Porcine Motilin Research Articles

Identification, characterization and distribution of motilin immunoreactivity in the rat central nervous system

Motilin-immunoreactivity was evaluated in rat brain using 15 different antisera and by combining gel filtration, high pressure gel filtration and reverse phase high pressure liquid chromatography with radioimmunoassay. Gel filtration chromatography demonstrated a high molecular weight and low molecular weight form of immunoreactive motilin. The high molecular weight form predominated in brain while the low molecular weight peptide was the predominant form of duodenum. The low molecular weight immunoreactive motilin was indistinguishable from synthetic porcine motilin by gel filtration and high molecular weight gel filtration. Low molecular weight rat motilin could, however, be distinguished from synthetic porcine motilin by high pressure liquid chromatography and certain antisera. Immunological results suggest that the slight structural difference may be in the N-terminal portion of the molecule. Immunoreactivity was measured in grossly and microdissected regions of the rat brain. The peptide had quite a unique distribution as highest concentrations are observed in the cerebellum. High concentrations were also observed in hypothalamic nuclei. Particularly high concentrations were noted in the organum vasculosum lamina terminalis. Lowest motilin concentrations in the rat brain were in the pons and the medulla. The distribution of motilin in rat brain suggests that it may have roles in regulating both neuroendocrine and neurological processes.

Motilin-antimotilin reaction reveals two antigenic determinants in motilin

The reaction of motilin with four rabbit antimotilin sera raised by immunization with synthetic porcine motilin-bovine serum albumin conjugate was studied with respect to various binding parameters and specificity. All four antisera exhibited an extremely high degree of specificity and high affinity (K greater than 10(11) M-1) for porcine motilin. Studying the various synthetic motilin fragments. These antisera appeared to contain binding sites reacting strongly with the N-terminal sequence in which the first three amino acid residues are essential for high affinity binding. One of the antisera, R-3-6, appeared to contain approximately 20% of its binding sites with high affinity for C-terminus-containing fragments. These results suggest that motilin possesses two antigenic domains along its primary structure; one contains the N-terminal tripeptide and the other contains the C-terminal nanopeptide as essential parts. Thus, in addition to heterogeneity in affinity, a given antibody preparation may be heterogenous with respect to the specificity along the sequence of a peptide. Gel filtration studies of the methanol extracts of human and dog plasma indicated that an immunoreactive motilin-like material with a molecular size similar to natural porcine motilin was measured by our routine radioimmunoassay.

Molecular forms of motilin in the mammalian and human gut and human plasma

Regional specific antibodies and gel permeation chromatography were used to analyze the molecular forms of motilin in the human gut and plasma and in the porcine gut. The same antibodies were also used for immunocytochemical localization of the cell of origin of motilin. Two main molecular forms of motilin were identified, peak I eluting at a volume suggesting a larger molecular weight substance (Kav = 0.16) and peak II coeluting with the porcine motilin standard (Kav = 0.67). Peak I accounted for over 50% of the total motilin immunoreactivity in the human gut, whereas in the porcine intestine it represented less than 5%. Peak I thus appeared to have a greater n-terminal sequence similarity with the porcine motilin standard. Chromatographic analysis of human plasma showed the presence of both immunoreactive peaks in approximately equal amounts which were both suppressed following infusion of somatostatin. Infusion of porcine motilin resulted in the expected rise of plasma immunoreactivity in the peak II position, whereas peak I was significantly depressed, suggesting the possibility of a negative feedback. Immunocytochemistry revealed that both antibodies immunostained equally the non argentaffin cell, which contained small round, electrondense granules. In addition some argentaffin enterochromaffin cells were stained predominantly with the n-terminal specific antibody.

Changes in plasma motilin concentration in response to manipulation of intragastric and intraduoduenal contents in man.

Immunoreactive plasma motilin concentrations were studied following a variety of stimuli in 24 healthy fasting subjects. Plasma motilin was measured by a radioimmunoassay using antibody GP 71 (J. C. Brown) and natural porcine motilin as standard. Basal motilin levels ranged from undetectable to 365 pg/mL. Antral and intraduodenal infusion of 50 mL o.1 N HCl (pH 1.2) at 5 mL/min failed to alter significantly plasma motilin levels but duodenal acid infusions at 17 mL/min caused a significant increase (70.8 +/- 29.5 pg/mL, mean +/- SEM; n = 6), maximal at 40 min. Duodenal alkalinization with 50 mL 0.3 M Tris buffer (pH 8.0) infused at 5 mL/min produced no change in plasma motilin. A mixed meal did not affect plasma motilin levels. Ingestion of 60 g fat significantly increased plasma motilin (n = 13; maximal increase 150.3 +/- 43.3 pg/mL at 30 min) but duodenal infusions of fat failed to increase plasma motilin levels. These results suggest that motilin secretion induced by fat requires that the fat be present initially within the stomach for secretion to occur. We conclude that ingested fat is a potent stimulus of motilin release. As duodenal acidification (50 mL 0.1 N HCl over 10 min) induces duodenal activity resembling migrating motor complexes but does not release motilin, our data argue against the release of motilin following duodenal acidification as a trigger for the initiation of these complexes in man.

ChemInform Abstract: SYNTHESIS OF PORCINE MOTILIN VIA ITS SULFOXIDES

AbstractDie Fragmente (I) und (II), die im wesentlichen unter Anwendung der REMA‐("repetitive excess mixed anhydrid"‐) Methode gewonnen werden, werden nach dem Mischanhydrid‐Verfahren unter Bildung von geschütztem Motilin‐S‐sulfoxid (IIIa) kondensiert.

Synthesis of porcine motilin via its sulfoxides

AbstractThe protected segments corresponding to sequences 1‐8 and 9‐22 proposed for porcine motilin were synthesized mainly by the repetitive excess mixed anhydride (REMA) method. Mixed anhydride coupling of the segments gave protected motilin sulfoxide (Scheme). Deprotection with liquid hydrogen fluoride yielded the crude motilin sulfoxides which, after purification by HPLC on a semi‐preparative scale, were quantitatively reduced by iodide in aqueous trifluoroacetic acid.According to Dr. Zen Itoh, Japan, the minimum dose of our REMA‐synthesized motilin required to induce interdigestive migrating contractions (IMC) in dogs was 0.03 μg/kg.h, which indicates a potency of about ten times the standard synthetic preparation used in the assay. In a comparison with natural porcine motilin by Dr. Pierre Poitras, U.S.A., REMA‐motilin showed at least 100% immunological cross‐reactivity.

Heterogeneity of immunoreactive motilin.

Motilin-like immunoreactivities were examined in extracts of duodena of pig, human, dog, rat, cat, rabbit and guinea pig with a combination of radioimmunoassays using three different antisera possessing distinct region-specificities. Antisera GP-1103 and R-1104 were rather C-terminal specific, but the immunological feature of GP-1103 was different from that of R-1104. In the case of antiserum GP-1103, the Glu residue in position 15 in the motilin molecule participated in antigen-antibody interaction, but not in the case of R-1104. Antiserum MBR-02 was N-terminal specific, with of the three assay systems, only a single major peak was detected in the elution profile from BioGel P-10 column of motilin-like immunoreactivity in the porcine duodenal extract, of which the elution volume corresponded to that for synthetic porcine motilin. On the other hand, the gel filtration profiles of the human and canine duodenal extracts consisted of two or three peaks of the immunoreactivity. The major peak emerged in the elution volume identical with that for synthetic porcine motilin and the other two peaks were eluted earlier. The peak which was eluted in the volume corresponding to that for BSA marker was detectable only by the assay system with N-terminal specific antiserum MBR-02. The immunoreactivity of the major peak component in the human, canine or rat extract was found to be lower than that in the porcine extract. The present data may indicate the existence of heterogeneity of motilin-like immunoreactivity and species variation of the structure of the immunoreactive motilin.

Amino acids and peptides. XXXI. Phosphorus in organic synthesis. XVIII. Synthesis of porcine motilin by the solid-phase method using diphenyl phosphorazidate(DPPA) and diethyl phosphorocyanidate(DEPC).

Synthesis of porcine motilin, which exhibits a gastric motor stimulating activity, was accomplished by the solid-phase method with diphenyl phosphorazidate (DPPA) and diethyl phosphorocyanidate (DEPC) as coupling reagents. The purified synthetic peptide showed characteristic contractile activity towards the duodenum, colon and jejunum in the rabbit. Its potency was almost the same as that of the natural material.

Motilin and the interdigestive migrating motor complex in man

In order to assess the possible role of the new candidate gut hormone, motilin, in controlling the interdigestive migrating motor complex (MMC) in man, 14 normal subjects were studied after an overnight fast by means of three pressure-recording catheters with orifices 25 cm apart in the upper small intestine. The typical aboral progressing bursts of pressure waves occurred at a mean interval of 137 minutes and were preceded by a peak motilin level 25 pmol/liter higher than the lowest level in the postactivity-front quiescent period. To study the effect of exogenous motilin, an infusion of pure porcine motilin at various dose levels was given to 16 normal volunteers shortly after the onset of the phase I quiescent period. Motilin infusion induced an activity front in 12 of the 16 subjects. The mean activity front interval was reduced to 46 min (P less than 0.001). This effect could be obtained even at the low dose level of 0.4 pmol/kg/min, which produced an increase in plasma motilin level of only 57 pmol/liter. These data suggest that a cyclic rise in plasma motilin levels is one of the factors involved in the production of the activity front of the migrating motor complex in man.

Immunohistochemical Localization of Motilin in Endocrine Non-Enterochromaffin Cells of the Small Intestine of Humans and Monkey

Motilin-immunoreactive cells in the human and monkey duodenum and upper jejunum were investigated by immunofluorescence (IF) and peroxidase-antiperoxidase (PAP) techniques using antibodies against synthetic 13-norleucine motilin and synthetic porcine motilin. Contrary to previous reports, we have demonstrated that motilin-immunoreactive cells are a distinct cell population that does not correspond to 5-hydroxytryptamine-containing enterochromaffin (EC-) cells. EC-cells, indentified by formaldehyde-induced fluorescence (FIF) or by argentaffinity (AA), do not react with either antisera. EC-cells of the monkey, utilizing one method (PAP), reacted to one antiserum very weakly. This reaction was also suppressed by absorption with 13-norleucine motilin. It is suggested that the EC-cells of the monkey contain either a very small amount of motilin or another peptide that exhibits a part of the amino acid sequence of motilin. The discrepancy between these results and those of other authors can be explained by the different reactivity of the antisera used.

Radioimmunoassay of motilin. Validation and studies on the relationship between plasma motilin and interdigestive myoelectric activity of the duodenum of dog.

A radioimmunoassay method of motilin was developed in our laboratory and was validated in dogs with a platinum monopolar electrode in the duodenum. We confirmed that a bolus infusion of 0.3 M tris-buffer solution or 0.1 N HCl solution in the duodenum produces a significant rise in plasma immunoreactive motilin (IRM) concentrations. This coincided with a marked increase in the percentage of spike potentials on slow waves of the duodenum, similar to phase III of interdigestive myoelectric-activity (MA). A possible relationship between plasma IRM and interdigestive MA of canine duodenum was studied. It was found that cyclic changes occurred in the fasting plasma IRM concentrations in dogs. While the peak motilin concentration was always observed in phase III, the lowest concentration of motilin was found in phase I of interdigestive MA in the duodenum. In dogs with the electrodes in the duodenum and jejunum, the peak IRM concentration did not correlate with phase III of interdigestive MA in the jejunum. A dose of synthetic porcine motilin, 0.06 μg/kg/hr, which produced the plasma IRM concentration comparable to the peak fasting motilin concentration, could induce an identical phase III in the duodenum. These observations indicate that there is a relationship between cyclic changes in plasma IRM concentrations and interdigestive MA of the duodenum. It is suggested further that motilin is a hormone which may play an important role in inducing phase III of interdigestive MA in the duodenum.

ChemInform Abstract: SYNTHESIS AND BIOLOGICAL ACTIVITY OF 8‐L‐ALA‐MOTILIN AND 8‐D‐ALA‐MOTILIN

AbstractAnaloga des Schweinemotilins (I) mit D‐ und L‐Alanin anstelle des Glycinrestes in Stellung 8 werden mit Hilfe der konventionellen Lösungsmethode über die Methioninsulfoxid‐Derivate synthetisiert.

Development of radioimmunoassay for motilin

A highly specific and sensitive radioimmunoassay for motilin has been developed. High titers of motilin antisera were produced in rabbits by repeated immunization with synthetic porcine motilin coupled to bovine serum albumin. Motilin was radioiodinated with Na[ 125I] by the chloramine-T method and was purified using a CM-Sephadex C-25 column eluted with ammonium acetate gradient at pH 5.9. A specific radioactivity of 300 to 400 μCl/μg was usually obtained. Untreated plasma showed significant nonspecific interference in the assay; however, the interference could be minimized by extracting the plasma with methanol. All of the known gastrointestinal hormones were tested and showed no cross-reactivity at 10 ng/ml. Plasma motilin levels in dogs were significantly increased within 3 min, after an intraduodenal bolus injection of 50 ml of 0,1 n HCl. This increase coincided with a marked increase in myoelectrical activities of the duodenum.

Effects of motilin and octapeptide of cholecystokinin on antral and duodenal myoelectric activity in the interdigestive state and during inhibition by secretin and gastric inhibitory polypeptide.

In conscious dogs prepared with gastric cannulae and antral and duodenal electrodes, intravenous administration of synthetic porcine motilin and CCK-OP in varying doses resulted in significant increases in the mean percent frequency of spike potentials on slow waves. Motilin was a more potent stimulant than CCK-OP. Motilin-stimulated spike potentials in both antrum and duodenum were significantly suppressed by either natural porcine secretin or synthetic porcine GIP. Secretin suppressed the spike potentials of the antrum stimulated by CCK-OP but not those of the duodenum. The spike potentials stimulated by CCK-OP could not be suppressed by GIP in the dosages employed.

Synthesis of porcine motilin and its D-Phe1-analog by the use of methanesulfonic acid.

Motilin and [D-Phe1]-motilin were synthesized via the corresponding sulfoxide (Met13). The protected Met (O)13-motilin and [D-Phe1, Met (O)13]-motilin, in which Met residue was substituted by Met (O) residue, were constructed from three suitable subunits, respectively. The protected peptides were deblocked by methanesulfonic acid in the presence of anisole to obtain Met (O)13-motilin and its D-Phe1-derivative, and the resulting Met (O)-peptides were finally reduced by thiol to give the desired docosapeptides, motilin and D-Phe1-motilin.

Synthesis and biological activity of 8-L-Ala-motilin and 8-D-Ala-motilin.

Two analogs of porcine motilin altered at position 8, [L-Ala8]-motilin and [D-Ala8]-motilin, were prepared by the conventional solution method via the corresponding sulfoxides (Met13) and evaluated in vitro for the ability to contract the duodenal muscle of rabbit. The D-Ala analog exhibited a higher potency than synthetic motilin, whereas substitution of glycine at position 8 with L-alanine decreased the biological activity to approximately 30% relative to the synthetic motilin.

ChemInform Abstract: SYNTHESIS OF THE HEXADECAPEPTIDE CORRESPONDING TO POSITIONS 1 THROUGH 16 OF PORCINE MOTILIN, A GASTRIC MOTOR ACTIVITY STIMULATING POLYPEPTIDE

AbstractDie Synthese des Hexadecapeptids (I), das der Sequenz 1‐16 von Motilin entspricht, wird beschrieben.

Structure-Activity Relationships of Gastrointestinal Hormones: Motilin, Gip, And [27-Tyr]CCK-PZ

The docosapeptide and the tritetracontapeptide corresponding to the entire amino acid sequence of porcine motilin and gastric inhibitory peptide were synthesized, and in addition, an unsulfated form of cholecystokinin-pancreozymin (CCK-PZ) was prepared to cast some light on the structure-activity relationships of these gastrointestinal hormones. In a series of motilin peptides, elimination of the pentapeptide from the amino terminus decreased the activity (in vitro contraction of rabbit duodenal muscle) to 1/250 of the whole molecule, and subsequent removal of the tripeptide Thr-Tyr-Gly resulted in the complete loss of its effects, In a series of gastric inhibitory peptides, two fragments corresponding to positions 1 to 28 and 26 to 43 were both inactive. However, the nonacosapeptide (15 to 43) retained one-fourth of the activity of the tritetracontapeptide (suppression of the gastric acid secretion stimulated by tetragastrin in Heidenhain pouch dogs). Synthetic [27-Tyr]CCK-PZ exhibited 1/250 of the activity of natural CCK-PZ (amylase release from rat pancreas). This compound was smoothly and efficiently labeled with 125I (specific activity 200 to 250 muc per mug).

ChemInform Abstract: SYNTHESIS OF PORCINE MOTILIN BY FRAGMENT CONDENSATION USING THREE PROTECTED PEPTIDE FRAGMENTS

AbstractSchweine‐Motilin (I) wird unter Verwendung seiner bereits synthetisierten 3 Teilsequenzen 1‐6, 7‐1 1 und 12‐22 dargestellt [1, Kondensation der Fragmente 7‐1 1 und 12‐22; 2. Kondensation des gebildeten Fragments 7‐22 mit dem Fragment 1‐6 (modifizierte Azidmethode nach Rudinger); Schutz der e‐ Aminogruppe von Lysin und der Guanidinogruppe von Arginin durch den Tosylrest; Schutzgruppenabspaltung beim erhaltenen geschützten Dokosapeptid mit Na/ flüssi‐ 1:14 gem NH3 ; Reinigung des Endproduktes durch Ionenaustausch‐Chromatographie (CM‐Sephadex, Amm0niumacetat)].

ChemInform Abstract: SYNTHESIS OF PROTECTED PEPTIDE FRAGMENTS RELATED TO PORCINE MOTILIN

AbstractDie Synthesen der 3 Peptidfragmente (I), (II) und (III), die den Aminosäuresequenzen 1‐6, 7‐1 1 bzw. 12‐22 von Schweine‐Motilin (IV) , einem die motorische Magenfunktion fördernden Polypeptid entsprechen, erfolgt durch schrittweisen Aufbau nach üblichen Verfahren (Methode der gemischten Anhydride, Azidmethode).