] and partially blocked by the NK3 receptor antagonist SR142801 [41]. ACh, acting via muscarinic receptors, also elicits slow depolarizing responses in myenteric IPANs [62]. However, a major component of the transmission by IPANs appears to occur via cholinergic fast EPSPs because the application of an antagonist of nicotinic ACh receptors, hexamethonium, at the site of the primary afferent to interneuron synapses reduces ascending reflexes in response to mucosal distortion [63]. Moreover, 5HT4 receptor activation facilitates ACh release in the myenteric plexus [64] and may initiate enteric reflexes by acting on the ending IPANs [65]. Therefore, the inhibitory effects of atropine [29], GR113808, adenosine, and SR142801 on ghrelin-induced gastric contractions suggest the importance of IPANs in the regulation of ghrelin activity in motility response. However, we cannot make a firm conclusion because the pharmacological properties of IPANs have been elucidated only for the intestines and not for the stomach thus far. Therefore, a future line of investigation will be to confirm the morphological presence of IPANs in the S. murinus stomach with co-localization of receptors for ghrelin, ACh, NK3, and 5HT4.
Finally, we concluded that the effect of ghrelin-induced gastric contractions is mediated through postganglionic and preganglionic cholinergic receptors, 5-HT3 receptors, α and α1 receptors, and opioid receptors; NO synthesis is also involved, and the final mediators of these ghrelin-induced gastric contractions are myenteric cholinergic neurons [28], [29]. Moreover, the mucosa plays an important role in the neural pathway of ghrelin-induced gastric contraction. The reason why pretreatment with a low dose of motilin (10−10 M) is needed to induce ghrelin contractions remains unknown. Our predicted mechanism for ghrelin-induced gastric contractions is demonstrated in Figure 6; motilin stimulates gastric contractions through the myenteric plexus [28], whereas ghrelin may stimulate muscarinic cholinergic receptors by passing signals from the mucosa to the myenteric plexus through the stimulation of 5HT4, ACh, and NK3 release from IPANs. Administration of low doses of motilin may open a gate so that ghrelin can induce gastric contractions in the S. murinus stomach. The confirmation of this mechanism may be an interesting finding in the near future. Moreover, the synergistic effects of motilin and ghrelin observed in the present study are probably species-dependent, and clarifying the potential clinical application of these motilin/ghrelin-derived compounds would be interesting.
Considered with the previous results that motilin (10−9 to 10−7 M) induces gastric contractions by activating the myenteric plexus [28], the present findings suggest that a very low dose of motilin (10−10 M) may initiate an “open gate” effect and restore ghrelin activity via the myenteric plexus. Consequently, ghrelin may transfer signals from the mucosa to the myenteric plexus through involvement of intrinsic primary afferent neurons (IPANs) and regulate gastric contractions through muscarinic cholinergic receptors.
Supporting Information
Figure S1.
Effect of adenosine and SR142801 pretreatment on the motilin-induced contractions. Both the adenosine (10-8.5 M; A) and SR142801 (10−7 M; B) has no effect on the motilin-stimulatory pathway. Each value is mean ± SEM (N = 4). •: Control; ▪: antagonist treatment.
https://doi.org/10.1371/journal.pone.0060365.s001
(TIF)
Figure S2.
Effect of GR113808 pretreatment on the motilin-induced contractions. The GR113808 (10−7 M) partially inhibited the motilin-stimulatory pathway. Each value is mean ± SEM (N = 12). •: Control; ▴: antagonist treatment.
https://doi.org/10.1371/journal.pone.0060365.s002
(TIF)
Table S1.
Comparison between the effects of different receptor antagonist on the motilin- and ghrelin-induced S. murinus gastric contractions.
https://doi.org/10.1371/journal.pone.0060365.s003
(DOCX)
Author Contributions
Conceived and designed the experiments: TS IS. Performed the experiments: AM SA CG. Analyzed the data: AM TS. Contributed reagents/materials/analysis tools: SO. Wrote the paper: AM.
References
- 1. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, et al. (1999) Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402: 656–660.
- 2. Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, et al. (2001) A role for ghrelin in the central regulation of feeding. Nature 409: 194–198.
- 3. Peeters TL (2005) Ghrelin: a new player in the control of gastrointestinal functions. Gut 54: 1638–1649.
- 4. Ariga H, Tsukamoto K, Chen C, Mantyh C, Pappas TN, et al. (2007) Endogenous acyl ghrelin is involved in mediating spontaneous phase III-like contractions of the rat stomach. Neurogastroenterology and motility: the official journal of the European Gastrointestinal Motility Society 19: 675–680.
- 5. Masuda Y, Tanaka T, Inomata N, Ohnuma N, Tanaka S, et al. (2000) Ghrelin stimulates gastric acid secretion and motility in rats. Biochemical and Biophysical Research Communications 276: 905–908.
- 6. Zheng J, Ariga H, Taniguchi H, Ludwig K, Takahashi T (2009) Ghrelin regulates gastric phase III-like contractions in freely moving conscious mice. Neurogastroenterol Motil 21: 78–84.
- 7. Ohno T, Kamiyama Y, Aihara R, Nakabayashi T, Mochiki E, et al. (2006) Ghrelin does not stimulate gastrointestinal motility and gastric emptying: an experimental study of conscious dogs. Neurogastroenterol Motil 18: 129–135.
- 8. Fujino K, Inui A, Asakawa A, Kihara N, Fujimura M, et al. (2003) Ghrelin induces fasted motor activity of the gastrointestinal tract in conscious fed rats. J Physiol 550: 227–240.
- 9. Fukuda H, Mizuta Y, Isomoto H, Takeshima F, Ohnita K, et al. (2004) Ghrelin enhances gastric motility through direct stimulation of intrinsic neural pathways and capsaicin-sensitive afferent neurones in rats. Scand J Gastroenterol 39: 1209–1214.
- 10. Date Y, Murakami N, Toshinai K, Matsukura S, Niijima A, et al. (2002) The role of the gastric afferent vagal nerve in ghrelin-induced feeding and growth hormone secretion in rats. Gastroenterology 123: 1120–1128.
- 11. Sakata I, Yamazaki M, Inoue K, Hayashi Y, Kangawa K, et al. (2003) Growth hormone secretagogue receptor expression in the cells of the stomach-projected afferent nerve in the rat nodose ganglion. Neurosci Lett 342: 183–186.
- 12. Asakawa A, Inui A, Kaga T, Yuzuriha H, Nagata T, et al. (2001) Ghrelin is an appetite-stimulatory signal from stomach with structural resemblance to motilin. Gastroenterology 120: 337–345.
- 13. Depoortere I, De Winter B, Thijs T, De Man J, Pelckmans P, et al. (2005) Comparison of the gastroprokinetic effects of ghrelin, GHRP-6 and motilin in rats in vivo and in vitro. Eur J Pharmacol 515: 160–168.
- 14. Kitazawa T, De Smet B, Verbeke K, Depoortere I, Peeters TL (2005) Gastric motor effects of peptide and non-peptide ghrelin agonists in mice in vivo and in vitro. Gut 54: 1078–1084.
- 15. Dass NB, Munonyara M, Bassil AK, Hervieu GJ, Osbourne S, et al. (2003) Growth hormone secretagogue receptors in rat and human gastrointestinal tract and the effects of ghrelin. Neuroscience 120: 443–453.
- 16. Bassil AK, Dass NB, Sanger GJ (2006) The prokinetic-like activity of ghrelin in rat isolated stomach is mediated via cholinergic and tachykininergic motor neurones. Eur J Pharmacol 544: 146–152.
- 17. Edholm T, Levin F, Hellstrom PM, Schmidt PT (2004) Ghrelin stimulates motility in the small intestine of rats through intrinsic cholinergic neurons. Regul Pept 121: 25–30.
- 18. Depoortere I, Thijs T, Thielemans L, Robberecht P, Peeters TL (2003) Interaction of the growth hormone-releasing peptides ghrelin and growth hormone-releasing peptide-6 with the motilin receptor in the rabbit gastric antrum. J Pharmacol Exp Ther 305: 660–667.
- 19. Kitazawa T, Ichikawa S, Yokoyama T, Ishii A, Shuto K (1994) Stimulating action of KW-5139 (Leu13-motilin) on gastrointestinal motility in the rabbit. Br J Pharmacol 111: 288–294.
- 20.
Itoh Z, Honda R, Hiwatashi K, Takeuchi S, Aizawa I, et al.. (1976) Motilin-induced mechanical activity in the canine alimentary tract. Scand J Gastroenterol Suppl 39: 93–110.
- 21. Janssens J, Vantrappen G, Peeters TL (1983) The activity front of the migrating motor complex of the human stomach but not of the small intestine is motilin-dependent. Regul Pept 6: 363–369.
- 22. Douady CJ, Douzery EJ (2003) Molecular estimation of eulipotyphlan divergence times and the evolution of “Insectivora”. Mol Phylogenet Evol 28: 285–296.
- 23. Murphy WJ, Pringle TH, Crider TA, Springer MS, Miller W (2007) Using genomic data to unravel the root of the placental mammal phylogeny. Genome Res 17: 413–421.
- 24. Tsutsui C, Kajihara K, Yanaka T, Sakata I, Itoh Z, et al. (2009) House musk shrew (Suncus murinus, order: Insectivora) as a new model animal for motilin study. Peptides 30: 318–329.
- 25. Ishida Y, Sakahara S, Tsutsui C, Kaiya H, Sakata I, et al. (2009) Identification of ghrelin in the house musk shrew (Suncus murinus): cDNA cloning, peptide purification and tissue distribution. Peptides 30: 982–990.
- 26. Suzuki A, Ishida Y, Aizawa S, Sakata I, Tsutsui C, et al. (2012) Molecular identification of GHS-R and GPR38 in Suncus murinus. Peptides 36: 29–38.
- 27. Sakahara S, Xie Z, Koike K, Hoshino S, Sakata I, et al. (2010) Physiological characteristics of gastric contractions and circadian gastric motility in the free-moving conscious house musk shrew (Suncus murinus). Am J Physiol Regul Integr Comp Physiol 299: R1106–1113.
- 28. Mondal A, Kawamoto Y, Yanaka T, Tsutsui C, Sakata I, et al. (2011) Myenteric neural network activated by motilin in the stomach of Suncus murinus (house musk shrew). Neurogastroenterol Motil 23: 1123–1131.
- 29. Mondal A, Xie Z, Miyano Y, Tsutsui C, Sakata I, et al. (2012) Coordination of motilin and ghrelin regulates the migrating motor complex of gastrointestinal motility in Suncus murinus. Am J Physiol Gastrointest Liver Physiol 302(10): G1207–15.
- 30. Ito H, Nishibayashi M, Kawabata K, Maeda S, Seki M, et al. (2002) Immunohistochemical demonstration of c-fos protein in neurons of the medulla oblongata of the musk shrew (Suncus murinus) after veratrine administration. Exp Anim 51: 19–25.
- 31. Liang SD, Vizi ES (1997) Positive feedback modulation of acetylcholine release from isolated rat superior cervical ganglion. J Pharmacol Exp Ther 280: 650–655.
- 32. Ahn SW, Kim SH, Kim JH, Choi S, Yeum CH, et al. (2010) Phentolamine inhibits the pacemaker activity of mouse interstitial cells of Cajal by activating ATP-sensitive K+ channels. Arch Pharm Res 33: 479–489.
- 33. Fontaine J, Lebrun P (1989) Contractile effects of substance P and other tachykinins on the mouse isolated distal colon. Br J Pharmacol 96: 583–590.
- 34. Cosola C, Albrizio M, Guaricci AC, De Salvia MA, Zarrilli A, et al. (2006) Opioid agonist/antagonist effect of naloxone in modulating rabbit jejunum contractility in vitro. J Physiol Pharmacol 57: 439–449.
- 35. Maggi CA, Patacchini R, Bartho L, Holzer P, Santicioli P (1994) Tachykinin NK1 and NK2 receptor antagonists and atropine-resistant ascending excitatory reflex to the circular muscle of the guinea-pig ileum. Br J Pharmacol 112: 161–168.
- 36. Amemiya N, Hatta S, Ohshika H (1997) Effects of ondansetron on electrically evoked contraction in rat stomach fundus: possible involvement of 5-HT2B receptors. Eur J Pharmacol 339: 173–181.
- 37. Javid FA, Naylor RJ (1999) Characterization of the 5-hydroxytryptamine receptors mediating contraction in the intestine of Suncus murinus. Br J Pharmacol 127: 1867–1875.
- 38. Kitazawa T, Onodera C, Taneike T (2002) Potentiation of motilin-induced contraction by nitric oxide synthase inhibition in the isolated chicken gastrointestinal tract. Neurogastroenterology and Motility 14: 3–13.
- 39. Furness JB, Kunze WA, Bertrand PP, Clerc N, Bornstein JC (1998) Intrinsic primary afferent neurons of the intestine. Prog Neurobiol 54: 1–18.
- 40. Prins NH, Shankley NP, Welsh NJ, Briejer MR, Lefebvre RA, et al. (2000) An improved in vitro bioassay for the study of 5-HT(4) receptors in the human isolated large intestinal circular muscle. Br J Pharmacol 129: 1601–1608.
- 41. Alex G, Kunze WA, Furness JB, Clerc N (2001) Comparison of the effects of neurokinin-3 receptor blockade on two forms of slow synaptic transmission in myenteric AH neurons. Neuroscience 104: 263–269.
- 42. Mondal A, Xie Z, Miyano Y, Tsutsui C, Sakata I, et al. (2012) Coordination of motilin and ghrelin regulates the migrating motor complex of gastrointestinal motility in Suncus murinus. Am J Physiol Gastrointest Liver Physiol 302: G1207–1215.
- 43. Clerc N, Furness JB (2004) Intrinsic primary afferent neurones of the digestive tract. Neurogastroenterol Motil 16 Suppl 124–27.
- 44. Furness JB, Jones C, Nurgali K, Clerc N (2004) Intrinsic primary afferent neurons and nerve circuits within the intestine. Prog Neurobiol 72: 143–164.
- 45. Poole DP, Xu B, Koh SL, Hunne B, Coupar IM, et al. (2006) Identification of neurons that express 5-hydroxytryptamine4 receptors in intestine. Cell Tissue Res 325: 413–422.
- 46. Pineiro-Carrero VM, Clench MH, Davis RH, Andres JM, Franzini DA, et al. (1991) Intestinal motility changes in rats after enteric serotonergic neuron destruction. Am J Physiol 260: G232–239.
- 47. Taniguchi H, Ariga H, Zheng J, Ludwig K, Mantyh C, et al. (2008) Endogenous ghrelin and 5-HT regulate interdigestive gastrointestinal contractions in conscious rats. Am J Physiol Gastrointest Liver Physiol 295: G403–411.
- 48. Bulbring E, Crema A (1958) Observations concerning the action of 5-hydroxytryptamine on the peristaltic reflex. Br J Pharmacol Chemother 13: 444–457.
- 49. Kirchgessner AL, Tamir H, Gershon MD (1992) Identification and stimulation by serotonin of intrinsic sensory neurons of the submucosal plexus of the guinea pig gut: activity-induced expression of Fos immunoreactivity. J Neurosci 12: 235–248.
- 50. Kirchgessner AL, Liu MT, Gershon MD (1996) In situ identification and visualization of neurons that mediate enteric and enteropancreatic reflexes. J Comp Neurol 371: 270–286.
- 51. Cooke HJ, Sidhu M, Wang YZ (1997) 5-HT activates neural reflexes regulating secretion in the guinea-pig colon. Neurogastroenterol Motil 9: 181–186.
- 52. Kunze WA, Bornstein JC, Furness JB (1995) Identification of sensory nerve cells in a peripheral organ (the intestine) of a mammal. Neuroscience 66: 1–4.
- 53. Bertrand PP, Kunze WA, Bornstein JC, Furness JB, Smith ML (1997) Analysis of the responses of myenteric neurons in the small intestine to chemical stimulation of the mucosa. Am J Physiol 273: G422–435.
- 54. Christofi FL, Wood JD (1994) Electrophysiological subtypes of inhibitory P1 purinoceptors on myenteric neurones of guinea-pig small bowel. Br J Pharmacol 113: 703–710.
- 55. Moneta NA, McDonald TJ, Cook MA (1997) Endogenous adenosine inhibits evoked substance P release from perifused networks of myenteric ganglia. Am J Physiol 272: G38–45.
- 56. Costa M, Brookes SJ, Steele PA, Gibbins I, Burcher E, et al. (1996) Neurochemical classification of myenteric neurons in the guinea-pig ileum. Neuroscience 75: 949–967.
- 57. Mann PT, Southwell BR, Ding YQ, Shigemoto R, Mizuno N, et al. (1997) Localisation of neurokinin 3 (NK3) receptor immunoreactivity in the rat gastrointestinal tract. Cell Tissue Res 289: 1–9.
- 58. Jenkinson KM, Morgan JM, Furness JB, Southwell BR (1999) Neurons bearing NK(3) tachykinin receptors in the guinea-pig ileum revealed by specific binding of fluorescently labelled agonists. Histochem Cell Biol 112: 233–246.
- 59. Lomax AE, Furness JB (2000) Neurochemical classification of enteric neurons in the guinea-pig distal colon. Cell Tissue Res 302: 59–72.
- 60. Takahashi T (2012) Mechanism of interdigestive migrating motor complex. J Neurogastroenterol Motil 18: 246–257.
- 61. Bertrand PP, Galligan JJ (1995) Signal-transduction pathways causing slow synaptic excitation in guinea pig myenteric AH neurons. Am J Physiol 269: G710–720.
- 62. Morita K, North RA (1985) Significance of slow synaptic potentials for transmission of excitation in guinea-pig myenteric plexus. Neuroscience 14: 661–672.
- 63. Johnson PJ, Bornstein JC, Yuan SY, Furness JB (1996) Analysis of contributions of acetylcholine and tachykinins to neuro-neuronal transmission in motility reflexes in the guinea-pig ileum. Br J Pharmacol 118: 973–983.
- 64. Kilbinger H, Wolf D (1992) Effects of 5-HT4 receptor stimulation on basal and electrically evoked release of acetylcholine from guinea-pig myenteric plexus. Naunyn Schmiedebergs Arch Pharmacol 345: 270–275.
- 65. Gershon MD (2005) Nerves, reflexes, and the enteric nervous system: pathogenesis of the irritable bowel syndrome. J Clin Gastroenterol 39: S184–193.