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Am J Physiol Gastrointest Liver Physiol 295: G403-G411, 2008. First published June 19, 2008; doi:10.1152/ajpgi.90260.2008
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NEUROREGULATION AND MOTILITY

Endogenous ghrelin and 5-HT regulate interdigestive gastrointestinal contractions in conscious rats

Hiroshi Taniguchi,1,2 Hajime Ariga,1 Jun Zheng,1,2 Kirk Ludwig,1,2 Christopher Mantyh,1 Theodore N. Pappas,1 and Toku Takahashi1,2

1Department of Surgery, Duke University Medical Center, Durham, North Carolina; and 2Department of Surgery, Medical College of Wisconsin and Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin

Submitted 27 March 2008 ; accepted in final form 18 June 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Endogenous ghrelin causes interdigestive contractions of the stomach in rats. In contrast, previous studies showed that 5-HT3 and 5-HT4 receptors were involved in regulating intestinal interdigestive contractions. We studied the possible role of endogenous ghrelin and 5-HT regulating interdigestive gastrointestinal (GI) contractions in rats. Four strain gauge transducers were implanted on the antrum, duodenum, and proximal and distal jejunum. After an overnight fast, GI contractions were recorded in freely moving conscious rats and ghrelin receptor antagonists [(D-lys3)GHRP6; 1 µmol/kg], 5-HT3 antagonists (Ondansetron; 0.5 mg/kg) and 5-HT4 antagonists (GR 125,487; 1 mg/kg) were administered (bolus iv). To evaluate the relationship between the luminal concentrations of 5-HT and phase III-like contractions of the duodenum, duodenal juice was collected via the intraduodenal catheter. 5-HT content of the duodenal juice was measured by HPLC. (D-lys3)GHRP6 significantly attenuated the occurrence and amplitude of phase III-like contractions of the antrum, but not the duodenum and jejunum. 5-HT4 antagonists significantly reduced spontaneous phase III-like contractions of the jejunum, without affecting those of the antrum and duodenum. In contrast, 5-HT3 antagonists did not affect phase III-like contractions in GI tract. Luminal concentration of 5-HT at the phase III-like contraction (36.0 ± 13.3 ng/ml, n = 9) was significantly higher than that at the phase I-like contractions of the duodenum (4.9 ± 1.6 ng/ml, n = 9, P < 0.05). It is suggested that released ghrelin from the gastric mucosa mediates gastric phase III-like contractions, whereas 5-HT released from enterochromaffin cells of the duodenal mucosa mediates intestinal phase III-like contractions via 5-HT4 receptors.

5-HT3 receptors; 5-HT4 receptors; enterochromaffin cells

DURING THE INTERDIGESTIVE state, the stomach and small intestine show a remarkable motor pattern, known as the migrating motor complex (MMC) in dogs (51) and humans (56). MMC consists of three phases: phase I (period of motor quiescence), phase II (period of irregular low-amplitude contractions), and phase III (period of regular high-amplitude contractions). Phase III contractions of MMC have physiological importance for clearance of secretions, debris, and microbes during fasting and let the stomach be ready to receive a next meal (56). Plasma motilin levels are highly associated with the appearance of gastric phase III in dogs (34). Motilin administration caused phase III contractions in human and dogs (33, 60).

The characteristic feature of gastric MMC is different among species. In humans and dogs, MMC is usually observed every 90–120 min the interdigestive state. In contrast, MMC cycle is short (less than 20 min) in rats and not so regular as that of humans and dogs (2, 18, 53). Since it is rather difficult to distinguish three phases in rats, these phases are called as phase I-like contractions and phase III-like contractions in rats (3, 18, 53).

Ghrelin, a 28-amino acid peptide, was discovered as the endogenous ligand for growth hormone secretagogue receptor (GHS-R) from the rat stomach (37). Because of a structural resemblance to motilin, ghrelin is known as a motilin-related peptide (4, 54). Although motilin administration does not affect gastric emptying and gastrointestinal (GI) transit in rats (13), ghrelin stimulates GI motility in rats. Thus it is well established that motilin regulates gastric phase III contractions in dogs, whereas ghrelin regulates gastric phase III-like contractions in rats.

Ghrelin administration increases gastric motility in a dose-dependent manner in urethane-anesthetized rats (42). Ghrelin administration also causes phase III-like contractions in the antrum and duodenum in conscious rats (18, 58). We have previously showed that plasma ghrelin levels were highly associated with the occurrence of phase III-like contractions of the rat stomach (3). We also showed that spontaneous phase III-like contractions of the antrum were abolished by a GHS-R antagonist. These suggest that the spontaneous phase III-like contractions of the antrum are mediated via endogenously released ghrelin in rats (3). However, it still remains unknown whether endogenous ghrelin regulates spontaneous phase III-like contractions of the small intestine in rats.

The gastric and intestinal phase III contractions are controlled by different mechanisms in dogs. Plasma motilin level is highly associated with the appearance of gastric phase III in dogs (34). In contrast, phase III contractions in the small intestine sometimes occur without a concomitant increase in plasma motilin concentration (47).

Motilin antiserum inhibits the occurrence of phase III contractions only in the stomach, not in the intestine (38). After duodenectomy, no obvious phase III contractions were seen in the antrum, but migrating phase III contractions were seen in the upper jejunum in dogs (50). Gastric phase III is vagal dependent, whereas intestinal phase III is vagal independent in dogs (33).

The role of serotonin (5-HT) in regulating GI motility has been well documented for over 30 years (46). It has been shown that intestinal migrating myoelectrical activity are regulated via an endogenous 5-HT in rats (5, 40, 48). 5-HT stimulates phase II-like contractions when administered during phase I of the small intestine in dogs (45). In humans, 5-HT reuptake inhibitor (paroxetine) shortened the MMC cycle and increased the propagation velocity of intestinal phase III (25). In addition to motilin, 5-HT initiates gastric phase III contractions via 5-HT3 receptors in dogs (32). However, it still remains unclear whether 5-HT is involved in mediating gastric phase III-like contractions in rats.

Intestinal migrating myoelectrical activity was reduced by a 5-HT3 antagonist, but not by a 5-HT4 antagonist, in rats (40). In contrast, others showed that intestinal migrating myoelectrical activity was reduced by a 5-HT4 antagonist in rats (5). Thus it remains unclear which 5-HT receptor subtypes are involved in mediating intestinal MMC in rats.

Although 5-HT acts as a neurotransmitter of the enteric nervous system (23), the majority of 5-HT is stored in enterochromaffin (EC) cells of epithelial cells. EC cells have been considered to release 5-HT mainly into the blood vessels and/or intrinsic nerve terminal via a basolateral border (21). In contrast, others showed that 5-HT is also released into the intestinal lumen (1, 15, 28, 35). We have recently demonstrated that 5-HT is released into the lumen, but not into the portal circulation in response to luminal pressure increase of the rat proximal colon (55).

Immunoelectron microscopic study showed the anatomical evidence that 5-HT is released from EC cells in response to increase of luminal pressure of the rat duodenum. 5-HT is stored in the secretary granules of EC cells and released into the cytoplasmic matrix. Then 5-HT particles diffuse or are transported into the intestinal lumen in response to intraluminal pressure increase (17). However, it still remains unclear whether luminally released 5-HT regulates intestinal MMC in rats.

In the present study, we investigated whether ghrelin and/or 5-HT receptors are involved in mediating spontaneous phase III-like contractions in GI tract in rats. We also studied the relationship between the luminal concentration of 5-HT and spontaneous phase III-like contractions of the duodenum in conscious rats.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals. Male Sprague-Dawley rats weighing 280–340 g were kept in group cages under conditions of controlled temperature (22–24°C), humidity, and light (12-h light cycle starting at 7:00 AM) with free access to laboratory chow and water. Protocols describing the use of rats were approved by the Institutional Animal Care and Use Committee of Duke University (Durham) and Zablocki Veterans Affairs Medical Center (Milwaukee) and carried out in accordance with the National Institutes of Health "Guide for the Care and Use of Laboratory Animals." All efforts were made to minimize animal suffering and to reduce the number of animal in experiments.

Animal preparation for GI motility recording. After overnight fasting, the rats were anesthetized with intraperitoneal injection of pentobarbital sodium (45 mg/kg). Through the midline laparotomy, strain gauge transducers were implanted on the serosal surface of the antrum, duodenum, and jejunum. Duodenal transducers were implanted at 5 cm distal from the pylorus. Jejunal transducers were implanted at 15 cm (the proximal jejunum: J-1) and 25 cm (the distal jejunum: J-2) distal from the pylorus, respectively. The wires from the transducer were exteriorized through the abdominal wall and run under the skin toward the back. The intravenous catheter was inserted into the right jugular vein and similarly exteriorized to the back, as previously reported (2, 3). The catheter was filled with heparinized saline (100 unit/ml) to prevent coagulation. Wires and a catheter were protected by a protective jacket (Star Medical, Tokyo, Japan). After the surgery, the rats were housed individual and were allowed to recover for 1 wk before the experiments.

We have previously studied the effect of fixed feeding on plasma ghrelin levels and interdigestive gastric contractions in rats. Rats were trained to the assigned meal-feeding regimen once daily at 12:00–4:00 PM. We demonstrated that fixed feeding augments interdigestive gastric contractions (2). We also showed that the plasma ghrelin levels of fasted state are higher than that of fed state in rats (3). To obtain regular phase III-like contractions of GI tracts, we utilized the fixed-fed regimen, as previously reported (2).

After the implantation of transducers, rats were given food once daily at 12:00–4:00 PM. Experiments of GI motility recording were started at 9:00 AM every day. The wires from the transducer were connected to the recording system (Power-Lab model 8SP, ADI instruments, Colorado Springs, CO). GI contractions were measured with free access to water in freely moving conscious rats. Spontaneous phase III-like contractions were observed for 2–3 h. Phase III-like contractions were defined as clustered contractions with amplitude of more than 4 g, followed by a silent phase (phase I-like contractions), as previously reported (53).

Effect of various antagonist on spontaneous phase III-like contractions in the interdigestive state. Two hours after recording the spontaneous contractions, various antagonists were administered. To study the involvement of ghrelin and 5-HT in mediating spontaneous phase III-like contractions, (D-lys3)GHRP-6 (GHS-R antagonist; 1.0 µmol/kg), ondansetron (5-HT3 receptor antagonist; 0.5 mg/kg), and GR125,487 (5-HT4 receptor antagonist; 1.0 mg/kg) were administered intravenously (bolus).

We have previously showed that (D-lys3)GHRP-6 (1.0 µmol/kg) almost completely abolished phase III-like contractions induced by exogenously applied ghrelin (3). We selected doses of ondansetron (0.5 mg/kg) (40) and GR125,487 (1.0 mg/kg) (9) on the basis of the previous reports.

Five animals were randomly treated with each antagonist in crossover design. Each study was performed at least 3 days apart. Motility index (MI), peak amplitude, and frequency of phase III-like contractions of GI tract were compared 30 min before and after the administration of (D-lys3)GHRP-6, ondansetron, and GR125,487. Saline injected rats served as controls.

Luminal concentration of 5-HT of the duodenum in the interdigestive state. To evaluate the relationship between the intraluminal concentration of 5-HT and phase III-like contraction of the duodenum, an intraduodenal catheter was inserted into the lumen of the duodenum. A transducer was also implanted on the duodenum. At 1 week after the surgery, duodenal juice (20 µl) was collected during the interdigestive state in a conscious state. 5-HT content of the duodenal juice was measured by HPLC, as previously reported (55).

Effects of vagotomy on phase III-like contractions in the interdigestive state. To investigate whether vagal pathways are involved in mediating phase III-like contractions, rats received truncal vagotomy, at the time of transducer implantation. As previously reported (44), the lower part of the esophagus was exposed and anterior and posterior branches of the vagal nerves were incised above the hepatic and celiac branches. In sham-operated rats, the vagal trunks were similarly exposed but not cut. One week after the surgery, interdigestive GI motility was recorded.

We have previously showed that continuous infusion of acyl ghrelin (active form; 0.8 µg·kg–1·min–1) augmented phase III-like contraction in the rat stomach (3). In our present study, acyl ghrelin (0.8 µg·kg–1·min–1) was continuously infused for 30 min in sham-operated rats and vagotomized rats.

Chemicals. Acyl ghrelin (Tocris Cookson, Ellisville, MO) and (D-lys3)GHRP-6 (Bachem, King of Prussia, PA) were kept in powder form at –70°C. Peptides were dissolved in saline immediately before use.

Statistical analysis. Results were shown as means ± SE. Statistical analysis of the data were performed by Student's t-test to assess the difference between groups. One-way ANOVA followed by Dunnett's post hoc test was also used to test the significance of differences among groups. A P value <0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Gastrointestinal contractions in the interdigestive state and postprandial state in conscious rats. It has been showed that spontaneous phase III-like contractions are observed at 12- to 15-min intervals of the stomach (3, 18, 53) in conscious rats.

In our present study, cyclic changes of contractions were detected in the antrum, duodenum, J-1, and J-2, including a quiescence period (phase I-like contractions) followed by a grouping of strong contractions (phase III-like contractions). Spontaneous phase III-like contractions were observed every 14.8 ± 2.0 min at the antrum, 14.1 ± 2.0 min at the duodenum, 14.6 ± 1.3 min at the J-1, and 14.3 ± 1.5 min at the J-2 (n = 12).

The fasted motor patterns in the antrum, duodenum, J-1, and J-2 were disrupted immediately after food intake and were replaced by the fed motor pattern. There were no silent phases (phase I-like contractions) in the antrum, duodenum, and jejunum during the fed state (Fig. 1 and Table 1).


Figure 1
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  		Fig. 1. Effect of feeding on gastrointestinal (GI) contractions in conscious rats. The fasted motor patterns in the antrum, duodenum, proximal jejunum (jejunum-1), and distal jejunum (jejunum-2) were disrupted by food intake and replaced by the fed motor pattern. Asterisks indicate spontaneous phase III-like contractions.

 

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  		Table 1. MI and peak amplitude of the GI tract between the fasted state and fed state

 
Effect of various antagonists on phase III-like contractions. After the administration of a GHS-R antagonist, spontaneous phase III-like contractions of the antrum were significantly attenuated for 30 min (Fig. 2). MI, peak amplitude, and frequency of phase III-like contractions were significantly reduced by a GHS-R antagonist in the antrum (Table 2, Dunnett's test, P < 0.05).


Figure 2
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  		Fig. 2. Effect of (D-lys3)GHRP-6 (1 mg/kg, bolus iv) on spontaneous phase III-like contractions of the GI tract. (D-lys3)GHRP-6 significantly inhibited phase III-like contractions at the antrum, without affecting the spontaneous phase III-like contractions of duodenum, jejunum-1, and jejunum-2. Asterisks indicate spontaneous phase III-like contractions.

 

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  		Table 2. Effect of (D-lys3)GHRP-6, ondansetron, and GR125487 on % changes of MI, peak amplitude, and frequency of phase III-like contractions of GI tract

 
In contrast, MI, peak amplitude, and frequency of phase III-like contraction at the duodenum, J-1, and J-2 were not significantly affected by a GHS-R antagonist (Fig. 2 and Table 2).

After the administration of an ondansetron, spontaneous phase III-like contractions were not affected in all of the antrum, duodenum, J-1, and J-2 (Fig. 3 and Table 2).


Figure 3
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  		Fig. 3. Effect of ondansetron (1 mg/kg, bolus iv) on spontaneous phase III-like contractions of GI tract. Phase III-like contractions of all of the GI tract were not affected by ondansetron. Asterisks indicate spontaneous phase III-like contractions.

 
After the administration of a GR125,487, spontaneous phase III-like contractions were not affected in the antrum and duodenum. In contrast, phase III-like contractions of J-1 and J-2 were significantly inhibited by GR125,487 (Fig. 4). GR125,487 significantly reduced MI, peak amplitude, and frequency of phase III-like contractions at the J-1 and J-2 (Table 2, Dunnett's test, P < 0.05).


Figure 4
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  		Fig. 4. Effect of GR125,487 (1 mg/kg, bolus iv) on spontaneous phase III-like contractions of GI tract. Phase III-like contractions at the antrum and duodenum were not affected by GR125,487. In contrast, GR125,487 significantly attenuated phase III-like contractions of jejunum-1 and jejunum-2. Asterisks indicate spontaneous phase III-like contractions.

 
To study the interaction between ghrelin and 5-HT, GR125,487 was administered 15 min before ghrelin (0.8 µg·kg–1·min–1) infusion. In the antrum and duodenum, augmented phase III-like contractions were observed in response to ghrelin infusion even in the presence of GR125,487. MI, peak amplitude, and frequency of phase III-like contraction were not significantly changed at the antrum and duodenum in the presence of GR125,487 compared with only ghrelin infusion (Fig. 5 and Table 2, Student's t-test, P < 0.05).


Figure 5
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  		Fig. 5. Effect of GR125,487 (1 mg/kg, bolus iv) on ghrelin (0.8 µg·kg–1·min–1 iv)-induced phase III-like contractions of the GI tract. GR125,487 was administered 15 min before the ghrelin infusion. In the antrum and duodenum, phase III-like contractions were observed in response to ghrelin infusion in the presence of 5-HT4 antagonists. In contrast, ghrelin failed to induce phase III-like contractions of jejunum-1 and jejunum-2 were in the presence of GR125,487. Asterisks indicate spontaneous phase III-like contractions.

 
In contrast, phase III-like contractions at J-1 and J-2 were significantly inhibited by GR125,487. MI, peak amplitude, and frequency of phase III-like contraction on ghrelin infusion in the presence of GR125,487 were not significantly changed at the J-1 and J-2 compared with only ghrelin infusion (Student's t-test, P < 0.05). Ghrelin failed to stimulate any spontaneous phase III-like contractions at J-1 and J-2 in the presence of GR125,487 (Fig. 5 and Table 2).

Luminal concentration of 5-HT of the duodenum. To evaluate the correlation between the luminal concentration of 5-HT and phase III-like contraction of the duodenum, duodenal juice was collected through the duodenal catheter during the period of phase I-like and phase III-like contractions of the duodenum. The luminal concentration of 5-HT at the phase III-like contraction (36.0 ± 13.3 ng/ml, n = 9) was significantly higher than that at the phase I-like contractions of the duodenum (4.9 ± 1.6 ng/ml, n = 9, Student's t-test, P < 0.05).

Effects of vagotomy on phase III-like contractions in the interdigestive state. In sham-operated rats, spontaneous phase III-like contractions were occurred every 15 min in the antrum and jejunum. In contrast, phase III-like contractions in the antrum were no more observed in vagotomized rats. In the jejunum, however, phase III-like contractions were still observed in vagotomized rats (Fig. 6).


Figure 6
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  		Fig. 6. Effect of ghrelin (0.8 µg·kg–1·min–1) on interdigestive contractions in sham-operated (A) and vagotomized rats (B). Spontaneous phase III-like contractions of the antrum, but not the jejunum, disappeared in vagotomized rats. Ghrelin infusion-induced gastric contractions were significantly attenuated in vagotomized rats (B).

 
In sham-operated rats, phase III-like contractions were augmented in response to ghrelin infusion at the antrum. In contrast, ghrelin-induced contractions were significantly attenuated in vagotomized rats. Peak amplitude of contractions at the antrum in vagotomized rats (6.4 ± 1.1 g) was significantly lower than that of sham-operated rats (12.2 ± 1.7 g, n = 5, Student's t-test, P < 0.05).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have previously showed that GHS-R antagonists significantly inhibited spontaneous phase III-like contractions in conscious rats (3), suggesting that endogenously released ghrelin regulates spontaneous phase III-like contractions of the rat stomach. We also showed the correlation between the plasma ghrelin levels and occurrence of gastric phase I- and III-like contractions of the antrum (3).

It is not clear whether intestinal phase III-like contractions are regulated by endogenously released ghrelin. Our present study showed that GHS-R antagonists inhibited phase III-like contractions in the antrum, but not the duodenum and the jejunum. This suggests that endogenously released ghrelin plays a major role to regulate gastric, but not intestinal, spontaneous phase III-like contractions in rats. Previous studies also showed that GHS-R antagonists did not affect phase III-like contractions in the duodenum (58).

It is well known that exogenously administered ghrelin augments phase-III like contractions of the antrum (3, 18). Our present study showed that exogenously administered ghrelin augmented phase-III like contractions in the jejunum as well as the antrum. In vitro study showed that ghrelin stimulates intestinal motility and that GHS-R is present in the enteric neurons of the rat jejunum (14). It is conceivable that exogenously administered ghrelin (pharmacological dose of ghrelin) causes phase-III like contractions at the jejunum, in addition to the stomach.

Ghrelin receptors are identified on the vagal sensory fibers of the gastric wall in rats (12). Ghrelin receptors are also localized to the nodose ganglia that project to the stomach in rats (49). It is highly likely that released ghrelin from the gastric mucosa stimulates primarily vagal afferents.

It still remains controversial whether the stimulatory effect of ghrelin on gastric motor function is mediated via intrinsic neurons or extrinsic neurons. In vitro muscle strip study showed that ghrelin itself (up to 10–5 M) failed to cause any motor activity of the spontaneous contraction in rats (11, 13, 19) and mice (36), whereas others showed that ghrelin itself (10–7 to 10–6 M) stimulates contractions in a dose-dependent manner in rats (39).

Ghrelin enhances (10–9 M to 10–5 M) electrical field stimulation-evoked contractions of the gastric muscle strips in rats (11, 13, 19). Although these studies suggest the direct action of ghrelin on the gastric myenteric plexus, it still remains unclear whether the pharmacological doses of ghrelin (10–9 to 10–5 M) used for in vitro study are compatible to the doses used for in vivo study.

Our present study showed that spontaneous phase III-like contractions of the antrum completely disappeared in vagotomized rats. This suggests that spontaneous phase-III like contractions, which are regulated by endogenously released ghrelin, are mediated by the vagal pathway. We also showed that ghrelin infusion-induced gastric contractions were significantly attenuated in vagotomized rats. Others also showed that ghrelin-induced acid secretion is abolished by vagotomy in rats (61). Thus the direct action of ghrelin on the gastric wall (myenteric plexus) should be minor in physiological conditions.

It has been shown that ghrelin secretion is reduced after vagotomy (31, 59). Therefore, we cannot exclude the possibility that impaired spontaneous phase III-like contractions after vagotomy is due to decreased ghrelin secretion.

Previous reports suggest that gastric and intestinal phase III contractions are regulated by the different mechanisms in dogs. Phase III contractions in the small intestine are independent of plasma motilin concentration (47). Motilin antiserum inhibits the occurrence of phase III contractions only in the stomach (38, 50). Acute vagal nerve blockade abolished gastric phase III without affecting intestinal phase III in dogs (29).

Our present study suggests that endogenously released ghrelin can regulate spontaneous phase III-like contractions of the stomach, but not the small intestine, in conscious rats. If so, what is the mediator regulating intestinal MMC in rats?

It has been demonstrated that 5-HT is involved in mediating interdigestive contractions of the small intestine in rats (46). Subcutaneous or intravenous administration of 5-HT can induce intestinal migrating myoelectrical activity in a dose-dependent manner in rats (40, 48).

Intestinal migrating myoelectrical activity was reduced by a 5-HT3 antagonist, but not by a 5-HT4 antagonist in rats (40). However, the same group, several years later, showed that intestinal migrating myoelectrical activity was reduced by a 5-HT4 antagonist as well as a 5-HT3 antagonist in rats (5).

Our present study showed that phase III-like contractions in the jejunum, not the antrum and duodenum, were significantly attenuated by 5-HT4 antagonists. In contrast, 5-HT3 antagonists did not affect phase III-like contractions in all of the upper GI tract. These suggest that spontaneous phase III-like contractions in the jejunum are mediated via 5-HT4 receptors, but not 5-HT3 receptors.

We also showed that phase III-like contractions were still observed in response to ghrelin infusion in the presence of 5-HT4 antagonists in the antrum and duodenum. In contrast, ghrelin failed to induce any phase III-like contractions at the jejunum in the presence of 5-HT4 antagonists. This further confirms that phase III-like contractions of the antrum are mediated via GHS-R and that phase III-like contractions of the small intestine are mediated via 5-HT4 receptors.

Although 5-HT acts as a neurotransmitter of the enteric nervous system (23), the majority of 5-HT is stored in EC cells of epithelial cells of GI tract. EC cells release 5-HT into the blood vessels and intrinsic nerve terminal via a basolateral border (21). 5-HT is also released into the intestinal lumen from EC cells (1, 15, 28, 35).

Luminal pressure increase stimulates 5-HT release into the lumen, but not into the portal circulation, of the rat proximal colon (55). Immunoelectron microscopic study also showed that 5-HT is released intraluminally from EC cells in response to luminal pressure increase of the rat duodenum (17).

Luminally applied 5-HT can move by passive diffusion across the intestinal wall of the guinea pig ileum (10). Recent study showed that 5-HT can cross the intestinal wall from the mucosa to the serosa (apical-to-basolateral direction) (41). Thus 5-HT into the intestinal lumen could reach the synaptic circuitry, resulting in stimulation of 5-HT receptors located on the lamina propria.

5-HT activates enteric afferent neurons to stimulate intestinal motor function (7, 16, 30). Intraduodenal administration of 5-HT also causes intestinal myoelectrical activity in rats (57). Luminal administration of 5-HT into the proximal colon increased the fecal pellet output (20) and accelerated colonic transit (55) in rats.

5-HT3 receptors (43) and 5-HT4 receptors (52) are located on the cholinergic neurons of the myenteric plexus as well as sensory neurons of the intestinal mucosa (22). Nerve endings of sensory neurons may well be the targets for the 5-HT released from EC cells (6). It is generally accepted that 5-HT stimulates intrinsic nerve fibers via 5-HT4 receptors (16), whereas 5-HT stimulates extrinsic nerve fibers via 5-HT3 receptors (8, 22) in rats. 5-HT3 receptors are located on the nerve terminal of vagal afferent of the duodenal mucosa in rats (24). In contrast, there is no evidence of 5-HT4 receptors located on the vagal afferent or nodose ganglia.

5-HT released by mucosal stimuli initiates peristalsis by activating 5-HT4 receptors on sensory CGRP neurons of the rat colon in vitro (27). Ascending contractions and descending relaxations were inhibited by selective 5-HT4, but not by selective 5-HT3 antagonists in human jejunum in vitro (26). These suggest that 5-HT4 receptors play a major role in mediating an intrinsic neural reflex.

Because phase III-like contractions of duodenum and jejunum were not attenuated by a GHS-R antagonist, we hypothesized that ghrelin-independent intestinal phase III-like contractions may originate at the duodenum. We further hypothesized that 5-HT released from EC cells from the duodenal mucosa mediates intestinal phase III-like contractions.

To prove the hypothesis, we measured luminal concentration of 5-HT of the duodenum during the period of phase I-like contractions and phase III-like contractions. We showed that luminal concentration of 5-HT at the time of phase III-like contractions was significantly higher than that of phase I-like contractions. These results suggest that 5-HT released from EC cells of the duodenal mucosa initiates intestinal phase III-like contractions in rats. Luminally released 5-HT from duodenal EC cells stimulates duodenal phase III-like contractions via 5-HT4 receptors located on intrinsic primary afferent neurons. Our present study showed that phase III-like contractions of the jejunum were not affected by vagotomy. This indicates that intestinal phase III-like contractions occur independently of vagal pathway.

Our rat study demonstrated that spontaneous phase III-like contractions are mainly regulated by ghrelin and its own receptors in the antrum, whereas spontaneous phase III-like contractions are regulated by 5-HT and 5HT4 receptors in the jejunum. Released ghrelin from the gastric mucosa initiates gastric phase III-like contractions. 5-HT is released from EC cells of the duodenal mucosa. Released 5-HT induces intestinal phase III-like contractions via 5-HT4 receptors in rats (Fig. 7).


Figure 7
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  		Fig. 7. Proposed mechanism of phase III-like contractions of GI tract in rats. Released ghrelin from the gastric mucosa initiates gastric phase III-like contractions via vagovagal reflex. Released 5-HT induces intestinal phase III-like contractions via 5-HT4 receptors located on intrinsic primary afferent neurons (IPAN). GHS, growth hormone secretagogue.

 
However, the stimulatory mechanism of ghrelin release from the stomach and 5-HT release from the duodenum remains to be investigated.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Takahashi, Zablocki VA Medical Center, 5000 W. National Ave., Milwaukee, WI 53295 (e-mail: ttakahashi{at}mcw.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ahlman H, DeMagistris L, Zinner M, Jaffe BM. Release of immunoreactive serotonin into the lumen of the feline gut in response to vagal nerve stimulation. Science 213: 1254–1255, 1981.[Abstract/Free Full Text]
  2. Ariga H, Imai K, Chen C, Mantyh C, Pappas TN, Takahashi T. Fixed feeding potentiates interdigestive gastric motor activity in rats: importance of eating habits for maintaining interdigestive MMC. Am J Physiol Gastrointest Liver Physiol 294: G655–G659, 2008.[Abstract/Free Full Text]
  3. Ariga H, Tsukamoto K, Chen C, Mantyh C, Pappas TN, Takahashi T. Endogenous acyl ghrelin is involved in mediating spontaneous phase III-like contractions of the rat stomach. Neurogastroenterol Motil 19: 675–680, 2007.[CrossRef][Web of Science][Medline]
  4. Asakawa A, Inui A, Kaga T, Yuzuriha H, Nagata T, Ueno N, Makino S, Fujimiya M, Niijima A, Fujino MA, Kasuga M. Ghrelin is an appetite-stimulatory signal from stomach with structural resemblance to motilin. Gastroenterology 120: 337–345, 2001.[CrossRef][Web of Science][Medline]
  5. Axelsson LG, Wallin B, Gillberg PG, Sjoberg B, Soderberg C, Hellstrom PM. Regulatory role of 5-HT and muscarinic receptor antagonists on the migrating myoelectric complex in rats. Eur J Pharmacol 467: 211–218, 2003.[CrossRef][Web of Science][Medline]
  6. Berthoud HR, Kressel M, Raybould HE, Neuhuber WL. Vagal sensors in the rat duodenal mucosa: distribution and structure as revealed by in vivo DiI-tracing. Anat Embryol (Berl) 191: 203–212, 1995.[Medline]
  7. Bertrand PP, Kunze WA, Furness JB, Bornstein JC. The terminals of myenteric intrinsic primary afferent neurons of the guinea-pig ileum are excited by 5-hydroxytryptamine acting at 5-hydroxytryptamine-3 receptors. Neuroscience 101: 459–469, 2000.[CrossRef][Web of Science][Medline]
  8. Blackshaw LA, Grundy D. Effects of 5-hydroxytryptamine (5-HT) on the discharge of vagal mechanoreceptors and motility in the upper gastrointestinal tract of the ferret. J Auton Nerv Syst 45: 51–59, 1993.[CrossRef][Web of Science][Medline]
  9. Clayton NM, Gale JD. 5-HT4 receptors are not involved in the control of small intestinal transit in the fasted conscious rat. Neurogastroenterol Motil 8: 1–8, 1996.[Web of Science][Medline]
  10. Cooke HJ, Montakhab M, Wade PR, Wood JD. Transmural fluxes of 5-hydroxytryptamine in guinea pig ileum. Am J Physiol Gastrointest Liver Physiol 244: G421–G425, 1983.[Abstract/Free Full Text]
  11. Dass NB, Munonyara M, Bassil AK, Hervieu GJ, Osbourne S, Corcoran S, Morgan M, Sanger GJ. Growth hormone secretagogue receptors in rat and human gastrointestinal tract and the effects of ghrelin. Neuroscience 120: 443–453, 2003.[CrossRef][Web of Science][Medline]
  12. Date Y, Murakami N, Toshinai K, Matsukura S, Niijima A, Matsuo H, Kangawa K, Nakazato M. The role of the gastric afferent vagal nerve in ghrelin-induced feeding and growth hormone secretion in rats. Gastroenterology 123: 1120–1128, 2002.[CrossRef][Web of Science][Medline]
  13. Depoortere I, De Winter B, Thijs T, De Man J, Pelckmans P, Peeters T. Comparison of the gastroprokinetic effects of ghrelin, GHRP-6 and motilin in rats in vivo and in vitro. Eur J Pharmacol 515: 160–168, 2005.[CrossRef][Web of Science][Medline]
  14. Edholm T, Levin F, Hellstrom PM, Schmidt PT. Ghrelin stimulates motility in the small intestine of rats through intrinsic cholinergic neurons. Regul Pept 121: 25–30, 2004.[CrossRef][Web of Science][Medline]
  15. Ferrara A, Zinner MJ, Jaffe BM. Intraluminal release of serotonin, substance P, and gastrin in the canine small intestine. Dig Dis Sci 32: 289–294, 1987.[CrossRef][Web of Science][Medline]
  16. Foxx-Orenstein AE, Kuemmerle JF, Grider JR. Distinct 5-HT receptors mediate the peristaltic reflex induced by mucosal stimuli in human and guinea pig intestine. Gastroenterology 111: 1281–1290, 1996.[CrossRef][Web of Science][Medline]
  17. Fujimiya M, Okumiya K, Kuwahara A. Immunoelectron microscopic study of the luminal release of serotonin from rat enterochromaffin cells induced by high intraluminal pressure. Histochem Cell Biol 108: 105–113, 1997.[CrossRef][Web of Science][Medline]
  18. Fujino K, Inui A, Asakawa A, Kihara N, Fujimura M, Fujimiya M. Ghrelin induces fasted motor activity of the gastrointestinal tract in conscious fed rats. J Physiol 550: 227–240, 2003.[Abstract/Free Full Text]
  19. Fukuda H, Mizuta Y, Isomoto H, Takeshima F, Ohnita K, Ohba K, Omagari K, Taniyama K, Kohno S. Ghrelin enhances gastric motility through direct stimulation of intrinsic neural pathways and capsaicin-sensitive afferent neurones in rats. Scand J Gastroenterol 39: 1209–1214, 2004.[CrossRef][Web of Science][Medline]
  20. Fukumoto S, Tatewaki M, Yamada T, Fujimiya M, Mantyh C, Voss M, Eubanks S, Harris M, Pappas TN, Takahashi T. Short-chain fatty acids stimulate colonic transit via intraluminal 5-HT release in rats. Am J Physiol Regul Integr Comp Physiol 284: R1269–R1276, 2003.[Abstract/Free Full Text]
  21. Gershon MD. Nerves, reflexes, and the enteric nervous system: pathogenesis of the irritable bowel syndrome. J Clin Gastroenterol 39: S184–S193, 2005.[CrossRef][Web of Science][Medline]
  22. Gershon MD. Review article: roles played by 5-hydroxytryptamine in the physiology of the bowel. Aliment Pharmacol Ther 13, Suppl 2: 15–30, 1999.[CrossRef][Web of Science][Medline]
  23. Gershon MD, Drakontides AB, Ross LL. Serotonin: synthesis and release from the myenteric plexus of the mouse intestine. Science 149: 197–199, 1965.[Abstract/Free Full Text]
  24. Glatzle J, Sternini C, Robin C, Zittel TT, Wong H, Reeve JR Jr, Raybould HE. Expression of 5-HT3 receptors in the rat gastrointestinal tract. Gastroenterology 123: 217–226, 2002.[CrossRef][Web of Science][Medline]
  25. Gorard DA, Libby GW, Farthing MJ. 5-Hydroxytryptamine and human small intestinal motility: effect of inhibiting 5-hydroxytryptamine reuptake. Gut 35: 496–500, 1994.[Abstract/Free Full Text]
  26. Grider JR, Foxx-Orenstein AE, Jin JG. 5-Hydroxytryptamine4 receptor agonists initiate the peristaltic reflex in human, rat, and guinea pig intestine. Gastroenterology 115: 370–380, 1998.[CrossRef][Web of Science][Medline]
  27. Grider JR, Kuemmerle JF, Jin JG. 5-HT released by mucosal stimuli initiates peristalsis by activating 5-HT4/5-HT1p receptors on sensory CGRP neurons. Am J Physiol Gastrointest Liver Physiol 270: G778–G782, 1996.[Abstract/Free Full Text]
  28. Gronstad K, Dahlstrom A, Florence L, Zinner MJ, Ahlman J, Jaffe BM. Regulatory mechanisms in endoluminal release of serotonin and substance P from feline jejunum. Dig Dis Sci 32: 393–400, 1987.[CrossRef][Web of Science][Medline]
  29. Hall KE, Greenberg GR, El-Sharkawy TY, Diamant NE. Vagal control of migrating motor complex-related peaks in canine plasma motilin, pancreatic polypeptide, and gastrin. Can J Physiol Pharmacol 61: 1289–1298, 1983.[Web of Science][Medline]
  30. Hicks GA, Coldwell JR, Schindler M, Ward PA, Jenkins D, Lynn PA, Humphrey PP, Blackshaw LA. Excitation of rat colonic afferent fibres by 5-HT(3) receptors. J Physiol 544: 861–869, 2002.[Abstract/Free Full Text]
  31. Hosoda H, Kangawa K. The autonomic nervous system regulates gastric ghrelin secretion in rats. Regul Pept 146: 12–18, 2008.[CrossRef][Web of Science][Medline]
  32. Itoh Z. Motilin and clinical application. Peptides 18: 593–608, 1997.[CrossRef][Web of Science][Medline]
  33. Itoh Z, Honda R, Hiwatashi K, Takeuchi S, Aizawa I, Takayanagi R, Couch EF. Motilin-induced mechanical activity in the canine alimentary tract. Scand J Gastroenterol Suppl 39: 93–110, 1976.[Medline]
  34. Itoh Z, Takeuchi S, Aizawa I, Mori K, Taminato T, Seino Y, Imura H, Yanaihara N. Changes in plasma motilin concentration and gastrointestinal contractile activity in conscious dogs. Am J Dig Dis 23: 929–935, 1978.[CrossRef][Web of Science][Medline]
  35. Kellum J, McCabe M, Schneier J, Donowitz M. Neural control of acid-induced serotonin release from rabbit duodenum. Am J Physiol Gastrointest Liver Physiol 245: G824–G831, 1983.[Abstract/Free Full Text]
  36. Kitazawa T, De Smet B, Verbeke K, Depoortere I, Peeters TL. Gastric motor effects of peptide and non-peptide ghrelin agonists in mice in vivo and in vitro. Gut 54: 1078–1084, 2005.[Abstract/Free Full Text]
  37. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402: 656–660, 1999.[CrossRef][Web of Science][Medline]
  38. Lee KY, Chang TM, Chey WY. Effect of rabbit antimotilin serum on myoelectric activity and plasma motilin concentration in fasting dog. Am J Physiol Gastrointest Liver Physiol 245: G547–G553, 1983.[Abstract/Free Full Text]
  39. Levin F, Edholm T, Ehrstrom M, Wallin B, Schmidt PT, Kirchgessner AM, Hilsted LM, Hellstrom PM, Naslund E. Effect of peripherally administered ghrelin on gastric emptying and acid secretion in the rat. Regul Pept 131: 59–65, 2005.[CrossRef][Web of Science][Medline]
  40. Lordal M, Hellstrom PM. Serotonin stimulates migrating myoelectric complex via 5-HT3-receptors dependent on cholinergic pathways in rat small intestine. Neurogastroenterol Motil 11: 1–10, 1999.[Medline]
  41. Martel F, Monteiro R, Lemos C, Vieira-Coelho MA. In vitro and in vivo effect of fluoxetine on the permeability of 3H-serotonin across rat intestine. Can J Physiol Pharmacol 82: 940–950, 2004.[CrossRef][Web of Science][Medline]
  42. Masuda Y, Tanaka T, Inomata N, Ohnuma N, Tanaka S, Itoh Z, Hosoda H, Kojima M, Kangawa K. Ghrelin stimulates gastric acid secretion and motility in rats. Biochem Biophys Res Commun 276: 905–908, 2000.[CrossRef][Web of Science][Medline]
  43. Miyata K, Kamato T, Nishida A, Ito H, Yuki H, Yamano M, Tsutsumi R, Katsuyama Y, Honda K. Role of the serotonin3 receptor in stress-induced defecation. J Pharmacol Exp Ther 261: 297–303, 1992.[Abstract/Free Full Text]
  44. Nakamura K, Takahashi T, Taniuchi M, Hsu CX, Owyang C. Nicotinic receptor mediates nitric oxide synthase expression in the rat gastric myenteric plexus. J Clin Invest 101: 1479–1489, 1998.[Web of Science][Medline]
  45. Ormsbee HS 3rd, Silber DA, Hardy FE Jr. Serotonin regulation of the canine migrating motor complex. J Pharmacol Exp Ther 231: 436–440, 1984.[Abstract/Free Full Text]
  46. Pineiro-Carrero VM, Clench MH, Davis RH, Andres JM, Franzini DA, Mathias JR. Intestinal motility changes in rats after enteric serotonergic neuron destruction. Am J Physiol Gastrointest Liver Physiol 260: G232–G239, 1991.[Abstract/Free Full Text]
  47. Poitras P, Steinbach JH, VanDeventer G, Code CF, Walsh JH. Motilin-independent ectopic fronts of the interdigestive myoelectric complex in dogs. Am J Physiol Gastrointest Liver Physiol 239: G215–G220, 1980.[Abstract/Free Full Text]
  48. Sagrada A, Brancaccio N, Schiavone A. 5-Hydroxytryptamine affects rat migrating myoelectric complexes through different receptor subtypes: evidence from 5-hydroxytryptophan administration. Life Sci 46: 1207–1216, 1990.[CrossRef][Web of Science][Medline]
  49. Sakata I, Yamazaki M, Inoue K, Hayashi Y, Kangawa K, Sakai T. Growth hormone secretagogue receptor expression in the cells of the stomach-projected afferent nerve in the rat nodose ganglion. Neurosci Lett 342: 183–186, 2003.[CrossRef][Web of Science][Medline]
  50. Suzuki H, Mochiki E, Haga N, Shimura T, Itoh Z, Kuwano H. Effect of duodenectomy on gastric motility and gastric hormones in dogs. Ann Surg 233: 353–359, 2001.[CrossRef][Web of Science][Medline]
  51. Szurszewski JH. A migrating electric complex of canine small intestine. Am J Physiol 217: 1757–1763, 1969.[Free Full Text]
  52. Talley NJ. Serotoninergic neuroenteric modulators. Lancet 358: 2061–2068, 2001.[CrossRef][Medline]
  53. Tatewaki M, Harris M, Uemura K, Ueno T, Hoshino E, Shiotani A, Pappas TN, Takahashi T. Dual effects of acupuncture on gastric motility in conscious rats. Am J Physiol Regul Integr Comp Physiol 285: R862–R872, 2003.[Abstract/Free Full Text]
  54. Tomasetto C, Karam SM, Ribieras S, Masson R, Lefebvre O, Staub A, Alexander G, Chenard MP, Rio MC. Identification and characterization of a novel gastric peptide hormone: the motilin-related peptide. Gastroenterology 119: 395–405, 2000.[CrossRef][Web of Science][Medline]
  55. Tsukamoto K, Ariga H, Mantyh C, Pappas TN, Yanagi H, Yamamura T, Takahashi T. Luminally released serotonin stimulates colonic motility and accelerates colonic transit in rats. Am JÅ;Physiol Regul Integr Comp Physiol 293: R64–R69, 2007.
  56. Vantrappen G, Janssens J, Hellemans J, Ghoos Y. The interdigestive motor complex of normal subjects and patients with bacterial overgrowth of the small intestine. J Clin Invest 59: 1158–1166, 1977.[Web of Science][Medline]
  57. Wagner C, Clayton MK, Gallegos J, Bass P, Oaks JA. Intraduodenal serotonin elicits non-propagating spike potentials in the small intestine of the rat. Comp Biochem Physiol A Mol Integr Physiol 136: 591–603, 2003.[CrossRef][Medline]
  58. Wang Y, Dong L, Cheng Y, Zhao P. Effects of ghrelin on feeding regulation and interdigestive migrating complex in rats. Scand J Gastroenterol 42: 447–453, 2007.[CrossRef][Web of Science][Medline]
  59. Williams DL, Grill HJ, Cummings DE, Kaplan JM. Vagotomy dissociates short- and long-term controls of circulating ghrelin. Endocrinology 144: 5184–5187, 2003.[Abstract/Free Full Text]
  60. Wingate DL, Ruppin H, Green WE, Thompson HH, Domschke W, Wunsch E, Demling L, Ritchie HD. Motilin-induced electrical activity in the canine gastrointestinal tract. Scand J Gastroenterol Suppl 39: 111–118, 1976.[CrossRef][Medline]
  61. Yakabi K, Ro S, Onouhi T, Tanaka T, Ohno S, Miura S, Johno Y, Takayama K. Histamine mediates the stimulatory action of ghrelin on acid secretion in rat stomach. Dig Dis Sci 51: 1313–1321, 2006.[CrossRef][Web of Science][Medline]



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