Porcine Motilin Research Articles

Effects of Mitemcinal (GM-611), an Acid-Resistant Nonpeptide Motilin Receptor Agonist, on the Gastrointestinal Contractile Activity in Conscious Dogs

The effects of mitemcinal (GM-611) on the gastrointestinal contractile activity were investigated using chronically implanted force transducers in conscious dogs and were compared with the effects of porcine motilin (pMTL), EM-523 and EM-574. In the interdigestive state, intravenous and oral administration of mitemcinal, EM-523 and EM-574 induced the gastrointestinal contractile activity in a manner similar to pMTL. The contractile activity caused by mitemcinal was suppressed by continuous intravenous infusion of a motilin receptor antagonist. In the digestive state, intravenous and oral administration of mitemcinal, EM-523 and EM-574 also stimulated the gastrointestinal contractile activity. Mitemcinal, EM-523 and EM-574 given intravenously increased the gastric contractile activity in a similar dose range; however, mitemcinal was approximately 10 times more potent than EM-523 and EM-574 when administered orally in the digestive state. These results indicate that the mitemcinal-induced gastrointestinal contractile activity operates via motilin receptors and possesses a higher activity than EM-523 and EM-574 when administered orally in conscious dogs in the digestive state. Mitemcinal may therefore be useful in the treatment of several gastrointestinal disorders involving dysmotility, such as gastroparesis and functional dyspepsia, even when administered orally.

In vitro Pharmacological Characterization of Mitemcinal (GM-611), the First Acid-Resistant Non-Peptide Motilin Receptor Agonist, in Smooth Muscle of Rabbit Small Intestine

The pharmacological properties of mitemcinal (GM-611), the first acid-resistant non-peptide motilin agonist, were investigated in the smooth muscle of the rabbit small intestine and compared with porcine motilin (pMTL), erythromycin A (EMA) and its derivatives (EM-523, EM-574 and ABT-229). Mitemcinal, pMTL, EMA, EM-523, EM-574 and ABT-229 produced concentration-dependent contractions with approximately the same maximum contractions in the isolated rabbit duodenum longitudinal strips. The contractile response to mitemcinal or pMTL was competitively inhibited by a selective motilin antagonist, GM-109. The pA₂ values for GM-109 as an antagonist of mitemcinal and pMTL showed approximately the same values. However, the concentration-dependent contractile responses to mitemcinal or pMTL were not affected by pretreatment with atropine, tetrodotoxin, hexamethonium, naloxone or tropisetron. The removal of calcium ions from the medium and pretreatment with verapamil greatly suppressed the contractions induced by mitemcinal and pMTL. The contractile response to mitemcinal was not affected by preincubation in acidic solutions, while those of EM-523, EM-574 and ABT-229 were strongly diminished in the same condition. Mitemcinal as well as other motilin agonists displaced ¹²⁵I-pMTL bound to a homogenate of the rabbit duodenum muscle tissue. The displacement curves of all these compounds were parallel. These results indicate that mitemcinal is a selective and full motilin receptor agonist in the smooth muscle of the rabbit small intestine and that this agent has an excellent acid-resistant property.

Effect of intraluminal distension or ischemic strangulation obstruction of the equine jejunum on jejunal motilin receptors and binding of erythromycin lactobionate

To determine whether inflammation of the jejunum of horses decreases the number of motilin receptors and amounts of motilin receptor mRNA and alters erythromycin lactobionate binding affinity to the motilin receptor in jejunal tissues. Jejunal segments in 6 adult horses. Each horse was anesthetized, and a ventral median celiotomy was performed; 2 segments of jejunum underwent a sham operation, 2 segments underwent ischemic strangulation obstruction (ISO), and 2 segments underwent intraluminal distension (ILD). Treatments were maintained for 120 minutes. From each segment, full-thickness biopsy samples were collected and smooth-muscle homogenates were prepared. Affinity and distribution of motilin binding to these preparations were determined by use of iodine 125 (125I)-labeled synthetic porcine motilin. Via displacement experiments, competition between 125I-labeled motilin and erythromycin lactobionate for binding to motilin receptors in the different segments was investigated. A quantitative real-time PCR technique was used to assess motilin receptor mRNA content in the muscle preparations. Compared with the ISO or ILD segments, the number of motilin receptors was significantly higher in the sham-operated segments; ILD segments contained the lowest number of motilin receptors. The expression of motilin receptor mRNA was significantly decreased in ILD segments but not in ISO segments. Erythromycin lactobionate displacement of 125I-labeled motilin from motilin receptors did not differ significantly among the jejunal segments. Results suggest that downregulation and decreased production of motilin receptors in inflamed jejunal tissue contribute to the altered prokinetic response to erythromycin in horses with gastrointestinal disease.

Effects of motilin and ursodeoxycholic acid on gastrointestinal myoelectric activity of different origins in fasted rats.

To investigate gastrointestinal migrating myoelectric complex (MMC) and the effects of porcine motilin and ursodeoxycholic acid (UDCA) on MMC of gastrointestinal tract of different origins in fasted rats. Three bipolar silver electrodes were chronically implanted on the antrum, duodenum and jejunum. Seven days later 24 experimental rats were divided into 2 groups. One group was injected with porcine motilin via sublingual vein at a dose of 20 microg/kg, the other group was perfused into stomach with UDCA. The gastrointestinal myoelectric activity was recorded 1 h before and 2 h after the test substance infusions into the rats. In all fasted rats a typical pattern of MMC was observed. Among the totally 68 activity fronts recorded in fasted rats under control, 67% started in duodenum, and 33% in antrum. MMC cycle duration and duration of phase III of antral origin were longer than those of duodenal origin. Administration of 20 microg/kg porcine motilin induced a premature antral phase III of antral origin. But perfusion into stomach with UDCA resulted in shorter MMC cycle duration, longer duration of phase III of duodenal origin, which were followed with shorter cycle duration and duration of antral phase III. In fasted rats, MMC could originate from antrum and duodenum respectively. The characteristics of MMC of different origins may contribute to the large variations within subjects. The mechanisms of different origins of phase III may be different. Porcine motilin and UDCA could affect MMC of different origins of the gastrointestinal tract in fasted state, respectively.

Binding of radiolabeled porcine motilin and erythromycin lactobionate to smooth muscle membranes in various segments of the equine gastrointestinal tract.

To identify and characterize motilin receptors in equine duodenum, jejunum, cecum, and large colon and to determine whether erythromycin lactobionate competes with porcine motilin for binding to these receptors. Specimens of various segments of the intestinal tracts of 4 adult horses euthanatized for reasons unrelated to gastrointestinal tract disease. Cellular membranes were prepared from smooth muscle tissues of the duodenum, jejunum, pelvic flexure, and cecum. Affinity and distribution of motilin binding on membrane preparations were determined by use of 125I-labeled synthetic porcine motilin. Displacement studies were used to investigate competition between 125I-labeled synthetic porcine motilin and erythromycin lactobionate for binding to motilin receptors in various segments of bowel. Affinity of 125I-labeled synthetic porcine motilin for the equine motilin receptor was estimated to be 6.1nM. A significantly higher number of motilin receptors was found in the duodenum than in the pelvic flexure and cecum. The jejunum had a significantly higher number of motilin receptors than the cecum. Erythromycin lactobionate displacement of 125I-labeled porcine motilin from the equine motilin receptor did not differ significantly among various segments of bowel. Motilin receptors were found in the duodenum, jejunum, pelvic flexure, and cecum of horses. The highest number of motilin receptors was in the duodenum, and it decreased in more distal segments of bowel. Erythromycin lactobionate competed with motilin binding in the equine gastrointestinal tract. This suggests that 1 of the prokinetic actions of erythromycin in horses is likely to be secondary to binding on motilin receptors.

In vitro evaluation of motilin agonism by macrolide immunosuppressive drugs.

The immunosuppressive drugs tacrolimus and sirolimus may have a stimulatory influence on gastric emptying, in view of their macrolide structure. The aim of this study was to investigate in vitro the possible interaction of tacrolimus and sirolimus with motilin receptors in the rabbit antrum and duodenum. Rabbit duodenum strips were mounted under a load of 1 g in 10 ml organ baths containing Krebs solution. Erythromycin, tacrolimus, cyclosporin, and sirolimus were added to the bathing solution in a cumulative way; in a second series, after incubation with cyclosporin (10(-7) mol/l), tacrolimus (10(-8) mol/l), or sirolimus (3 x 10(-8) mol/l), a cumulative concentration-response curve to erythromycin was obtained. The effect of cumulatively added tacrolimus and nle(13)-porcine motilin on the contractile response to electrical field stimulation was tested in strips from the rabbit gastric antrum. Displacement by tacrolimus of (125)I-nle(13)-porcine motilin bound to its receptor was tested with crude homogenates of the smooth-muscle layer of the rabbit antrum. In rabbit duodenum strips, carbachol (10(-5) mol/l) induced a stable and reproducible contraction. Erythromycin induced concentration-dependent contractions. Expressed as a percentage of the response to carbachol, the maximal attained effect was 78%; the EC(50) was 4.8 x 10(-7) mol/l. Tacrolimus (10(-8) to 10(-5) mol/l), cyclosporin (10(-8) to 10(-5) mol/l), and sirolimus (10(-8) to 3 x 10(-5) mol/l) had no influence. The response to erythromycin (10(-8) to 3 x 10(-5) mol/l) in the presence of cyclosporin (10(-7) mol/l), tacrolimus (10(-8) mol/l), or sirolimus (3 x 10(-8) mol/l) did not differ from that obtained with erythromycin alone, except for a decrease of the EC(50) in the presence of tacrolimus (2.2 x 10(-7) mol/l) (P<0.05 vs erythromycin alone). Motilin enhanced the response to electrical field stimulation of rabbit antral strips, but tacrolimus had no stimulatory effect. Tacrolimus weakly displaced motilin bound to its receptor. The pIC(50) was 4.97. As opposed to erythromycin, neither tacrolimus nor sirolimus showed a contractile effect in the rabbit duodenum. Tacrolimus did not activate the neural motilin receptor of the rabbit gastric antrum and had low affinity for the smooth-muscle motilin receptor. It is thus unlikely that these macrolide immunosuppressive drugs possess gastroprokinetic effects via motilin agonism.

Design and synthesis of motilin antagonists derived from the [1-4] fragment of porcine motilin.

A series of cyclic peptides having the general structure H-Phe-c[-N(epsilon)-Lys-X-NH-(CH(2))(n)-CO-] were designed on the basis of structure-activity relationship studies of motilin. All were motilin antagonists. The cyclic peptides, in which X is a 3-tert-butyl-substituted tyrosine residue (H-Phe-c[-N(epsilon)-Lys-Tyr(3-tBu)-beta Ala-] (3), H-Phe-c[-N(epsilon)-Lys-Tyr(3-tBu)-Gly-] (6), H-Phe-c[-N(epsilon)-Lys-Tyr(3-tBu)-Abu-] (7), and H-Phe-c[-N(epsilon)-Lys-Tyr(3-tBu)-Ahx-] (8)) showed potent motilin receptor antagonistic activity in the rabbit smooth muscle (pA(2) > 7). The 3-tert-butyl Tyr was found to be the moiety responsible for enhanced binding to the motilin receptor, while the size of the ring had little importance.

Demonstration of a functional motilin receptor in TE671 cells from human cerebellum

Background: Our laboratory has described the presence of motilin receptors in the rabbit cerebellum. We discovered its presence in the human TE671 cell line, which is of cerebellar origin. Methods: Cytosolic Ca 2+ fluxes were monitored on a confocal microscope in cells loaded with Indo-1 and stimulated with motilin under various conditions. Binding studies were performed with 125I-[Nle 13]porcine motilin. Using primers, PCR for the motilin receptor was performed. Results: Cells responded to motilin after 45±20 s. At different concentrations of motilin (10 −8, 10 −7, 10 −6.5, 10 −6 and 10 −5 M) the percentage of responding cells was 0±0, 0.6±1.5, 4.9±4.7, 21.7±15 and 35.7±12, respectively. The response was blocked by the motilin antagonists [Phe 3, Nle 13]po-motilin (0.8±1.8%) and GM-109 (0.0±0.0%) and mimicked by the agonist ABT-229 (23.6±15%). After stimulation with motilin, ABT-229 or [Phe 3,Leu 13]po-motilin, but not with the antagonist GM-109, cells were desensitized. The response to motilin persisted in Ca 2+-free solution (22.8±14.7%), was not affected by nifedipine (44±11%) but was abolished by incubation with thapsigargin (0±0%). Neither ryanodine, nor a previous stimulation with caffeine (0±0%) in Ca 2+-free Krebs, nor both could block the response to motilin (28, 32.0±5.7, 41.3±6.1%, respectively). Binding studies revealed two binding sites for motilin, with a p K d of 8.9±0.05 and 6.11±0.61 ( n=4). There were 100 times more low than high affinity receptors per cell. The presence of receptor mRNA was confirmed by PCR. Conclusion: Functional motilin receptors are present in TE671 cells. The response requires intracellular IP 3-sensitive Ca 2+ stores. These cells may serve as a model of the central motilin receptor.

Design and Synthesis of N-terminal Cyclic Motilin Partial Peptides: A Novel Pure Motilin Antagonist.

Motilin antagonist was designed and synthesized on the basis of the structure-activity relationship analysis of porcine motilin that we reported recently. The drug design was performed on a specific concept to reduce a flexibility of peptide conformation of porcine motilin partial peptide by its cyclization. The cyclic peptide was synthesized using Boc (tert-butyloxycarbonyl) solid phase methodology, followed by cyclization using the azide procedure, and tested for the binding activity to motilin receptor and smooth muscle contractile activity. The cyclic peptides 3, 4, and 5 showed antagonistic property on contraction assay (pA2 [the negative logarithm of molar concentration of antagonist causing a 2-hold shift to the right of the concentration-response curve for motilin]: 4.5, 4.34, and 4.04, respectively, in rabbit duodenum) and no contractile activity even at high concentration.

EM574, an erythromycin derivative, improves delayed gastric emptying of semi-solid meals in conscious dogs

The gastroprokinetic effects of de( N-methyl)- N-isopropyl-8,9-anhydroerythromycin A 6,9-hemiacetal (EM574), a non-peptide motilin receptor agonist, were investigated in conscious dogs in a normal state and with experimentally-induced gastroparesis. Gastric emptying of semi-solid meals was assessed indirectly from acetaminophen absorption with simultaneous recording of gastric antral motility. In the normal state, post-prandial intraduodenal administration of EM574 (30 mg/kg) stimulated antral motility and significantly enhanced gastric emptying as potently as did intravenous porcine motilin (0.003 mg/kg/h). Intraduodenal cisapride at 1 mg/kg denal cisapride at 1 mg/kg elicited antral contractions and tended to accelerate gastric emptying but at 3 mg/kg, gastric emptying was not enhanced despite a further increase in the motor index. In dogs with gastroparesis induced by intraduodenal oleic acid or intravenous dopamine, EM574 (0.03 mg/kg) increased antral motility and reversed the delayed gastric emptying completely. Cisapride (1 mg/kg) partially ameliorated the impaired emptying under these conditions. In atropinized dogs, no acceleration of gastric emptying by EM574 was observed. These results indicate that EM574 potently accelerates gastric emptying of caloric meals in dogs in a normal state and with experimentally-induced gastroparesis, and also suggest that the effect is mediated through stimulation of a cholinergic neural pathway.

Structure-Activity Study of Intact Porcine Motilin.

Biologically important sites on intact porcine motilin (pMTL) were explored using its partial peptides. The partial peptides were synthesized using Fmoc (9-fluorenylmethyloxycarbonyl) solid phase methodology, and tested for the binding activity to motilin receptor and the smooth muscle contractile activity. The results were as follows: important residues for the contractile activity were found to be Phe1, Ile4, and Tyr7, and an open space existed beyond the N-terminus between motilin and its receptor. On the model of interaction between motilin and motilin receptor evolved from these results, the three points of interaction, due to Phe1, Ile4, and Tyr7, and the presence of an open space were expected. The motilin agonist and antagonist, designed on this model, will help the inquiry into motilin associated diseases.

Motilin in human milk: Identification and stability during digestion

The presence of motilin in human milk and the influence of human milk on the degradation of [ 125I][Nle 13] porcine motilin by gastric and duodenal fluids were investigated. Milk and plasma samples were collected from 14 mothers, and motilin was measured by radioimmunoassay. Plasma levels were 416 ± 37 pg/mL. In 8 defatted samples the motilin level was 105 ± 14 pg/mL, in the six others levels were above 1000 pg/mL but dilution curves were non-linear. After solid-phase extraction milk levels were 108 ± 21 pg/mL in 13 samples, in 1 sample the dilution curve was still non-linear. The stability of motilin after ingestion was studied in vitro by incubating [ 125I][Nle 13] porcine motilin with gastric and intestinal juices obtained from newborns (10 times diluted). Incubations were performed at 37 °C at pH 1.8, 3.2 and 5.8 for the gastric fluid and at pH 7.4 for the duodenal fluid. After different times of intervais (5, 10, 20 and 30 minutes) intact motilin was precipitated with trichloroacetic acid and the radioactivity of the supernatant was determined. Motilin was rapidly degraded by gastric juice. The breakdown was greatest at pH 3.2 (74 % after 30 minutes) and lowest at pH 5.8 (29 %), the pH after milk feeding in neonates. Degradation by intestinal juice at pH 7.4 was also very rapid (77 % after 30 minutes). Human milk and BSA inhibited partially the gastric digestion at pH 3.2 (17 and 29 %, respectively). Digestion by intestinal juice was not affected by human milk and BSA. These results suggest that digestion of motilin in the stomach may be sufficiently retarded by human milk in the newborn to exert a biological role.

Isolation and sequencing of the cDNA encoding the motilin precursor from sheep intestine

The nucleotide sequence of sheep prepromotilin has been determined from cDNA clones. The nucleotide sequence revealed an open reading frame of 345 nucleotides encoding 115 amino acids. The amino acid sequence deduced from the nucleotide sequence consists of a 25 amino acid signal peptide, followed by the 22 amino acid motilin sequence, an endoproteinase cleavage site (Lys 23–Lys 24) and a 66 amino acid motilin associated peptide (MAP). Compared with human and pig motilin we observed two substitutions at positions 10 (Leu→Val) and 19 (Asn→Tyr). The second one may explain the poor cross-reactivity of ovine motilin with C-terminally directed antibodies against porcine motilin. The sheep motilin precursor exhibits the same structure as the motilin precursors from rabbit, pig and man. However, while there is considerable identity in the amino acid sequences as well as in the nucleotide sequences of the signal peptide and motilin, the MAP strongly differs between the species. This may be a result of `mosaic evolution' at the molecular level.

Functional characterization of neural and smooth muscle motilin receptors in the chicken proventriculus and ileum

To characterize the motilin receptors present in the chicken, the effects of chicken motilin (Phe-Val-Pro-Phe-Phe-Thr-Gln-Ser-Asp-Ile-Gln-Lys-Met-Gln-Glu-Lys-Glu-Arg-Asn-Lys-Gly-Gln), Leu 13 porcine motilin, canine motilin and three erythromycin derivatives (EMA, EM523, GM611) on the contractility of the chicken gastrointestinal (GI) smooth muscles were investigated in vitro and compared with those in the rabbit duodenum. In the proventriculus longitudinal and circular muscle layers, chicken motilin (3 nM–1 μM) caused an atropine- and a tetrodotoxin-sensitive contraction (EC 50=39–49 nM), and potentiated the EFS-induced contraction without affecting the responsiveness of acetylcholine. EM523 and GM611 (3–100 μM) contracted the proventriculus longitudinal muscle, and the maximum amplitudes of contraction were about 60% of that induced by chicken motilin. Chicken motilin (0.1 nM–100 nM) also caused contraction of the ileum (EC 50=7 nM) through direct action on the smooth muscle cells. On the other hand, erythromycin derivatives showed only a weak contractile efficacy (about 20% of the maximum response of chicken motilin) even at high concentrations (10–100 μM). The rank order of potency in the ileum was chicken motilin>canine motilin≥Leu 13 porcine motilin>>GM611≥EM523≥EMA. GM109 slightly inhibited the idea contractions induced by Leu 13 porcine motilin at 100 μM (pA2=3.86). In the rabbit duodenum, chicken motilin was a full agonist with the same intrinsic activity as Leu 13 porcine motilin, canine motilin and the erythromycin derivatives. However, the rank order of potency (Leu 13 porcine motilin≥canine motilin>chicken motilin>GM611≥EM523>EMA) was different from that in the chicken ileum. In conclusion, chicken motilin causes an excitatory response in the chicken GI tract through activation of neural (proventriculus) and smooth muscle motilin receptors (ileum). The motilin receptor present in the ileum is different from that demonstrated in the rabbit intestine, because of a different rank order of motilin peptides in producing the contraction, low contracting activity of erythromycin derivatives and low antagonistic efficacy of GM109. Different pharmacological characteristics of the mechanical response induced by motilin peptides and erythromycin derivatives between the proventriculus and the ileum are discussed.

Three-dimensional structure and position of porcine motilin in sodium dodecyl sulfate micelles determined by 1H NMR.

The solution structure of the porcine gastrointestinal peptide hormone motilin was determined in the presence of sodium dodecyl sulfate (SDS) micelles at 28 degrees C using 1H nuclear magnetic resonance, full relaxation matrix analysis, and structure calculations based on restrained molecular dynamics. The structure of motilin in SDS micelles is described by a reverse gamma-turn and a beta-turn of type II in the N terminal end, an alpha-helical region in the middle of the molecule, and an extended structure at the C terminus. The position of the motilin molecule relative to the SDS micelles was probed by adding spin-labeled stearic acids, containing 12-doxyl or 5-doxyl spin-labels. We observed selective broadening of the proton resonances of residues 3-5 and concluded that they must be located in the interior of the micelle. These experiments suggest a structural model in which the hydrophobic N terminus consists of two well-defined turns buried in the interior of the micelle, whereas the amphiphilic alpha-helical part is located at the surface of the micelle. Spectral density mapping using a 13C label on the alphaC of Leu10 gave overall rotational correlation times taum of 6.6 and 4.5 ns at 35 and 45 degrees C, respectively. The long correlation time in combination with a high order parameter (S = 0.92) indicates that motilin has a rigid structure in the complex with the SDS micelle.

EM574, an erythromycin derivative, is a motilin receptor agonist in the rabbit

This study was performed to examine whether an erythromycin derivative, de( N-methyl)- N-isopropyl-8,9-anhydroerythromycin A 6,9-hemiacetal (EM574) is a motilin receptor agonist in the rabbit gastrointestinal tract. EM574 and porcine motilin induced contractions in segments of isolated rabbit intestine with pEC 50 values of 8.26±0.04 and 8.69±0.07, respectively, but not in rat or guinea pig preparations. The sensitivity and efficacy of the response to both compounds in rabbits decreased aborally and was insensitive to pretreatment with atropine or tetrodotoxin, but was markedly suppressed under Ca 2+-free conditions. EM574 and porcine motilin specifically displaced [ 125I-Tyr 23]canine motilin bound to gastric antral smooth muscle homogenates with pIC 50 values of 8.21±0.13 and 9.20±0.11, respectively. The pEC 50 value for the contractile response and pIC 50 value for the receptor binding for motilin, EM574, erythromycin A and three other derivatives correlated well ( r=0.94, P<0.01). Tissue section autoradiography in the antrum revealed that specific labeled motilin binding sites were localized in the circular muscle layer and myenteric plexus, and could be reduced in the presence of an excess of EM574. These results indicate that EM574 is a potent motilin receptor agonist in the rabbit gastrointestinal tract. © 1997 Elsevier Science B.V. All rights reserved.

Exogenous motilin stimulates endogenous release of motilin through cholinergic muscarinic pathways in the dog

BACKGROUND & AIMS: Exogenous motilin is believed to stimulate endogenous release of motilin, but this has not been studied in detail. The aim of this study was to investigate whether and by what mechanism exogenous motilin stimulates endogenous release of motilin in the dog. METHODS: Gastric and duodenal contractile activity in conscious dogs was monitored by chronically implanted force transducers. Plasma canine motilin (c-motilin) concentrations in response to exogenous porcine motilin (p-motilin) were determined by specific radioimmunoassay for c- motilin. The release of motilin from motilin cells obtained from canine duodenal mucosa was studied in vitro using a perifusion system. RESULTS: In vitro, c-motilin release was stimulated by carbachol but not by p-motilin, and the carbachol-induced c-motilin release was inhibited by atropine. In vivo, exogenous p-motilin stimulated endogenous c-motilin release and gastric and duodenal phase III-like contractions; this motilin-induced motilin release was inhibited by atropine, hexamethonium, and a 5-hydroxy-tryptamine 3 receptor antagonist. CONCLUSIONS: In the dog, exogenous motilin stimulates endogenous motilin release through muscarinic receptors on motilin- producing cells via preganglionic pathways involving 5- hydroxytryptamine 3 receptors. (Gastroenterology 1996 Dec;111(6):1456-64)

Vagus-Dependent and Vagus-Independent Mechanisms of Action of the Erythromycin Derivative EM574 and Motilin in Dogs

The motor-stimulating action of de(N-methyl)-N-isopropyl-8,9-anhydroerythromycin A 6,9-hemiacetal (EM574) on the upper gastrointestinal tract was studied in fasted conscious dogs using chronically implanted force transducers and compared with those of porcine motilin and cisapride. EM574 induced gastric phase Ill-like migrating contractions and increased the plasma motilin levels slightly. The gastric motility induced by low doses of EM574 and motilin was abolished by a 5HT3-receptor antagonist ondansetron and acute vagal blockade, whereas under these conditions, high doses of both agents induced contractions, which were abolished by atropine. Cisapride-induced gastric motility was inhibited by atropine and acute vagal blockade, but not by ondansetron. EM574 did not stimulate gastric secretion in the basal state. These results indicate that EM574- and motilin-induced gastrointestinal motility is attributable mainly to motor-stimulating vagal cholinergic neurons, and 5HT3-receptors are probably involved in the process. At high doses, EM574 and motilin also appear to stimulate cholinergic neurons in a non-vagal pathway, probably the enteric nervous system.

Antagonistic properties of [Phe 3,Leu 13]porcine motilin

We describe the antagonistic properties due to the replacement of Pro 3 by phenylalanine in porcine motilin. The analogue, [Phe 3,Leu 13]porcine motilin (OHM-11526), displaces iodinated [Nle 13]porcine motilin bound to a homogenate of rabbit antral smooth muscle tissue. The dissociation constant (p K d) was 9.26 ± 0.04, versus 9.11 ± 0.01 for motilin and 8.24 ± 0.06 for ANQ-11125, the (1–14) fragment of OHM-11526. The Hill coefficient was close to one and Schild plot analysis confirmed the competitive nature of the interaction. In the tissue bath OHM-11526 was unable to induce contractions of segments of rabbit duodenum. At a concentration of 10 −6 M, OHM-11526 inhibited the effect of maximally effective doses of porcine motilin and of the erythromycin derivative, EM-523, but was without effect on contractions induced by acetylcholine, substance P and serotonin. Increasing doses of OHM-11526 shifted the dose-response curves of motilin and EM-523 to the right, but caused a depression of the maximal response as well. From the motilin curves, and assuming a dual competitive and non-competitive interaction, the pA 2 was 7.79 ± 0.08, the pD′ 2 6.91 ± 0.08. The EM-523 curves yielded comparable data (pA 2 = 8.10 ± 0.12 and pD′ 2 = 7.06 ± 0.13). OHM-11526 also blocked the motilin responses observed with smooth muscle strips from the rabbit and human antrum. However, in a preparation of the chicken small intestine, OHM-11526 was a full agonist with a potency (pD 2 = 6.84) comparable to that of porcine motilin (pD 2 = 6.71). Our data confirm the interaction of motilides with the motilin receptor. Due to its increased affinity for the motilin receptor, OHM-11526 will be a valuable tool for studying the physiology of motilin and the pharmacology of motilin and motilides.

Excitatory action of [Leu13]motilin on the gastrointestinal smooth muscle isolated from the chicken

The effects of a porcine motilin analogue, [Leu13]motilin (LMT) on the smooth muscle preparations isolated from the chicken gastrointestinal (GI) tract were investigated in vitro. In the proventriculus, LMT (100 nM to 30 microM) caused an atropine-sensitive contraction and enhanced the electrical field stimulation (EFS)- or 1,1-dimethyl-4-phenyl-piperazinium (DMPP)-induced contraction without affecting the response to acetylcholine (ACh). LMT also caused a concentration-dependent contraction of the intestinal tract (duodenum, jejunum, ileum, and colon). The responsiveness to LMT was strongest in the jejunum and weakest in the colon. The responses to LMT in the intestinal segments were not affected by tetrodotoxin, atropine, hexamethonium, pyrilamine, spantide, and 5-hydroxyltryptamine-induced desensitzation, but significantly decreased by verapamil or removal of external Ca2+. LMT did not enhance the EFS- or DMPP-induced contraction in the ileum. Canine motilin also contracted the intestinal segments in a similar concentration range to LMT with an equal potency, but erythromycin A (EMA) and N-ethyl-N-demethyl-8,9-anhydroerythromycin A, 6-9-hemiketal (EM523) showed only a weak contractile activity even at high concentration (up to 100 microM), indicating that motilin receptors in the chicken intestine were somewhat different from those of mammals. In conclusion, LMT produces an excitatory response in the chicken GI tract with a different sensitivity from region to region. The mechanisms of the action were different between the proventriculus and small intestine; that is, LMT contracts the small intestine through the direct action on the smooth muscle cells, but this peptide acts on the enteric cholinergic neurones and stimulates ACh release, and thus regulates autonomic neuroeffector transmission in the proventriculus.