Motilin receptors in rabbit stomach and small intestine

doi:10.1016/0167-0115(86)90084-4

Regulatory Peptides

Volume 15, Issue 2, September 1986, Pages 143-153

https://doi.org/10.1016/0167-0115(86)90084-4 Get rights and content

Abstract

Motilin receptors in rabbit antral and duodenal smooth muscle tissue were characterized by direct binding technique using ¹²⁵I-labeled porcine motilin as a tracer ligand. Binding at 30°C was maximal at 90 min, was saturable and partially reversible. Displacement studies with natural porcine motilin, synthetic leucine-motilin or norleucine-motilin indicated a dissociation constant ( $K_{d}$ ) of $1.1 ± 0.3 nM$ and a maximal binding capacity ( $B_{max}$ ) of $42 ± 10 fmol/mg$ protein. Binding was unaffected by glucagon, pancreatic polypeptide and somatostatin, but substance P interfered via an unknown mechanism. By density gradient centrifugation motilin receptors were shown to be present in plasma membranes. Binding could only be demonstrated in preparations from antrum and upper duodenum.

These observations provide evidence for a localized target region for motilin in the gastrointestinal tract, and for a direct interaction of motilin with gastrointestinal smooth muscle tissue.

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Citation Excerpt :
Motilin is a 22-amino acid hormone, responsible for normal regulation of gastrointestinal (GI) motility.1,2 Motilin’s activity is mediated through a G-protein coupled receptor found on smooth muscle and enteric neurons of the GI tract.3–6 Erythromycin A 1, a 14-membered macrolide antibiotic, also acts as an agonist of the motilin receptor (EC50 ∼1 μM).1,7
A series of derivatives of the amine of 9-dihydro-9-O-ethylamino-N-desmethyl-N-isopropyl erythromycin A derivatives were synthesized as motilin agonists. The compounds were developed for potency without showing antibacterial activity and inhibition of the hERG potassium channel. The formamide of the amide series was found to show the optimal combination of properties relative to carbamates, ureas, thioureas, and amines. This prompted an investigation of heterocyclic isosteres for the amide. In this series the triazole had the optimal combination of properties. From the study, two compounds met the criteria for detailed pharmacokinetic studies.
An orally active motilin receptor antagonist, MA-2029, inhibits motilin-induced gastrointestinal motility, increase in fundic tone, and diarrhea in conscious dogs without affecting gastric emptying
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The pharmacological properties of MA-2029, a selective and competitive motilin receptor antagonist, were investigated in conscious dogs after oral administration. Gastrointestinal contractile activity was recorded by chronically implanted force transducers. The proximal gastric volume was measured with a barostat under constant pressure. Gastric emptying was examined using the paracetamol absorption test. MA-2029 (0.3–10 mg/kg, p.o.) administered in the interdigestive state inhibited gastrointestinal contractions induced by motilin (3 µg/kg, i.v.) in a dose-dependent manner. MA-2029 (0.3–3 mg/kg, p.o.) also inhibited the occurrence of spontaneous phase III contractions, even though MA-2029 had no effect on basal gastrointestinal motility or basal gastric emptying even at 10 and 30 mg/kg p.o. The inhibitory effect of MA-2029 on motilin-induced gastrointestinal motility corresponded to its plasma concentration. Motilin (0.3 µg/kg/h, i.v. infusion) reduced the proximal gastric volume by about 50% of control during isobaric distension. This effect was also inhibited by MA-2029 (1–10 mg/kg, p.o.) in a dose-dependent manner. In the digestive state, injection of motilin (3 µg/kg, i.v.) induced diarrhea in 9 of 11 dogs. MA-2029 (1–30 mg/kg, p.o.) reduced the incidence of diarrhea induced by motilin in a dose-dependent manner. The results indicate that MA-2029 inhibits hypermotility induced by motilin in conscious dogs without having an effect on the basal gastrointestinal tone or gastric emptying rate. MA-2029 may be useful in treating gastrointestinal disorders in which the pathogenesis involves the elevation of circulating motilin.
Motilin and ghrelin as prokinetic drug targets
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Motilin is a hormone released by the endocrine cells of the duodenal mucosa during fasting to stimulate gastrointestinal motility. Ghrelin, the closest family member of motilin, was discovered 10 years ago from the rat stomach as the long-awaited endogenous ligand of the growth hormone secretagogue receptor. Ghrelin has now emerged as a multifunctional hormone with important effects on energy homeostasis but also on gastrointestinal motility. Like motilin, it induces hunger contractions in the fasting state and acts postprandially to accelerate gastric emptying. While the development of motilin agonists for the treatment of hypomotility disorders has been going on for more than 15 years, the development of ghrelin agonists is still in its infancy. The failure of the first generation of motilin agonists in clinical trials has been largely due to problems of desensitization and worsening of symptoms due to effects on gastric accommodation. These issues are being taken care of with the second generation of motilin agonists that are currently under evaluation. Ghrelin agonists have the same potential as motilin agonists to treat hypomotility disorders but their effects on appetite may even be a bonus to treat disorders such as functional dyspepsia while ghrelin's anti-inflammatory effects may make it superior to motilin to treat post-operative ileus. Nevertheless the important endocrine activities of ghrelin may result in side effects which are not encountered with motilin. Future studies will need to point out whether the motilin–ghrelin receptor family will make it as a new class of gastroprokinetics.
Mode of Action of Macrolides with Motilin Agonistic Activity-Motilides
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The effect of Erythromycin A (EM) on canine gastrointestinal motility is quite similar to motilin. Based on this finding, a number of EM derivatives were synthesized and their biological activities were evaluated, resulting in the discovery of very unique and potent peptide receptor agonists, called motilides. The chemical structures of both motilin and the motilides are different but however they share the same binding site on the receptor. The human motilin receptor is a family of G-protein-coupled receptors. It exhibits 52% overall amino acid sequence identity to the receptor for growth hormone secretagogues. It is observed in the chapter that identification of the human motilin receptor promotes the analysis of the interaction between the receptor and its ligands. The studies on motilin receptors effectively help in understanding the physiological role of motilin in humans and animals. The identification of human motilin receptors can also facilitate the development of selective agonists and antagonists with chemical structures different from that of EM.
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A synthetic gene for the human motilin receptor containing 33 unique restriction sites was designed and stably coexpressed in HEK293 cells with the bioluminescent Ca²⁺ indicator protein aequorin. The dose-dependent response of the receptor to motilin was demonstrated using transient transfections, and a stable cell line was selected. [¹²⁵I]Motilin binding was used to estimate receptor expression level for the stable cell line, and titration of a membrane preparation indicated a K_d value of 0.8 nM. The same cell line was used to evaluate a panel of erythromycin-derived agonists and provided EC₅₀ values for receptor activation that agree closely with data obtained in contractility assays. The peptide antagonist ANQ11125 (Phe3Leu13 motilin 1–14) inhibited motilin induced response with a K_i value of 10 nM. The system is well-suited for the screening of compound libraries and receptor mutagenesis studies.
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A series of novel tetra-peptide motilin agonists, having the general structure H-Phe-Val-X-Ile-NH₂, were designed, on the basis of structure–activity relationship studies of motilin. Peptides, in which X is a side chain substituted tryptophan residue, have agonistic activity. H-Phe-Val-Trp(2′-CH₂CH₂OH)-Ile-NH₂ (7), H-Phe-Val-Trp(2′-SCH₃)-Ile-NH₂ (8), and H-Phe-Val-Trp(2′-SCH₂CH₂CH₃)-Ile-NH₂ (9), showed an EC₅₀ for contractile activity in the rabbit smooth muscle of 14.1±3.2, 12.9±4.1, and 4.6±1.6 μM, respectively. Interaction of the tryptophan aliphatic side chain with motilin receptor appears to influence the signal transduction via motilin receptor.

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^☆: A preliminary account of this investigation was presented at the 5th International Symposium on Gastrointestinal Hormones in Rochester, MN, 30 September–3 October 1984

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Motilin receptors in rabbit stomach and small intestine☆

Abstract

Gastroenterology

Gastroenterology

Gastroenterology

Life Sci.

Gastroenterology

J. Biol. Chem.

Biochim. Biophys. Acta

Methods Enzymol.

Jpn. J. Pharmacol.

Motilin, a gastric motor activity-stimulating polypeptide: the complete amino acid sequence

Can. J. Biochem.