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Keywords:

  • acetylcholine;
  • gastric motility;
  • ghrelin;
  • guinea pig;
  • nitric oxide;
  • vago-vagal reflex

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Competing interests
  8. References

Background  Ghrelin stimulates gastric motility in rats, mice and humans. Although ghrelin and the ghrelin receptor are known to be expressed in the guinea-pig gastrointestinal tract, the effects of ghrelin on gastric motility have not been examined. Aim of the present study was to clarify the motor-stimulating action of ghrelin in the guinea-pig stomach.

Methods  Gastric motility was measured as intraluminal pressure changes using a balloon inserted in the stomach of urethane-anaesthetized guinea pigs. The effects of ghrelin on gastric muscle contraction and [3H]-efflux from [3H]-choline-loaded strips were investigated in vitro.

Key Results  Ghrelin (0.3–30 μg kg−1, i.v.) increased gastric motility in a dose-dependent manner but des-acyl ghrelin was ineffective. The action of ghrelin was completely inhibited by hexamethonium and d-Lys3-growth-hormone releasing peptide-6. Atropine partially decreased the stimulatory action of ghrelin. In capsaicin-pretreated guinea pigs, the ghrelin-induced response was markedly decreased. Ghrelin (1 μmol L−1) did not affect [3H]-efflux in non-stimulated preparations but significantly decreased electrical field stimulation (EFS)-induced [3H]-efflux. l-Nitro arginine methylester (l-NAME) attenuated the inhibition of [3H]-efflux by ghrelin. Ghrelin did not cause any mechanical changes in gastric strips. Electrical field stimulation caused relaxation of gastric strips, which changed to atropine-sensitive contraction in the presence of l-NAME. Relaxation induced by EFS was slightly potentiated, but the EFS-induced contraction was not affected by ghrelin.

Conclusions & Inferences  Ghrelin stimulates gastric motility of the guinea pig through activation of capsaicin-sensitive vago-vagal reflex pathway including efferent cholinergic neurons. Peripheral ghrelin receptors on enteric nitrergic nerves might affect the ghrelin-induced gastric action by releasing nitric oxide.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Competing interests
  8. References

Ghrelin is an endogenous ligand for the growth hormone secretagogue-receptor (ghrelin receptor). Although ghrelin is mainly produced in the oxyntic mucosa of the stomach and possesses potent growth hormone releasing activity, other biological activities of ghrelin include stimulation of gastrointestinal motility and gastric acid secretion, modulation of food intake and energy metabolism, and regulation of endocrine and exocrine functions.1–3 The multifunctional faces of ghrelin are supported by wide expression of mRNA/protein for the ghrelin receptor in the central nervous system and in several peripheral tissues.1,2

The physiological role of ghrelin in the regulation of gastrointestinal motility has been investigated in small experimental animals and humans. In conscious rats and mice, ghrelin accelerates gastric emptying and enhances intestinal transit.4–7 Ghrelin also accelerates gastric emptying in healthy and idiopathic gastroparalytic humans.8 Measurement of gastric motility in conscious rats and mice indicated that ghrelin augmented phase III-like contractions, while ghrelin receptor antagonists and atropine abolished the occurrence of phase III-like contractions.9–11 Ghrelin also induced a premature phase III motor complex in humans.12 Ghrelin-induced gastric phase III-like contractions and increase in gastric emptying of rats were abolished by vagotomy or treatment with capsaicin, suggesting that a vago-vagal reflex pathway is involved in the motor-stimulating action of ghrelin9,13 as in ghrelin-induced growth hormone release and feeding.14 Besides a vago-vagal reflex pathway, the functional role of peripheral enteric ghrelin receptor has been demonstrated in isolated gastrointestinal strips of rats and mice.6,7,13,15–17 However, ghrelin did not stimulate canine gastrointestinal motility in vivo18 and ghrelin did not affect neural contraction of the rabbit stomach in vitro.19 These species-dependent different gastrointestinal motor-stimulating actions of ghrelin are similar to those of a ghrelin-related peptide, motilin. Motilin stimulated gastrointestinal motility of rabbits both in vivo and in vitro20 but had no effect on the gastrointestinal motility of mice and rats, the most classical experimental animals, in contrast to ghrelin.

The guinea pig is another experimental animal species and is widely used for analysis of gastrointestinal motor function because of easy separation of longitudinal and circular muscles and dense network of enteric neurons. Guinea-pig motilin has been identified, and the expression of motilin and the motilin receptor in the enteric nervous systems of the guinea pig have been demonstrated.21,22 Although electrical physiological study indicated the definite action of motilin on enteric neurons,23 motilin did not cause marked contraction24 and modification of neural responses in guinea-pig intestine.25 In addition to expression of motilin and its receptor, presence of ghrelin and ghrelin receptor have been also demonstrated in enteric neurons of the guinea-pig intestine.22 However, functional studies to clarify the effect of ghrelin on gastrointestinal motility have not been carried out in this species.

In the present experiments, to investigate ghrelin-induced motor actions, effects of ghrelin on gastric motility were observed in an anaesthetized guinea pig. The action of ghrelin on the peripheral ghrelin receptor was also examined in vitro using isolated gastric muscle strips. Effects of ghrelin on acetylcholine release were examined using [3H]-choline-loaded gastric muscles to characterize the action on enteric neurons.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Competing interests
  8. References

Animals

Hartley guinea pigs of both sexes (weighing 200–250 g) obtained from Sankyo Lab Service (Sapporo, Japan) were used in the present experiments. All procedures were approved by the Medical Ethics Committee of Rakuno Gakuen University. Guinea pigs were housed in stainless-steel cages at a controlled temperature (22 ± 2 °C) and at 60–65% relative humidity with a normal 12 : 12 h light/dark cycle.

In vivo study

Guinea pigs were anaesthetized with urethane (1.3 g kg−1, i.p.). After anaesthesia had been completed, a polyethylene cannula was inserted into the left carotid vein for systemic application of ghrelin and other drugs. The abdominal cavity was opened by midline incision, and the stomach was exposed. To measure gastric motility, intraluminal pressure change was detected by a rubber balloon inserted into the gastric antral region. After each experiment, the stomach was cut open and the positioning of the balloon in the antrum was verified. The balloon was filled with distilled water (23–24 °C), with pressure applied to the balloon usually set at 10 cmH2O, and was connected to a pressure transducer equipped with an ink-writing polygraph (RM-6200, Nihon Kohden, Tokyo, Japan) and computer-aided analysis system (Mac Lab, Japan Bioresearch Center, Gifu, Japan). The animals were allowed to equilibrate for 60 min, at which time a steady baseline of gastric motility was established. Drugs including ghrelin were dissolved in saline and administered through the carotid vein. The gastric motor actions evoked by ghrelin and des-acyl ghrelin and the inhibitory effects of antagonists on ghrelin-induced responses were examined. For quantitative analysis, the motor index was calculated by measurement of the area surrounded by the contraction waves and baseline during a 5-min period before and after administration of ghrelin. The increase in motility was calculated by the following equation: Increase in motor index (%) = 100 × [(motor index after stimulation/motor index before stimulation) − 1].

Capsaicin treatment is carried out to destroy afferent sensory fibres in vagus nerves. Capsaicin was injected into guinea pigs as previously reported.26 In brief, capsaicin (l0 mg mL−1) was dissolved in 10% ethyl alcohol and 10% Tween 80, and diluted in saline. Multiple subcutaneous injections were given over a period of 2 days (4 times per day) starting with a dose of 0.3 mg kg−1, and the total dose of capsaicin was 55 mg kg−1. To counteract capsaicin-induced respiratory impairment, the animals were pretreated with aminophylline (4 mg kg−1), diphenhydramine (2.5 mg kg−1) and atropine (1.5 mg kg−1) (i.p.), 20 min before each capsaicin injection.26

In vitro contraction study

Gastric strips isolated from the antrum were suspended vertically in an organ bath (3 mL) to measure circular muscle contraction. The organ bath contained warmed (37 °C) Krebs solution (mmol L−1): NaCl, 118; KCl, 4.75; MgSO4, 1.2; KH2PO4, 1.2; CaCl2, 2.5; NaHCO2, 25 and glucose, 11.5 equilibrated with 95%O+ 5%CO2 (pH = 7.4). Mechanical activity of preparations was measured with an isometric force transducer and recorded on an ink-writing recorder and computer-aided analysis system. The initial load was set at 0.5 g for each preparation. The organ bath was rinsed with Krebs solution every 15 min and allowed to equilibrate for 1 h. Prior to the addition of ghrelin and electrical field stimulation (EFS), the preparations were subjected to three or four stimulations with 50 mmol L−1 KCl until a reproducible contraction was obtained. After observing the spontaneous contraction and settled tonus, ghrelin was applied to investigate the action on non-stimulated preparations. Electrical field stimulation (1, 3 and 10 Hz; 15 s at 5-min intervals; 0.5-ms duration) was applied to the preparation through a pair of platinum ring electrodes fixed on the top and bottom of the bath. In the normal condition, EFS caused a frequency-dependent relaxation, and phasic off-contraction was sometimes evoked after cessation of EFS at 3 and 10 Hz. Treatment with l-nitro-arginine methylester (l-NAME, 100 μmol L−1) reversed the relaxation to contractile responses. Effects of ghrelin on both EFS (1 and 3 Hz)-induced relaxation and contraction were examined in the present study. Briefly after observing three EFSs (for 15 s) at 5-min intervals, ghrelin (1 μmol L−1) was applied in an organ bath after 2.5 min of last EFS, and 2.5 min later test EFS was applied three times at 5-min intervals. Amplitude of contraction or relaxation was normalized using the responses evoked by first EFS in the absence of ghrelin to evaluate the ghrelin-induced action.

In vitro release study

The effects ghrelin on release of acetylcholine were examined in guinea-pig antral circular muscle preparations loaded with [3H]-choline. The isolated preparations were incubated with 140 nmol L−1 [3H]-choline for 60 min in warmed Krebs solution (37°C), equilibrated with 95% O+ 5% CO2. After washing in fresh Krebs solution for 30 min, the preparations were immersed in 2 mL Krebs solution containing hemicholinium-3 (10 μmol L−1). The incubation medium (37 °C, bubbled with gas mixture) was sequentially changed at 5-min intervals. First, to investigate the effect of ghrelin on [3H]-outflow of a non-stimulated strip, the preparation was stimulated with 1 μmol L−1 ghrelin for 5 min and [3H]-effluxes before and after stimulation were compared. Ghrelin has been reported to stimulate nitric oxide release from nitrergic nerves in the rat stomach.27 Therefore, ghrelin-induced action was also examined in l-NAME-pretreated gastric preparations. Next, the effect of ghrelin on EFS-evoked [3H]-efflux was investigated to clarify modification of neurally evoked acetylcholine release by ghrelin. The protocols were as follows; first, EFS (S1, 4 Hz for 2 min, 30 V, 1-ms duration) was applied through two platinum ring electrodes fixed on the top and bottom of the preparations at 35 min of the start of experiments, and a second stimulation (S2) was applied 60 min after S1 in the absence (control) and presence of 1 μmol L−1 ghrelin. At the end of each experiment, the tissue was dissolved in Soluene (500 μL) and the radioactivity in the tissue and that in the incubation medium were measured in a scintillation counter. The [3H]-outflow was expressed as the fractional rate, in which the amount of radioactivity in the incubation medium was divided by the total radioactivity present in the tissue, at the same collection period. The [3H]-content of the tissue at each period was calculated by cumulatively adding the amount of [3H] in each fraction to the [3H]-content of the tissue at the end of the experiments. The inhibitory or excitatory effect of ghrelin on [3H]-efflux was evaluated by comparison of the S2/S1 ratio in the absence (control) and presence of ghrelin (1 μmol L−1).

Chemicals

The following chemicals were used in the present experiments: atropine sulphate (Sigma, St Louis, MI, USA), capsaicin (Wako, Osaka, Japan), des-n-octanoyl rat ghrelin (Des-acyl rat ghrelin, Peptide Institute. Inc. Osaka, Japan), d-Lys3-growth-hormone releasing peptide-6 (d-Lys3-GHRP-6, Funakoshi, Tokyo, Japan), rat ghrelin (Peptide Institute, Inc.), hexamethonium chloride (Tokyo Chemical Industry, Tokyo, Japan), hemicholinium-3 (Sigma), l-nitro-arginine methylester (l-NAME, Sigma), aminophylline (Eisai, Tokyo, Japan) and diphenhydramine (Mitsubishi Tanabe Pharma Corporation, Osaka, Japan).

Statistics

The results of experiments are expressed as means ± SEM of at least three experiments using animal and gastric strips from different guinea pigs. The significance of differences between two groups (single comparison) was determined by Student’s t-test (paired and unpaired). A P value of 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Competing interests
  8. References

In vivo study

Intravenously applied ghrelin (0.3–30 μg kg−1) increased gastric motility in a dose-dependent manner. The excitatory actions were evoked immediately after injection of ghrelin, continued for 3–5 min, and returned to the control level within 10 min after application (Figs 1 and 2A). Dose–response curve for ghrelin was constructed using the motor index from 0 to 5 min after application. Increases in motility index reached a plateau at 10 μg kg−1 and tended to decrease at 30 μg kg−1 (bell-shaped concentration–response curve, Fig. 2B). N-Octanoyl modification of Ser3 is essential for the biological activity of ghrelin. In the present experiments, the same doses of ghrelin and des-acyl ghrelin (10 μg kg−1) were injected and the responses were compared. Des-acyl ghrelin did not produce any stimulating actions in the guinea-pig stomach (motor index = 0.1 ± 0.04%, n = 4) (Fig. 3). The effect of d-Lys3-GHRP-6 (300 nmol kg−1) was investigated to confirm that the ghrelin receptor was involved in the motor-stimulating action. d-Lys3-GHRP-6 itself did not cause motor activity changes (7 ± 5.8%, n = 5), but treatment with d-Lys3-GHRP-6 almost completely abolished the ghrelin (10 μg kg−1) induced action (motor index = 0.2 ± 0.18%, n = 4) (Fig. 3).

image

Figure 1.  Typical mechanical actions of five increasing doses of ghrelin on gastric motility of an anaesthetized guinea pig. Traces were obtained in one guinea-pig preparation and ghrelin was applied at 30-min intervals. Each dose of ghrelin (0.3–30 μg kg−1) was applied intravenously at the point indicated by (bsl00066).

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image

Figure 2.  Time course and dose-dependency of ghrelin-induced gastric motor-stimulating action in anaesthetized guinea-pigs. (A) Each symbol indicates time-dependent changes in gastric motor activity induced by five different doses of ghrelin (0.3, 1, 3, 10 and 30 μg kg−1). Ordinate: Motor index expressed as percentage increase from baseline activity. Abscissa: Time (min) after application of each dose of ghrelin. (B) Dose–response curves of ghrelin in control (bsl00001) and capsaicin-treated guinea-pigs (•). Ordinate: Motor index expressed as percentage increase from baseline activity (from 0 to 5 min). Abscissa: dose of ghrelin (μg kg−1). Symbols and vertical bars are means and SEM of four experiments. *P < 0.05, significantly different from corresponding control values.

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image

Figure 3.  Effects of d-Lys3-GHRP-6, hexamethonium and atropine on the motor-stimulating actions of ghrelin in anaesthetized guinea-pigs. Motor-stimulating action of ghrelin (10 μg kg−1) was almost abolished by d-Lys3-GHRP-6 (d-Lys3, 300 nmol kg−1) and hexamethonium (Hexa, 2 mg kg−1). However, atropine (Atr, 0.5, 2.5mg kg−1) only partially inhibited the responses to ghrelin. The action of des-acyl ghrelin (10 μg kg−1) was also indicated for comparison with ghrelin. Columns and vertical bars are means and SEM of four experiments. *P < 0.05 compared with the control values.

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Effects of atropine and hexamethonium on the response to ghrelin were examined to estimate the mechanisms involved in the gastric motor-stimulating action of ghrelin. Neither atropine (0.5 and 2.5 mg kg−1) nor hexamethonium (2 mg kg−1) caused a marked decrease in gastric motility (0.5–1% of control level). Atropine (0.5 mg kg−1) significantly decreased the ghrelin (10 μg kg−1)-induced contraction, but a part of the contraction (motor index = 7.7 ± 2.9%, n = 4, control = 19.2 ± 2.4%) remained in the presence of atropine. Almost the same magnitude of ghrelin-induced contraction was remained in the presence of a high dose of atropine (2.5 mg kg−1, 6.8 ± 2.4%, n = 4) (Fig. 3). Atropine-resistant gastric action of ghrelin (10 μg kg−1) was abolished by additional application of d-Lys3-GHRP-6 (motor index = −0.3 ± 1.4%, n = 3). Treatment with hexamethonium (2 mg kg−1) completely inhibited the ghrelin (10 μg kg−1)-induced motor- stimulating action (motor index = 0.1 ± 0.02%, n = 4) (Fig. 3).

In capsaicin-treated guinea pigs, ghrelin did not cause any mechanical responses and the dose–response curve was shifted downward (Fig. 2B).

In vitro contraction study

Isolated antrum circular muscle strips contracted spontaneously. High-K+(50 mmol L−1) caused contraction of stomach strips. Ghrelin (1 μmol L−1) did not cause any mechanical changes and did not modify the spontaneous contractility and amplitude of high-K+-induced contraction (Fig. 4A).

image

Figure 4.  Effects of ghrelin in isolated gastric antral muscle strips from guinea-pigs. (A) Typical mechanical responses to ghrelin (1 μmol L−1) and high-K+ (50 mmol L−1). (B) Electrical field stimulation (1–10 Hz) caused frequency-dependent relaxation in the normal condition (control), but the EFS-induced relaxation was changed to frequency-dependent contraction in the presence of l-NAME (100 μmol L−1).

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Electrical field stimulation was applied to gastric strips to stimulate the enteric neurons. In the control condition, EFS caused a frequency-dependent relaxation (1–10 Hz) during stimulation. After cessation of stimulation, phasic off-contraction was sometimes observed at 3 and 10 Hz but the off-contraction varied among preparations. In the presence of l-NAME, the EFS-induced relaxation was changed to contractile responses (Fig. 4B). Tetrodotoxin (1 μmol L−1) abolished the both EFS-induced relaxation and contraction. Atropine (1 μmol L−1) abolished the EFS-induced contraction of gastric strips (1–10 Hz) (data not shown). Ghrelin (1 μmol L−1) slightly, but significantly increased the EFS-induced relaxation at 1 Hz. The relative relaxations were105 ± 7% just before application of ghrelin (control, n = 5) and 129 ± 8% (n = 5) after 2.5 min of the application (P = 0.047). The ghrelin-induced increase was transient and the EFS-induced relaxation returned to the control level at 7.5 min (113 ± 7%, n = 5, P = 0.42 vs control) and at 12.5 min (115 ± 7%, n = 5, P = 0.22 vs control) after ghrelin application. 3 Hz EFS-induced relaxation also tended to increase by ghrelin but the increase was not significant (control = 103 ± 8%, ghrelin = 116 ± 9%, n = 3, P = 0.25). The effect of ghrelin on the EFS-induced contraction was also examined in l-NAME (100 μmol L−1) treated muscle strips. EFS-induced contractions in the absence and presence of ghrelin (1 μmol L−1, 2.5 min) were 106 ± 7.4% and 107 ± 6.1% (n = 8, P = 0.42 vs control) for 1 Hz, 102 ± 2% and 104 ± 4% (n = 3, P = 0.11 vs control) for 3 Hz respectively.

In vitro release study

To investigate the effect of ghrelin on acetylcholine release from enteric cholinergic neurons, [3H]-efflux from [3H]-choline-loaded gastric strips was measured. Ghrelin (1 μmol L−1) did not affect [3H]-efflux from gastric strips in the absence and presence of l-NAME. Fractional rates before and after treatment with ghrelin were 0.43 ± 0.05% and 0.38 ± 0.07% (n = 4), respectively, in control condition and 0.18 ± 0.01% and 0.19 ± 0.02% (n = 4), respectively, in l-NAME-treated strips. The effect of ghrelin on EFS-induced [3H]-efflux was also investigated. The fractional rates of [3H]-efflux induced by S1 and S2 in control conditions were 1.93 ± 0.37% and 1.77 ± 0.27% (n = 4), respectively (S2/S1 = 0.94 ± 0.05, n = 4) (Fig. 5A). As shown in Fig. 5B, ghrelin (1 μmol L−1) significantly decreased the S2/S1 ratio (0.80 ± 0.02, n = 4) compared with the control level (P = 0.035). Ghrelin-induced decrease in the S2/S1 ratio recovered to the control level in the presence of l-NAME (100 μmol L−1, S2/S1 = 0.96 ± 0.10, n = 4). The S1/S2 ratio in the presence of l-NAME was calculated to be 0.87 ± 0.06 (n = 4), which was not significantly different from that in the presence of both l-NAME and ghrelin (1 μmol L−1), indicating that ghrelin did not stimulate [3H]-efflux in l-NAME-treated gastric strips (Fig. 5B).

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Figure 5.  Effects of ghrelin on EFS-induced [3H]-efflux in isolated guinea-pig gastric antral strips. (A) EFS (4 Hz for 2 min, 30 V, 1-ms duration)-induced reproducible increase of [3H]-efflux in the normal condition. First, EFS (S1) was applied at fraction 7 and a second EFS (S2) was applied at fraction 20. Fractional rates of S1 and S2 were calculated and S2/S1 was obtained (S2/S1 = 0.94 at control). When the effect of ghrelin was investigated, ghrelin (1 μmol L−1) was contained in fraction 20 (bsl00001) and the strips were stimulated. Ordinate: [3H]-efflux expressed as fractional rate. Abscissa: fraction number (5 min). (B) Effects of ghrelin on S2/S1 ratio in guinea-pig gastric strips. S2/S1 ratio was significantly decreased by ghrelin (1 μmol L−1) in the normal condition. In the presence of l-NAME (100 μmol L−1), ghrelin-induced inhibition on S2/S1 ration was abolished. Ordinate: [3H]-efflux expressed as fractional rate. Values are means and SEM of four experiments.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Competing interests
  8. References

In the present study, we showed that ghrelin stimulated gastric motility in an anaesthetized guinea pig. This is the first study showing motor stimulatory action of ghrelin in the guinea-pig stomach. The excitatory action of ghrelin might be mediated by activation of capsaicin-sensitive vago-vagal reflex pathway including efferent cholinergic neurons. However, contribution of peripheral enteric ghrelin receptors to the gastro-stimulatory action of ghrelin is small.

Ghrelin is a stomach-derived peptide hormone that stimulates gastric functions including motility.2,3,28 To characterize the species-related variation of ghrelin-induced actions in the gastrointestinal tract, the effect of ghrelin on gastric motility was investigated in the guinea pig, a widely used small experimental animal species. Ghrelin applied intravenously stimulated gastric motility in a dose-dependent manner. Effective doses of ghrelin in the guinea pig (0.3–30 μg kg−1, i.v.) were consistent with those reported in urethane-anaesthetized rats (0.8–20 μg kg−1, i.v.)29 and conscious rats (1–10 μg kg−1, i.v.)9. Plasma ghrelin levels in rats were reported to be 500–2000 pmol L−1 30. Bolus injection of 0.3–30 μg kg−1 ghrelin in guinea pigs resulted in a calculated plasma concentration of 1.2–120 nmol L−1 ghrelin. Although plasma ghrelin levels in guinea-pigs have not been reported yet and the anaesthesia by urethane might affect responsiveness to ghrelin, the present findings suggest that the concentrations we have used are likely to be within or upper range of physiological levels and our findings are of possible biological relevance of ghrelin in regulation of guinea-pig gastric motility. Acylation with octanoyl acid on Ser3 is essential for binding with the ghrelin receptor and for induction of biological activity.7,31,32 In the present study, des-acyl ghrelin did not stimulate gastric motor activity. In addition, d-Lys3-GHRP-6, a ghrelin receptor antagonist, completely inhibited the ghrelin-induced gastric motor action. Taken together, these results suggest that rat ghrelin evokes motor-stimulating action of the guinea-pig stomach through activation of ghrelin receptor.

Functional analysis of ghrelin-induced gastrointestinal motor-stimulating actions both in vivo and in vitro indicated that there are two main mechanisms of ghrelin-induced responses. Fujino et al.9 showed that peripheral ghrelin stimulates fasted intestinal motor activity in rats by activating neuropeptide Y neurons in the brain through ghrelin receptors on vagal afferent nerves. Gastric motor-stimulating action of ghrelin in rats was also inhibited by atropine29 and by capsaicin13. Evidence that ghrelin receptor was expressed in the nodosa ganglion as confirmed by RT-PCR and in situ hybridization histochemistry14,33 and evidence that ghrelin modifies the discharges of afferent vagal neurons34 support the essential role of a vago-vagal reflex pathway in ghrelin-induced responses. Another mechanism is activation of the peripheral enteric nervous system. A ghrelin-induced fasted motor pattern was also observed in vagotomized rats.9 Ghrelin caused intestinal contraction and enhanced the EFS-induced contraction in isolated gastrointestinal strips from rats and mice.6,7,13,15−17,35 In the guinea-pig stomach, gastric motor-stimulating action of ghrelin was almost completely inhibited by treatment with capsaicin and hexamathonium. As capsaicin selectively destroys afferent sensory neurons and hexamethonium is an antagonist of cholinergic ganglionic transmission from presynaptic vagal efferent to enteric neurons, inhibition of ghrelin-induced responses by both agents suggests that ghrelin stimulates gastric motility through activation of a vago-vagal reflex pathway as demonstrated in rats.9,13 The effect of atropine was examined to characterize the efferent neurons of the vago-vagal reflex pathway. Atropine significantly decreased the ghrelin-induced mechanical responses, but a part of the responses was resistant to atropine (about 40%) in contrast to the ghrelin-induced responses of the rat stomach.29,36 Although these results suggest involvement of both cholinergic and non-cholinergic neural pathways in ghrelin-induced gastric contraction of guinea pigs, Bogeski et al.37 indicated the resistance of cholinergic transmission in rat intestine to bolus application of atropine and atropine-resistant contraction induced by EFS was not observed in the present in vitro study. Therefore, further experiments are needed to confirm the presence of non-cholinergic excitatory neurons and involvement of them in ghrelin-induced actions of guinea-pig stomach.

Immunohistochemical studies have shown the expression of ghrelin receptors in the enteric neurons of rats, humans and guinea-pigs.15,22,27 Enhancement of enteric nerve-evoked mechanical responses by ghrelin6,7,15−17 suggests the expression of functional ghrelin receptors in enteric neurons. In the present study, ghrelin did not cause any changes in mechanical activity and [3H]-efflux of non-stimulated strips. EFS caused a reproducible increase in [3H]-efflux (S2/S1 = 0.94) and ghrelin significantly decreased the S2/S1 ratio (0.8). However, in the presence of l-NAME, ghrelin did not produce significant change of the S2/S1 ratio (control = 0.87, ghrelin = 0.96). Since nitric oxide has been demonstrated to decrease acetylcholine release in the rat stomach,38 the present results suggest that ghrelin activates nitrergic neurons and that the released nitric oxide decreases [3H]-efflux. The ghrelin receptor has been demonstrated to be expressed on nitrergic neurons in the rat stomach, and ghrelin-induced nitric oxide release was confirmed by using microdialysis techniques.27 In the present study, EFS (1–10 Hz) caused relaxation of the gastric antrum, which was changed to contraction by l-NAME. Electrical field stimulation-induced contraction was decreased by atropine, indicating the presence of at least two types of enteric neurons (nitrergic and cholinergic neurons). Ghrelin increased the EFS-induced relaxation (especially at 1 Hz) but did not affect the EFS-induced contraction in the presence of l-NAME. Taken together, the results of in vitro studies suggest that ghrelin acts on ghrelin receptors on nitrergic neurons and stimulates nitric oxide release. Released nitric oxide decreases the acetylcholine release from cholinergic neurons and acts on smooth muscles directly and finally enhances the nitric oxide-mediated relaxation by EFS. In the present in vivo study, gastric motor-stimulating action of ghrelin tended to decrease at a high dose (30 μg kg−1). It is speculated that nitrergic inhibition is involved in the ghrelin-induced mechanical responses at high doses. On the other hand, ghrelin is not effective for potentiating cholinergic contraction in the presence of l-NAME. Therefore, it is considered that the ghrelin receptor is not present on cholinergic neurons of the guinea-pig stomach, consistent with the results of an in vivo study showing that ghrelin was almost ineffective in capsaicin- or hexamethonium-treated animals and a [3H]-efflux study showing that ghrelin did not affect S2/S1 ratio in the presence of l-NAME. However, the conclusion derived from the present in vitro study needs to be treated with caution considering the results of an in vitro study showing that ghrelin interacts with both cholinergic and tachykininergic neurons but not with nitrergic neurons in rat gastric strips.17

In summary, ghrelin stimulates gastric motility of the anaesthetized guinea pig through activation of vago-vagal reflex pathways including efferent cholinergic neurons. Peripheral ghrelin receptors on enteric nitrergic neurons might affect the gastric motor-stimulating action of ghrelin by releasing nitric oxide.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Competing interests
  8. References