Concentration-dependent stimulation of cholinergic motor nerves or smooth muscle by [Nle13]motilin in the isolated rabbit gastric antrum
Introduction
The 22 amino acid peptide motilin is able to induce gastrointestinal motor activity and has been implied in the regulation of phase III of the migrating motility complex in the mammalian gastrointestinal tract. Motilin is secreted by neuro-endocrine cells in the duodenal mucosa and is thought to have a hormonal action on the contractility of the gastric antrum and the proximal intestine. For a recent review see Poitras (1994). In vitro organ bath studies in man, rabbit and cat demonstrated that the contractile effects of motilin are mediated through a direct action on a smooth muscle receptor, since they are not antagonized by muscarinic blockade or by inhibition of axonal conductance with tetrodotoxin (Strunz et al., 1975; Adachi et al., 1981; Lüdtke et al., 1989; Depoortere et al., 1993, Kitazawa et al., 1994). Specific binding sites for motilin have been identified in homogenates of the antral smooth muscle layers of man, rabbit and cat (Bormans et al., 1986; Peeters et al., 1988; Depoortere et al., 1993) and biotinylated motilin binds specifically to isolated rabbit smooth muscle cells (Sakai et al., 1994).
In vivo studies, however, have demonstrated that motilin exerts its effects on gut motility predominantly by activating excitatory neuronal pathways. In the dog, a species in which motilin has no effects in vitro, motilin-induced contractions in vivo are mediated by cholinergic nerves in the antrum and by cholinergic and non-cholinergic nerves in the duodenum (Fox et al., 1983; Itoh, 1990). However, also in man and rabbit, species with a smooth muscle motilin-receptor, the effect of motilin in the gastric antrum in vivo, is blocked by atropine (Boivin et al., 1995; Kitazawa et al., 1994). Therefore, a discrepancy exists between the in vivo and in vitro mechanisms of motilin action in man and rabbit. Furthermore, this discrepancy also exists for the macrolide antibiotic erythromycin, and for its more potent derivatives, the motilides. In vitro erythromycin and the motilides EM-523 and EM-574, act in rabbit and man on a smooth muscle motilin receptor (Peeters et al., 1989; Depoortere et al., 1989; Satoh et al., 1990, Satoh et al., 1994) as was definitively demonstrated by the development of the first motilin antagonist (Peeters et al., 1994; Depoortere et al., 1995). However, in dog and man the induction of gastric activity fronts by erythromycin and motilides is inhibited by atropine (Sarna et al., 1991; Shiba et al., 1995; Coulie et al., 1996).
Recently, neurally mediated effects were also detected in vitro. In the duodenum of the rabbit the tonic response to motilin appears to have a cholinergic component and motilin enhances the release of acetylcholine (Kitazawa et al., 1993). In the same species erythromycin in low doses has neurally mediated, chronotropic effects on betanechol and substance-P induced contractions in the gastric antrum and stimulates inhibitory neurotransmission to the pyloric muscle (Parkman et al., 1995, Parkman et al., 1996). On the other hand, in the chicken proventriculus motilin enhanced the response to electrical field stimulation (Kitazawa et al., 1995). However, these effects required concentrations higher than those needed to stimulate smooth muscle tissue directly.
The aim of the present study was to investigate whether in vitro, motilin influences the excitatory neurotransmission in the upper gastrointestinal tract. Since the gastric antrum is the main target for therapeutic use of motilides, modulation of excitatory enteric nerve function by motilin was studied in the rabbit antrum. This species was chosen, since previous studies from our and other groups have shown that contractile effects of motilin demonstrated in the rabbit in vitro, have a high degree of correlation with findings in man.
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Animals
Adult New Zealand white rabbits of either sex (2.5 to 3.0 kg) were killed by cervical dislocation and bleeding. After midline laparotomy, the stomach was quickly removed and washed with saline. All experiments were approved by the Animal Ethics Committee of the University of Leuven.
Materials
The norleucine13 analogue of porcine motilin ([Nle13]motilin) was obtained from Novabiochem (Laüfelfingen, Switzerland) and tetrodotoxin from Serva (Heidelberg, Germany). This analogue has been shown to be equipotent
Characterization of the response to electrical field stimulation
In preliminary experiments performed under conditions normally used in our laboratory with duodenal preparations, spontaneous phasic contractions of the antral neuromuscular strips interfered with induced twitch responses. The spontaneous activity was not present in every preparation and was tetrodotoxin and atropine insensitive (data not shown). Spontaneous activity was suppressed by lowering the Ca2+ concentration from 2.50 to 1.25 mM and all subsequent experiments were therefore performed
Discussion
This is the first in vitro study of the biological activity of motilin, providing evidence for high affinity motilin receptors on neural pathways. Our data demonstrate that motilin influences the contractility of the gastric antrum in the rabbit in vitro through the interaction with both postganglionic cholinergic nerves and gastric smooth muscle cells. However, the action of motilin on cholinergic nerves occurs at low concentrations (10−9–10−8 M), which are unable to induce the phasic-tonic
Acknowledgements
This study was supported by grants from the Belgian National Science Foundation (NFWO grant No. 3.0187.96) and the Belgian Ministry of Science (GOA 92/96-04 and IUAP P4/16). G.V.A. is a doctoral fellow and I.D. is a postdoctoral research fellow of the Belgian National Research Foundation.
References (38)
- et al.
Mechanism of the excitatory action of motilin on isolated rabbit intestine
Gastroenterology
(1981) - et al.
Motilin receptors in rabbit stomach and small intestine
Regul. Pept.
(1986) - et al.
Distribution and characterization of motilin receptors in the cat
Peptides
(1993) - et al.
Antagonistic properties of [Phe3, Leu13]porcine motilin
Eur. J. Pharmacol.
(1995) - et al.
Excitatory action of [Leu13]motilin on the gastrointestinal smooth muscle isolated from the chicken
Peptides
(1995) - et al.
Migrating motor complex recorded spontaneously and induced by motilin and erythromycin in an ex vivo rabbit intestinal preparation
Peptides
(1994) - et al.
Effect of different Ca2+ modulators on motilin-induced contractions of the rabbit duodenum. Comparison with acetylcholine
Regul. Pept.
(1988) - et al.
Erythromycin inhibits rabbit pyloric smooth muscle through neuronal motilin receptors
Gastroenterology
(1996) - et al.
Comparison of the biological activity of canine and porcine motilin in rabbit
Regul. Pept.
(1986) - et al.
Comparison of motilin binding to crude homogenates of human and canine gastrointestinal smooth muscle tissue
Regul. Pept.
(1988)