Concentration-dependent stimulation of cholinergic motor nerves or smooth muscle by [Nle13]motilin in the isolated rabbit gastric antrum

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Abstract

In man, rabbit and cat, the effects of motilin and motilides are neurally mediated in vivo, whereas in vitro binding and contractility studies suggest the presence of a smooth muscular receptor. The aim of this study was to investigate in vitro interactions of motilin with the enteric excitatory neurotransmission in the gastric antrum of the rabbit. Circular muscle strips from the pre-pyloric antrum were subjected to electrical field stimulation (1 ms, 1–32 Hz, 10 s train) and muscle twitch responses were recorded isometrically. Induced twitch responses were frequency dependent (1–32 Hz) and entirely neurogenic (tetrodotoxin sensitive). [Nle13]motilin dose-dependently (10−9–10−8 M) enhanced the amplitude of, atropine sensitive, evoked contractions. At 4 Hz the response, expressed as a % of the response to 32 Hz, increased from 15.5±4.1% (control) to 28.1±5.8% (motilin 10−9 M), and to 45.8±3.6% (motilin 10−8.5 M) (P<0.05). This effect was not inhibited by hexamethonium (10−3.3 M) but was abolished by the motilin receptor antagonist GM-109 (10−5 M). In unstimulated strips, motilin induced phasic-tonic contractions with a threshold concentration of 10−8 M and an pEC50 of 7.48, which were also inhibited by GM-109 (10−5 M) but not by tetrodotoxin (10−5.5 M). The maximal tension, frequency and dose-dependency of carbachol-induced contractions were not influenced by motilin (pEC50, carbachol: 6.48±0.06 (control), 6.49±0.07 (motilin)). In conclusion, motilin enhances contractions induced by electrical field stimulation in the rabbit antrum by a post-ganglionic interaction with the cholinergic neurotransmission in vitro at low doses and interacts directly with antral smooth muscle at high doses. This model is an accurate reflection of the in vivo effects of motilin and provides a tool to study neurogenic and myogenic actions of motilin and motilides in vitro.

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.

Section snippets

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.

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