Immunoreactive Motilin Research Articles

Distribution and electrophysiological effects of motilin in Purkinje cells

Evidence exists that motilin immunoreactivity is highly expressed in Purkinje cells. In this study, immunohistochemistry and whole-cell patch-clamp recording were performed to investigate the spatial distribution and electrophysiological effects of motilin receptors in the cerebellum. We show here that motilin receptors are strongly expressed in Purkinje cells of the human and rat cerebellum. Motilin at 10 nM depolarized Purkinje cells of the rat cerebellum, and this was mimicked by the motilin receptor agonist erythromycin. The depolarization evoked by motilin persisted in the presence of tetrodotoxin, glutamate and gamma-amino-n-butyric acid receptor antagonists, indicating that motilin excited the Purkinje cells by activating the receptor expressed on the neurons recorded. We suggest that motilin may serve specific neural functions in the cerebellum.

Sequence and characterization of cDNA encoding the motilin precursor from chicken, dog, cow and horse. Evidence of mosaic evolution in prepromotilin.

Motilin is involved in the regulation of the fasting motility pattern in man and in dog, but may have a different role in other species. Immunoreactive motilin has been demonstrated in several species, but the sequence is mostly unknown. The aim of this study was to isolate and sequence the cDNA encoding the motilin precursor from several mammalian species and from chicken. Total RNA was isolated from the duodenal mucosa of the chicken, dog, cow and horse. In each case single stranded cDNA was synthesized. Motilin cDNA fragments were amplified by PCR, ligated into a plasmid and cloned. Clones which were positive after screening with an appropriate (32)P-labeled probe were sequenced. The 5'- and 3'-ends were determined by the rapid amplification of cDNA ends (RACE) method. Analysis of the cDNAs revealed an open reading frame coding for 115 (chicken and cow), or 117 (dog and horse) amino acids. It consists of a 25 amino acid signal peptide, motilin itself, and a 68 (chicken and cow) or 70 (dog and horse) amino acid motilin associated peptide (MAP). As in all motilin precursors already sequenced (man, monkey, pig and rabbit), an endoproteinase cleavage site is present at Lys(23)-Lys(24). Comparison of all known sequences shows considerable identity in amino acid and nucleotide sequence of the signal peptide and motilin. However, the MAPs differ not only in length but also, more strongly, in amino acid and nucleotide sequence. Our study demonstrates that the N- and C-terminal regions of the motilin precursor have evolved at different rates, which is evidence for 'mosaic evolution'.

Involvement of somatostatin, bombesin and serotonin in the origin of the migrating myoelectric complex in sheep

Antroduodenal myoelectric activity was recorded in conscious sheep by electrodes chronically implanted in the muscular wall. Furthermore, plasma immunoreactive (i.r.) motilin, somatostatin and bombesin concentrations were determined by RIA. The intravenous infusion of somatostatin (20 ng/kg/min), bombesin (10 ng/kg/min) or serotonin (5-HT, 4 μg/kg/min) for 5 min, induced a duodenal myoelectric activity front followed by a period of quiescence. These duodenal events were concomitant with an antral inhibition. This pattern resembled that observed in a spontaneous migrating myoelectric complex (MMC) in sheep. Bombesin and 5-HT evoked an additional and transient increase in antral activity simultaneously with the duodenal activity front. On the other hand, plasma i.r. motilin levels did not show any fluctuations during spontaneous MMC cycles or after somatostatin, bombesin or 5-HT infusions. Likewise, plasma i.r. bombesin levels remained unchanged during spontaneous MMC or after administration of somatostatin. However, 5-HT-induced duodenal activity fronts were closely associated with a sharp peak in plasma i.r. bombesin. Finally, plasma i.r. somatostatin concentrations rose at the end of spontaneous phase III and peaked in phase I. A similar pattern of somatostatin release in plasma was found while the duodenal activity front and quiescence period developed after either 5-HT or bombesin infusions. These results do not indicate a role for motilin in the control of MMCs in sheep, although a definitive conclusion cannot be drawn until synthetic sheep motilin is available. However, our data suggest that somatostatin and bombesin-like peptides as well as 5-HT, acting in a coordinated manner, could be involved in the regulation of cyclical antroduodenal motor events in sheep.

Mechanism of motilin-induced contractions in isolated perfused canine stomach

Background: Motilin is known to induce gastric phase III contractions via neural pathways in vivo, but the local mechanism of action is not clearly determined. Methods: An isolated perfused canine stomach was used to demonstrate the mechanism of motilin. Synthetic canine motilin at doses of 0.1, 0.3, 1.0, and 3.0 μg/h was infused intra-arterially, and effects of several receptor antagonists on motilin-induced contractions were examined. Results: The immunoreactive motilin concentration of venous effluent showed that motilin at doses of 0.1 and 0.3 μg/h was within the physiological range. Each dose of motilin induced phasic contractions in the isolated stomach, and a dose-related increase in frequency was observed, but not their mean amplitude. Atropine, hexamethonium, ICS205-930, BRL43694, phentolamine, yohimbine, and propranolol significantly inhibited motilin-induced contractions. Naloxone, methysergide, and timolol did not affect the response of motilin. Prazosin significantly increased the mean amplitude of motilin-induced contractions. Conclusions: Physiological dose of motilin can initiate phasic contractions in the stomach independently of the presence of the extrinsic nerves. The results suggest that cholinergic pathway, 5-hydroxytryptamine (HT)3 receptors, and α receptors are involved in the motilin-induced contractions.

Mechanism of gastroprokinetic effect of EM523, an erythromycin derivative, in dogs

Background: The pharmacological properties of EM523, a nonpeptide motilin agonist, have not been well characterized. Methods: The prokinetic effect of EM523 on motor-stimulating activity in the stomach, duodenum, and jejunum in seventeen conscious dogs was studied using force transducers implanted long term. EM523 (0.3–10.0 μg/kg) and receptor antagonists were injected intravenously during the interdigestive state. Results: EM523 induced phase III-like contractions in a dose-dependent manner, and the contractions were inhibited dose dependently by pretreatment with cholinergic and 5-HT3 receptor antagonists and dopamine but not by adrenoceptor and opiate antagonists or methysergide. The plasma immunoreactive motilin level was increased after EM523 to 60% of the mean maximum value during the spontaneous phase III contractions. Pretreatment with anti-canine motilin serum inhibited EM523-induced contractions by 19.2% in the motor index, but the contractile pattern was not affected. Conclusions: EM-523-induced phase III-like contractions are brought about through the cholinergic neural pathway and 5-HT3 receptors, and endogenous motilin release is partially involved.

Disseminated rectal carcinoid tumor with production of immunoreactive motilin.

This report presents a patient with a rectal carcinoid tumor of small size ( 14 mm in diameter), with typical growth pattern, localized in the mucosa. Despite these microscopically good prognostic features the patient died from metastatic disease 30 months later. The tumor had an unusual hormone profile with main secretion of immunoreactive motilin and serotonin. Immunocytochemically these substances were localized in separate tumor cell populations; the majority of tumor cells were motilin-immunoreactive and a minority were serotonin-immunoreactive. The patient was first treated interventionally by hepatic arterial embolizations and later medically with octreotide. The treatment resulted in long periods of good palliation related to reduced levels of tumor markers and weight gain. The plasma concentrations of motilin were analyzed with a N-terminal-specific assay before and during treatment.

Slow transit chronic constipation (Arbuthnot Lane's disease)

Seven patients (6 women, 1 man) with severe idiopathic chronic constipation, who underwent surgery with subtotal colectomy and ileorectal anastomosis, were investigated for the occurrence and density of nerve fibres, immunoreactive to different neuropeptides in the mucosa, submucosa, ganglia and smooth muscle in fresh specimens from the colon ascendens, the colon transversum and the colon descendens-sigmoideum. The following substances were studied: enkephalin, substance P, somatostatin, neuropeptide Y, vasoactive intestinal polypeptide, calcitonin gene-related peptide, bombesin, motilin, tyrosine hydroxylase, dynorphin and galanin. Nerve fibres immunoreactive to CGRP occurred in large numbers in the myenteric ganglia of the patients with severe idiopathic chronic constipation, whereas in the myenteric ganglia of the control cases they only occurred in low numbers. In two patients there was no detectable motilin immunoreactivity and in one patient only sparse in the mucosa and the smooth muscle. The other neuropeptides investigated occurred in the density and distribution previously reported in the normal gut. With the present technique there were indications that patients with severe idiopathic chronic constipation have a significant difference in the occurrence of immunoreactive nerve fibres to CGRP and motilin compared to control patients.

Characterization of immunoreactive motilin from the rat small intestine

Immunocytochemistry, radioimmunoassay, chromatography, and biological assay using a rabbit isolated duodenal muscle strip preparation were used in attempting to characterize motilin from the rat small intestine. Several different antisera and monoclonal antibodies directed against natural porcine motilin were used. A variety of fixation techniques using Bouin's, paraformaldehyde, and benzoquinone with different staining methods including, fluorescein-conjugated second antibody, peroxidase-antiperoxidase or peroxidase-conjugated second antibody techniques were used. All methods failed to detect immunoreactive motilin cells in the rat small intestine. The same antisera were used in radioimmunoassays for motilin to evaluate extracts of rat intestinal tissue. Two of these detected immunoreactive motilin in gut extracts, and these antisera showed a different distribution for the peptide. Samples containing immunoreactive motilin obtained from cation exchange chromatography on SP-Sephadex-G25 were concentrated and assayed for biological activity in a rabbit duodenal muscle strip preparation. Desensitization of duodenal tissue to porcine motilin could be demonstrated by pretreatment with this peptide. The biological activity of partially purified rat intestinal immunoreactive motilin was not prevented by pretreatment of the tissue with motilin. Further purification of this preparation on Bio-Gel P-10 yielded an immunoreactive motilin peak that co-eluted with natural porcine motilin. Rat intestinal immunoreactive motilin did not co-elute with natural porcine motilin following high pressure liquid chromatography on a Waters microBondapak C18 reversed-phase column using a linear gradient of water-acetonitrile (10-45%) over 30 min. Although of similar molecular size, rat motilin is probably structurally dissimilar to other mammalian motilins.

Variations of plasma immunoreactive motilin, pancreatic polypeptide, gastrin and somatostatin along the duodenal motility cycle in the pig

The peripheral plasma concentrations of immunoreactive motilin, pancreatic polypeptide (PP), somatostatin and gastrin were measured in 7 pigs fasted to 24 h and subsequently fed a standard meal. Plasma motilin peaked during the last part of phase II activity of the migrating myoelectric complex (MMC) sequence (25.2 ± 2.3 pM), the lowest value being recorded during phase I (10.6 ± 1.5 pM) after a 24 h fast. Plasma motilin remained at a low level during the digestive pattern of duodenal activity, no fluctuation occurring when the first postprandial MMC recurred. At variance analysis, gastrin and PP were not released phasically with MMC in the fasting state, while at autocovariance both peptides tended to fluctuate during the MMC sequence with positive and negative peaks at regular intervals along MMC cycles. No variation of plasma somatostatin was observed in the fasting animals. These findings argue against a major role of circulating PP, gastrin and somatostatin-like components in the control of fasted and post absorptive duodenal motility in pigs while the role of motilin remains equivocal.

Central administration of motilin stimulates feeding in rats

Peripheral administration of motilin has been found to stimulate feeding behavior in rats. Since motilin immunoreactivity has been found in discrete brain sites, we tested the effect of motilin administered intracer-ebroventricularly on feeding in rats. Injection of 1 μg of motilin significantly increased food consumption at 2 hours, 22 hours, and at 24 hours in animals tested either at or 2 hr prior to lights out. Motilin also significantly increased food consumption in animals maintained under continuous lights-on at 2 hours (488% of control), 22 hours (128% of control), and at 24 hours (140% of control).

Motilin and human pancreatic polypeptide measured in CSF from alcoholic and non-alcoholic neurological patients

Thirty-one CSF samples from alcoholics and non-alcoholic neurological patients were assayed for immunoreactive motilin and human pancreatic polypeptide (HPP). Both peptides were detected in all samples. Alcoholics without liver disease had significantly higher levels of motilin and lower levels of HPP than neurological controls.

Erythromycin induces migrating motor complex in human gastrointestinal tract.

Fifteen healthy subjects, fasted at least 8 hr, were studied by means of an infused manometric method. Twenty minutes after termination of the natural phase III activity in the duodenum, erythromycin or normal saline was administered intravenously for 15 min. When normal saline (N = 5) was infused, the next migrating motor complex (MMC) was initiated 151.2 +/- 42.1 min after the infusion. On the other hand, when erythromycin was infused at a rate of 1.0 mg/kg/hr (N = 5) or 3.0 mg/kg/hr (N = 5), MMC-like contractions were initiated at shorter intervals, ie, 47.8 +/- 40.9 min (P less than 0.005) or 23.0 +/- 13.0 min (P less than 0.001) respectively. The duration, frequency, amplitude, and migrating velocity of the naturally occurring MMC (N-MMC) were not significantly different from those of the erythromycin-induced contractions except for the duration of the phase III contractions in the stomach; the duration (5.3 +/- 2.2 min) of the erythromycin-induced contractions being significantly (P less than 0.05) longer than that (3.2 +/- 0.9 min) of the naturally occurring MMC. The immunoreactive motilin (IRM) concentration did not increase significantly after the infusion of erythromycin, when compared to that after infusion of normal saline. It is concluded that erythromycin at a dose of 1-3 mg/kg/hr for 15 min during the interdigestive state, similar to motilin, has a significant influence upon the initiation of MMC in the human gastrointestinal tract, but further investigations are required to confirm whether endogenous motilin is involved or not.

Effect of Haemorrhagic Shock and Duodenal Instillation of Blood on the Plasma Profiles of Somatostatin and Motilin in Pigs: A Study of Molecular Plasma Components

A severe haemorrhage inducing a deep hypovolaemic shock in six anaesthetized pigs effected a plasma somatostatin increase in the superior caval and the portal vein together with a plasma motilin decrease in the portal vein (p less than 0.05). In contrast, the shed blood volume simultaneously instilled intraduodenally in another six anaesthetized pigs in haemodynamic balance induced only an insignificant bimodal somatostatin increase and a modest but significant motilin decrease in plasma from the portal vein (p less than 0.05). Plasma somatostatin immunoreactivity was eluted in four peaks from a Sephadex G-50 Fine column, of which the first peak probably represents somatostatin bound to plasma proteins; the second peak, aggregates of somatostatin-14 and somatostatin degradation products; the third peak, somatostatin-28; and the fourth peak, somatostatin-14. Plasma motilin immunoreactivity eluted in two peaks, of which the first peak possibly represents motilin bound to plasma proteins, and the second peak free plasma motilin. The major changes in portal vein plasma somatostatin and motilin concentrations during the haemorrhagic shock occurred in their free molecular fractions.

Motilin and the vagus in dogs.

The aim of this work was to determine the influence of the vagus on the circulating levels of immunoreactive (IR) motilin. Five mongrel dogs were equipped with chronically implanted electrodes in the small intestine to record the myoelectrical activity. The release of IR motilin during fasting, after a meal, and during an infusion of insulin was studied before and after truncal vagotomy at the diaphragmatic level. When tested at least two weeks after the operation, the motility pattern of the small intestine and the secretion of IR motilin remained unaltered by vagal section. Cyclic increases in IR motilin associated with phase III's of the interdigestive myoelectric complexes were still observed after vagotomy (maximum levels of IR motilin: 250 +/- 37 versus 239 +/- 19 fmol X mL-1, not significant), and they were still abolished by feeding or by insulin. However, an inhibitory influence can probably be mediated by the vagus since, in normal animals, vagal stimulation by a "modified sham feeding" (tease feeding or presentation of food) at the beginning of a period of phase III activity promptly interrupted this part of the complex and decreased significantly the release of IR motilin by about 20%. The release of motilin is not chronically altered by distal vagotomy in dogs.

Radioimmunoassayable Plasma Motilin in Man

In normal humans, significant motilin increases were found after meal ingestion, intraduodenal infusion of fat, and intraduodenal infusion of physiological HCl doses. Only a non-significant plasma motilin increase was found in response to intraduodenal infusion of cattle bile. Plasma motilin decreased significantly after an intravenous insulin injection. During routine cardiac catheterization in a group of 10 patients plasma motilin was significantly lower in the renal vein than in the femoral vein, femoral artery, right atrium, and hepatic vein, suggesting that the kidneys participate in the removal of motilin from the circulation. Fasting and oral fat-stimulated plasma motilin immunoreactivity eluted in two peaks on a Sephadex G-50 Fine column. The two peaks behaved identically with porcine motilin in dilution series.

The mechanism of motilin excitation of the canine small intestine

Close intraarterial injections of motilin to the small intestine of the anaesthetized dog produce prolonged phasic contractions. Tetrodotoxin infused intraarterially blocked field stimulated contractions and abolished the response to motilin as did treatment with a combination of hexamethonium and atropine. Atropine alone increased the dose of motilin required to induce responses. Hexamethonium alone similarly increased the dose of motilin required in the jejunum, but not for the ileum. These results suggest that motilin acts to contract small intestine by stimulation of intrinsic excitatory nerves, some of which are post-ganglionic cholinergic and some of which are not, but are activated by a pathway with a nicotinic synapse. The ED 50 for ileal contractions was greater than that for the jejunum and the time to reach maximum contractions longer suggesting a decreased responsiveness of the lower small intestine to motilin as compared to the upper gastro-intestinal tract. These results and the lesser quantity of immunoreactive motilin in the ileum than in the jejunum may explain the lack of relationship of the activity front of the migrating motor complex in the lower small intestine to venous motilin concentrations.

The development of motilin, cholecystokinin and vasoactive intestinal peptide immunoreactivity in the forebrain and hindbrain of the rat, as determined by radioimmunoassay

Forebrain and brainstem motilin, cholecystokinin (CCK) and vasoactive intestinal peptide (VIP) were determined in rats from 4 days before to birth to 180 days after. CCK and VIP develop postnatally, as previously reported. Motillin concentrations in both areas, however, were highest 4 days before birth, decreasing after birth. In the oldest animals, motilin, CCK and VIP levels were decreased. The presence of substantial motilin immunoreactivity in the brain before birth is suggestive of a possible role for motillin in the regulation of development.

Cholinergic control mechanisms for immunoreactive motilin release and motility in the canine duodenum.

The relationship of immunoreactive (IR) motilin release from the duodenum to duodenal motility changes was investigated in anaesthetized dogs. Stimulation of one or both vagi at 5 or 15 Hz or field stimulation of intrinsic duodenal nerves produced significant increases in duodenal vein IR motilin concentrations and accompanying increases in duodenal motility. However, only stimulation of both vagi at 15 Hz produced significant changes in peripheral venous concentrations of IR motilin. These occurred after a delay at a time when both the duodenum and the antrum were quiescent. Either hexamethonium or atropine blocked IR motilin release induced by stimulation of intrinsic or extrinsic nerves while only atropine inhibited the release induced by intraarterial carbachol. The response stimulated by carbachol and blocked by atropine was tetrodotoxin insensitive and the muscarinic receptor involved was presumably located on a nonneural structure. The site sensitive to hexamethonium was presumably the neural pathway which terminated at the muscarinic receptor. Concomitant studies of duodenal motility responses to vagal and field stimulation suggested a conventional neural pathway with preganglionic cholinergic nerves in the vagus, postganglionic cholinergic nerves in the duodenum (activated by field stimulation) and a smooth muscle muscarinic receptor. Activation of antral motility by stimulation of the abdominal vagus or intraarterial carbachol injections to the antrum increased duodenal IR motilin release in the absence of duodenal motility. Thus activation of the intrinsic nerves which cross the pylorus initiated IR motilin release as well as inhibited duodenal motility.(ABSTRACT TRUNCATED AT 250 WORDS)

Does motilin control interdigestive pepsin secretion in the dog?

Pepsin output in the Heidenhain pouch, plasma motilin concentration, and contractile activity in the pouch and the main stomach were investigated in five dogs. During the interdigestive state, the pepsin output was significantly increased with a cyclic increase in contractile activity in both the pouch and main stomach at approximately 100-min intervals. The plasma immunoreactive motilin (IRM) concentration fluctuated during the interdigestive state, and, peaks of IRM concentration coincided with the maximum pepsin secretory activity. Exogenous administration of motilin (0.5 μg/kg-hr) increased contractile activity in the main stomach and pouch quite similar to the natural interdigestive migrating contractions (IMC), and increased pepsin output significantly. Atropine pre-treatment suppressed the naturally-occuring and motilin-induced pepsin output and contractions in the pouch. It is concluded that pepsin output and contractions in the Heidenhain pouch increase in close association with the IMC in the main stomach during the interdigestive state and these cyclic motor and secretory events in the vagally denervated fundic pouch are most likely regulated by motilin through the intramural cholinergic pathway.

Circadian variations in plasma immunoreactive motilin concentrations

Circadian variations in plasma immunoreactive motilin concentrations