Effect of peripheral obestatin on food intake and gastric emptying in ghrelin-knockout mice.

BACKGROUND AND PURPOSE: The finding that obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin's stimulatory effect on food intake and gastric emptying has been questioned. The effect of obestatin has been mostly investigated in fasted rodents, a condition associated with high blood levels of ghrelin which may mask the effect of obestatin. We therefore investigated the effect of obestatin on food intake, gastric emptying and gastric contractility in ghrelin knockout mice. EXPERIMENTAL APPROACH: The effect of obestatin on 6-h cumulative food intake was studied in fasted wildtype (ghrelin+/+) and ghrelin knockout (ghrelin-/-) mice. In both genotypes, the effect of obestatin and/or ghrelin was studied in vivo on gastric emptying measured with the (14)C-octanoic acid breath test and in vitro on neural responses elicited by electrical field stimulation (EFS) of fundic smooth muscle strips. KEY RESULTS: Administration of obestatin did not influence fasting-induced hyperphagia or gastric emptying in both genotypes. Injection of ghrelin accelerated gastric emptying in ghrelin+/+ and ghrelin-/- mice but the effect was not reversed by co-injection with obestatin. In fundic strips from ghrelin+/+ and ghrelin-/- mice, ghrelin increased EFS-induced contractions, but obestatin was without effect. However, co-administration with obestatin tended to reduce the excitatory effect of ghrelin in both genotypes. CONCLUSIONS AND IMPLICATIONS: In ghrelin-/- mice, obestatin failed to affect food intake and gastric motility. These results suggest that endogenous ghrelin does not mask the effect of obestatin and confirm that obestatin administered peripherally is not a major regulator of satiety signalling or gut motility.

Effect of peripheral obestatin on gastric emptying and intestinal contractility in rodents.

Obestatin has recently been discovered in the rat stomach. It is encoded by the ghrelin gene and has been claimed to be a functional opponent of ghrelin and to be the natural ligand of the GPR39 receptor. The latter could not be confirmed by Holst et al. (Endocrinology, 2006). Yet, in GPR39 knockout mice, gastric emptying is accelerated. We verified the effects of obestatin on gastric emptying and intestinal contractility in rodents. Gastric emptying was measured with the (14)C octanoic breath test in mice. In vitro, the effect of obestatin was studied on electrically stimulated and non-stimulated strips from the fundus and small intestine of mice and rats. Obestatin (60, 125, 250 nmol kg(-1)) did not affect gastric emptying parameters (T(half) and T(lag)) and did not inhibit the prokinetic effects of ghrelin. Mouse and rat intestinal and fundic smooth muscle strips did not respond to obestatin either in the absence or in the presence of electrical field stimulation. Obestatin (125 nmol kg(-1)) did not inhibit fasting-induced hyperphagia. Our results suggest that peripheral obestatin is not a satiety signal that plays a role in the regulation of gastric emptying and do not support the concept that obestatin is a physiological opponent of ghrelin.

Role of endogenous ghrelin in the hyperphagia of mice with streptozotocin-induced diabetes.

Ghrelin is an orexigenic peptide involved in the regulation of energy homeostasis. To investigate the role of ghrelin in the hyperphagia associated with uncontrolled streptozotocin-induced diabetes, food intake was followed in diabetic ghrelin knockout (ghrelin(-/-)) and control wild-type (ghrelin(+/+)) mice and diabetic Naval Medical Research Institute noninbred Swiss mice treated with either saline or the ghrelin receptor antagonist, D-Lys3-GH-releasing peptide-6 (D-Lys3-GHRP-6) for 5 d. In diabetic ghrelin(-/-) mice, hyperphagia was attenuated, and the maximal increase in food intake was 50% lower in mutant than in wild-type mice. The increased food intake observed during the light period (1000-1200 h) in ghrelin(+/+) mice was abolished in mutant mice. Diabetic ghrelin(-/-) mice lost 12.4% more body weight than ghrelin(+/+) mice. In diabetic ghrelin(+/+) mice, but not in ghrelin(-/-) mice, the number of neuropeptide Y (NPY)-immunoreactive neurons was significantly increased. Diabetic Naval Medical Research Institute noninbred Swiss mice were hyperphagic and had increased plasma ghrelin levels. Treatment with D-Lys3-GHRP-6 reduced daily food intake by 23% and reversed the increased food intake observed during the light period. The change in the number of NPY- (2.4-fold increase) and alpha-MSH (1.7-fold decrease)-immunoreactive hypothalamic neurons induced by diabetes was normalized by D-Lys3-GHRP-6 treatment. Our results suggest that enhanced NPY and reduced alpha-MSH expression are secondary to the release of ghrelin, which should be considered the underlying trigger of hyperphagia associated with uncontrolled diabetes.

Induction of secondary structure in the peptide hormone motilin by interaction with phospholipid vesicles.

Motilin is an intestinal peptide hormone that binds to a membrane bound receptor located in the gut tissue. Circular dichroism (CD) was used to study the interaction between either porcine or rabbit motilin or a 1-16 fragment of porcine motilin, with model systems of lipid membranes: sodium dodecyl sulphate (SDS), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The CD measurements show significant induction of secondary structure in both motilins and the fragment when negatively charged vesicles (DOPG) or negatively charged micelles (SDS) were present. In contrast, neutral DOPC vesicles did not induce any change in the secondary structure compared to water, in which a random-like secondary structure dominates. The induced secondary structure in the presence of DOPG vesicles is very close to that induced by a mixed aqueous solution containing 30% hexafluoroisopropanol, in which previous NMR-studies have resulted in a three-dimensional solution structure of porcine motilin. In both porcine and rabbit motilin the alpha-helix content is about 50%. This is in agreement with the presence of an amphipathic helix in the C-terminal half of motilin interacting with phospholipid membranes. The interaction appears to be mainly electrostatic in nature, and does not induce any significant alterations in the vesicle, as monitored by EPR studies of spin labels located at the fifth carbon atom of the backbone in a stearic acid molecule. In the 1-16 fragment the alpha-helical content induced by DOPG and SDS is only about 20%.

Somatostatin analog octreotide (SMS 201-995) prevents the decrease in blood pressure after oral glucose loading in the elderly.

In elderly subjects blood pressure (BP) may fall after a meal. The mechanism of this phenomenon is unknown, but it has been suggested that it may be mediated by insulin and/or vasoactive gut hormones. We studied in normo- and hypertensive elderly subjects the effects of the synthetic long-acting somatostatin analog octreotide (SMS 201-995) on the BP reduction that follows oral glucose administration in subjects who are recumbent and on their postglucose plasma vasoactive intestinal polypeptide (VIP) and insulin concentrations. After placebo treatment, mean arterial pressure fell by 15 +/- 1 mm Hg (P less than 0.001) in the 10 hypertensive subjects and by 7 +/- 2 mm Hg (P less than 0.01) in the 10 normotensive subjects. In contrast, when 50 micrograms octreotide were given sc, BP did not change significantly in either group. Oral glucose did not induce a rise in plasma VIP after either octreotide or placebo administration. The postglucose rises in plasma glucose concentrations were similar after octreotide and placebo treatments in both groups. After placebo administration the postglucose plasma insulin levels increased from 79 to 519 pmol/L in the hypertensive subjects and from 63 to 464 pmol/L in the normotensive subjects, whereas after octreotide treatment plasma insulin increased little in either group. These data indicate that treatment with octreotide holds promise for patients with symptomatic postprandial hypotension, and that VIP does not seem to play a role in this phenomenon.

Identification and expression of the motilin precursor in the guinea pig.

Motilin has never been isolated from rodents, the most frequently used laboratory animals, despite several attempts. We have isolated and sequenced the motilin precursor from duodenal mucosa of guinea pig (GenBank accession number AF323752) and studied its expression in several tissues. The percent homology with human motilin is the lowest yet observed due to several unique substitutions in the C-terminal end. As expected, the precursor was present in the gut mucosa with the exception of the gastric corpus. It was also present in medulla oblongata, nucleus of the solitary tract, hypophysis, spinal cord, hypothalamus, and cerebellum but not in the cerebral cortex. For the first time we demonstrated motilin expression in the thyroid.

Isolation and sequence of cDNA encoding the motilin precursor from monkey intestine. Demonstration of the motilin precursor in the monkey brain.

The motilin precursor cDNA has been isolated and sequenced from a cDNA library prepared from monkey small intestine. The sequence indicates a 345 bp open reading frame, a 63 bp 5' untranslated region and a 154 bp 3' untranslated region. The sequence encodes a 115 amino acid motilin precursor composed of a 25 amino acid signal peptide, the 22 amino acid motilin peptide and a 68 amino acid motilin associated peptide (MAP). Compared with the human motilin precursor cDNA, there are two amino acid substitutions in the signal peptide, one in motilin and four in the MAP. The presence of the motilin precursor in hypothalamus, hippocampus and cerebellum was demonstrated by RT-PCR.

GM-611 (Chugai Pharmaceutical).

GM-611 is an erythromycin derivative that acts as an agonist at the motilin receptor. It is being developed by Chugai as a potential treatment for gastric motility disorder [169036], as well as reflux esophagitis, non-ulcer dyspepsia and diabetic gastroparesis [347963]. GM-611 is in phase II trials in the US for reflux esophagitis [322624], [347955], [399349]. GM-611 acts by a novel mechanism whereby it stimulates and promotes peristalsis in the stomach and other segments of the gastrointestinal tract [334994]. The drug was shown to produce a dose-dependent sustained depolarization of rabbit duodenal smooth muscle. Depolarization appeared to be associated with activation of monovalent cation-selective channels [273336]. In December 2000, Credit Suisse First Boston predicted that successful development of GM-611 could lead to sales over $500 million [400228].

The influence of food temperature on postprandial blood pressure reduction and its relation to substance-P in healthy elderly subjects.

Blood pressure (BP) in the elderly may decrease after a meal or oral glucose loading. The mechanism of this phenomenon is still unclear. In addition, the effect of the temperature of a meal on postprandial BP is unknown. However, it has been suggested that vasoactive gastrointestinal peptides are involved in the etiology of postprandial BP reduction. Therefore, we studied the effects of a cold and a warm glucose solution on BP, heart rate, plasma glucose, insulin, and substance-P levels in 15 healthy elderly subjects with a mean age of 74 +/- 3 (SD) years. With an interval of at least 2 days, a warm (50 degrees C) and a cold (5 degrees C) solution (75 g glucose/300 mL water) were given in random order. After the cold glucose loading mean arterial pressure increased by a maximum of 3.9 +/- 1.3 mmHg (P less than 0.01). In contrast, BP decreased after the warm solution by a maximum of 8.0 +/- 1.1 mmHg (P less than 0.001). Neither test had an influence on plasma substance-P levels. Our data suggest that postprandial blood pressure reduction in the elderly is dependent on food temperature. Substance-P does not seem to play a role in this phenomenon.

Distribution and characterization of motilin receptors in the cat.

We demonstrate binding of [125I][Nle13-po]motilin to homogenates of cat gastric and small intestinal, but not to colonic smooth muscle tissue. The density was (Bmax in fmol/mg protein): 0 (fundus); 12 +/- 2 (corpus); 22 +/- 3 (antrum); 55 +/- 12 (duodenum); 44 +/- 10 (jejunum); 17 +/- 1 (ileum); 0 (colon). A significant (p < 0.05) difference was found between the dissociation constant for motilin in the stomach (pKd = 8.84 +/- 0.06) and in the small intestine (pKd = 8.58 +/- 0.08). The motilides erythromycin-A (EM-A), EM-523, and EM-A N-oxide displaced labeled [Nle13-po]motilin bound to cat duodenal receptor with potencies (pKd) of 5.47 +/- 0.23, 7.60 +/- 0.24, and < 4.3, respectively. Studies with [Leu13-po]motilin fragments showed that the N-terminus of motilin interacts with the receptor. In the tissue bath, duodenal strips mounted in the longitudinal direction responded to motilin, EM-523, and EM-A (pEC50: 8.29 +/- 0.08; 7.12 +/- 0.12; 5.99 +/- 0.15). The compounds had a comparable intrinsic activity (83 +/- 3%; 80 +/- 5%; 82 +/- 5% of the response to ACh), which was unaffected by atropine, TTX, hexamethonium, and zacopride but reduced by verapamil and calcium-free medium. Cat stomach and small intestine possess smooth muscle motilin receptors, which have comparable properties as those found in man and in rabbit.

Motilin receptors of the rabbit colon.

Binding studies with iodinated motilin revealed that in the small intestine motilin receptor density decreased aborally, disappeared in the caecum but returned in the colon and rectum. The highest density was in the distal colon (112 +/-/11 fmol/mg protein). The dissociation constant was the same in all regions (overall mean 1.10 +/- 0.22 nM). The ability of erythromycin-A (EM-A) and of two derivatives, EM-A N-oxide and EM-523, to displace motilin showed no difference between the tissues studied. Their order of potency was: motilin greater than EM-523 greater than EM-A greater than EM-A N-oxide. Proximal circular colonic smooth muscle strips showed maximal contractile responses towards motilin, EM-523 and EM-A of, respectively, 80 +/- 3%, 78 +/- 4% and 84 +/- 2% relative to the maximum obtained with acetylcholine. In proximal longitudinal muscle only a response of +/- 20% was obtained. Similar responses were obtained in the distal colon. The order of potency to induce contractions as reflected in the pED50 values was: motilin (8.03 +/- 0.1) greater than EM-523 (7.55 +/- 0.03) greater than EM-A (5.84 +/- 0.04) in proximal circular colon. The responses were not blocked by TTX (10(-6) M) or atropine (10(-6) M), but were reduced by verapamil (10(-6)M). The abundance of motilin receptors in colonic smooth muscle, if applicable to other species, opens new perspectives for the therapeutic applications of macrolides with motilin agonist properties.

The erythromycin derivative EM-523 is a potent motilin agonist in man and in rabbit.

Erythromycin may stimulate gastrointestinal motor activity via its effect upon motilin receptors. We have studied the ability of the derivative EM-523 [de(N-methyl)-N-ethyl-8,9-anhydroerythromycin A 6,9-hemiacetal] to induce contractions in duodenal smooth muscle strips and to displace labeled motilin bound to antral smooth muscle, in man and in rabbit. In both species EM-523 approached the potency of motilin for inducing contractions. Thus pED50 values were 7.84 +/- 0.11 and 8.69 +/- 0.12 for motilin in, respectively, man and rabbit, against 6.08 +/- 0.13 and 8.19 +/- 0.10 for EM-523. In rabbit the efficacy of both compounds decreased in parallel aborally, the responses to EM-523 could not be blocked by atropine (10(-7) M) or TTX (10(-7) M), and both compounds were unable to further enhance the maximum effect to the other compound. In binding studies the order of potency was the same as in the contraction studies. The pIC50 values were: motilin (8.84 +/- 0.31, 9.17 +/- 0.20) greater than EM-523 (7.89 +/- 0.1, 8.40 +/- 0.10). A Schild plot revealed that EM-523 was a competitive inhibitor of motilin receptor binding in man and in rabbit. We conclude that EM-523 is a potent motilin agonist.

Isolation, sequence, and bioactivity of chicken motilin.

Motilin was isolated from acid extracts of the small intestine of chickens by a combination of gel filtration chromatography, ion-exchange, and reverse-phase HPLC. The purification was monitored using a radioreceptor assay. The sequence of chicken motilin is FVPFFTQSDIQKMQEK-ERNKGQ. Although the six residues differing from porcine motilin (4, 7-10, and 12) are mostly in the pharmacophore of porcine motilin, the affinity of chicken motilin and of the (1-14) fragment of chicken motilin for the motilin receptor of rabbit antral smooth muscle is not much reduced (pKds of 8.90 and 8.45), compared with the affinity of [Nle13]porcine motilin (pKd 9.12). With smooth muscle tissue of the chicken, however, receptors could not be demonstrated with binding studies. In the tissue bath chicken motilin induced a dose-dependent tonic contraction, which was most pronounced with muscle strips prepared from chicken jejunum. This response was blocked by the Ca2+ antagonist verapamil, but atropine, TTX, L-NNA, guanethidine, prazosin, and yohimbine had no effect. The pEC50 for chicken motilin in the chicken jejunum was 7.41. Motilins from other species had lower potencies, and [Phe3, Leu13]porcine motilin, an antagonist in the rabbit, was an agonist in the chicken. The motilin agonists erythromycin A and EM-523 were almost without effect. Tested against rabbit duodenum, chicken motilin had a smaller potency than mammalian motilins. Thus, chicken motilin and the chicken motilin receptor differ from their mammalian counterparts.

Identification of motilin mRNA in the brain of man and rabbit. Conservation of polymorphism of the motilin gene across species.

The data regarding the identity of motilin-like immunoreactivity in the central nervous system are controversial. The aim of the present study was to clarify whether motilin mRNA is present in the brain of rabbit and man. Total RNA, prepared from several regions of the rabbit brain, was subjected to RT-PCR aimed at amplifying a 294 bp cDNA fragment of the rabbit motilin precursor. The amplified product was subcloned and sequenced. The sequence showed 7 differences compared to the one reported for the duodenal precursor (1). However the duodenal precursor from the rabbit used in the present study revealed identical substitutions. One of these, involving amino acid -11 of the signal peptide, was shown to be due to gene polymorphism, as has also been described at this site in man. By radioimmunoassay the highest concentration of motilin (fmol/mg protein) was detected in the hippocampus (4788 +/- 295), the lowest in the telencephalon (2127 +/- 221). Using a similar approach, but starting from commercial human brain mRNA, the sequence of a comparable cDNA fragment of the human brain motilin precursor was obtained. Its sequence was identical with the one published for the human intestinal precursor (41). Our study demonstrates that motilin mRNA is present in the brain of man and rabbit. Together with our recent findings of central motilin receptors, they suggest a central role for motilin.

Synthesis and in vitro evaluation of [Leu13]porcine motilin fragments.

Several peptide fragments representing N-terminal, C-terminal, and internal sequences of [Leu13]porcine motilin ([Leu13]pMOT) were synthesized using Fmoc solid phase methodology. Peptides were assayed for motilin receptor binding activity in a rabbit antrum smooth muscle preparation and for stimulation of contractile activity in segments of rabbit duodenum. In vitro activity was directly correlated with motilin receptor binding affinity for all [Leu13]pMOT fragments examined. N-Terminal fragments of just over half the length of the native peptide are nearly equipotent as full-length motilin. These results suggest that the N-terminal segment, together with residues from the mid-portion of the molecule, constitutes the bioactive portion of pMOT. The C-terminal segment, in contrast, contributes little to receptor binding affinity or in vitro activity.

D-amino acid and alanine scans of the bioactive portion of porcine motilin.

A recent systematic study of porcine motilin fragments has clearly shown that biological activity resides in the amino-terminal end. The amino-terminal tetradecapeptide retains more than 90% of the potency of the full molecule. We now examined the effect of replacement of residues 1 through 11 by either their D-isomer or by alanine in [Leu13]pMOT(1-14). Peptides were synthesized using Fmoc solid phase methodology, purified by HPLC, and assayed for their ability to displace bound motilin (rabbit antral smooth muscle homogenate) and to induce contractions (isolated rabbit duodenal segments). The negative logarithm of the concentration displacing 50% of the tracer (pIC50), or producing 50% of the maximal contractile response (pEC50), was determined. All compounds were still full agonists. A reduction in potency of more than two log units was seen for the compounds in which residues 1 (Phe), 4 (Ile), and 7 (Tyr) were replaced by Ala and residues 3 (Pro), 4 (Ile), and 6 (Thr) by their D-isomer. The largest drop was noted for the analogs substituted at position 4. For all compounds there was an almost perfect correlation between the pIC50 and the pEC50 values (r = 0.96), although the pEC50 was consistently smaller. These results show that the biological activity of motilin is mainly determined by the first seven residues. The pharmacophore consists of the aromatic rings from Phe1 and Tyr7 and the aliphatic side chains from Val2 and Ile4. Pro3, Phe5, and Thr6 may stabilize the bioactive conformation.

Transduction mechanism of motilin and motilides in rabbit duodenal smooth muscle.

The present study was undertaken to explore motilin's transduction pathway in the rabbit. Guanine nucleotides inhibited 125I-motilin binding in rabbit antral tissue and increased the dissociation of motilin from its receptor. Motilin, the motilin agonist erythromycin A enol ether (EM-201) and carbachol (taken as control) increased the production of inositol phosphates in rabbit duodenal smooth muscle strips labeled with myo-[2-3H]inositol. The effect of carbachol was blocked by atropine. Dose-response curves revealed that 50% of this effect was obtained with 3.9 nM motilin, 170 nM EM-201, 0.54 microM carbachol. Chromatographic separation of the inositol phosphate metabolites showed significant increases in the levels of [3H]inositol bisphosphate and of [3H]inositol trisphosphate. The three substances were without effect upon the metabolism of cAMP, nor did they modulate the rise in cAMP induced by GTP. We propose that motilin's transduction pathway uses a G protein that causes an increase in inositol trisphosphate which is rapidly metabolized, and which may release calcium from intracellular stores.

Effect of erythromycin and of octreotide on motilin receptor density in the rabbit.

Recent studies have shown that erythromycin lactobionate (EMLB) acts as a motilin agonist and is able to accelerate gastric emptying in diabetic gastroparesis. Using the rabbit as a model, we have studied the changes in motilin receptor density induced by EMLB (a motilin agonist) and octreotide (a somatostatin analogue and an inhibitor of motilin secretion). Binding studies were performed with antral smooth muscle tissue homogenates using iodinated nor-leucine13-porcine-motilin, and binding parameters were obtained from computerized fits to displacement curves. The contractile capacity towards motilin (10(-7) M) and EMLB (10(-5) M) was measured isotonically on duodenal segments and the response was expressed relative to the maximum obtained with ACh (10(-4) M). The first hours after the last i.v. administrations of EMLB (50 mg/day given on 3 consecutive days), motilin binding was completely abolished to 0.02 +/- 0.006 fmol/mg protein, compared to the control group (0.64 +/- 0.12 fmol/mg protein). The effect was dose-related: total doses of 17.5 mg, 87.5 mg, 175 mg EMLB reduced motilin binding and contractility towards motilin and EMLB to respectively 95 +/- 10, 82 +/- 5%; 36 +/- 9, 38 +/- 9%; 3 +/- 1, 24 +/- 2% of the control values. The effect was also long lasting: binding was still reduced to 60% of the control value 48 h after the highest dose. In contrast, octreotide induced a marked but short lasting upregulation. After 3 daily s.c. injections of 5 micrograms, Bmax rose to 13.6 +/- 1.9 fmol/mg protein (P less than 0.05). It was already obtained 1 h after 3 x 2.5 micrograms/24 h. The changes in receptor-density were not related to changes in affinity. We conclude that motilin receptors can be regulated by EMLB and octreotide presumably because one compound mimicks hypermotilinemia, the other one induces hypomotilinemia.

Comparison of motilin binding to crude homogenates of human and canine gastrointestinal smooth muscle tissue.

Pharmacological studies indicate that in man and in rabbit, but not in dog, motilin has a direct influence upon gastrointestinal smooth muscle. In accordance with this hypothesis we have presented direct biochemical evidence for the presence of motilin receptors on rabbit smooth muscle tissue. We have now extended our studies to human and canine tissue. Tissue homogenates were studied in binding experiments with iodinated porcine [Leu13]motilin and iodinated canine motilin. It was ascertained that the iodination procedure had little effect on the biological activity of the porcine analogue. In the human antrum specific binding of the iodinated porcine analogue was only found in the smooth muscle layer. It was absent in mucosal or serosal preparations. At 30 degrees C and pH 8.0, binding was maximal after 60 min of incubation, and was reversed by the addition of unlabeled porcine motilin. Binding was enhanced in the presence of calcium and magnesium ions. At a concentration of 10 mM MgCl2, binding was 220% of the binding observed in its absence. Displacement studies with synthetic porcine [Leu13]motilin or synthetic natural porcine motilin indicated a dissociation constant (Kd) of 3.6 +/- 1.6 nM and a maximal binding capacity (Bmax) of 77 +/- 9 fmol per mg protein. Canine motilin displaced iodinated porcine motilin with an apparent Kd of 2.2 +/- 0.9 nM. Compared to antral binding, receptor density decreased aborally and orally, and was absent in jejunum and ileum. In dog specific binding could not be demonstrated in antral and duodenal tissue, neither with labeled porcine nor with labeled canine motilin. However, labeled canine motilin was equipotent to labeled porcine motilin in binding studies with human tissue: the dissociation constant was 0.9 +/- 0.6 nM. The present studies therefore demonstrate the existence of a specific motilin receptor in the antroduodenal region of the human gut. Apparently, such receptors are not present in the canine gut. Our data support the hypothesis that in the human gastrointestinal tract, the gastroduodenal area is motilin's target region.

Motilin receptors in rabbit stomach and small intestine.

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 degrees 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 (Kd) of 1.1 +/- 0.3 nM and a maximal binding capacity (Bmax) 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.