Motilin--an update

Motilin isolated in 1971 from the porcine gastrointestinal tract and localized there to endocrine cells, now appears to have a CNS neural origin by RIA and immunohistochemistry. In most species motilin releases neurotransmitters in the CNS to both increase and decrease neural transmission and in the gastrointestinal tract to increase motor activity. In the fasting animal, motilin initiates premature activity fronts of the migrating motor complex (MMC) in the upper gastrointestinal tract by an atropine or tetrodotoxin-sensitive mechanism. Immunoreactive motilin-release from the gut can be correlated with the passage of these fronts through the upper gut. In the dog, the associated events of this MMC, i.e. motor activity of the duodenum extrinsic and intrinsic neural activity and emptying of biliary and pancreatic secretions into the duodenum, all appear to contribute to the peaks in peripheral plasma immunoreactive motilin concentrations. In man, there appears to be a close association of motilin secretion with biliary and pancreatic secretions being emptied into the duodenum and less evidence for motor activity releasing motilin. Only in the dog is there strong evidence for an absolute requirement of motilin for the consolidation of the motor activity of the upper gut into the MMC. In man, the evidence is less convincing although motilin may facilitate the process and in the pig, motilin appears to have little or no role in MMC generation. No pathological consequences of hypermotilemia have been described although elevated motilin levels have been found to be associated with some diarrheal states, renal failure, and in the first week following abdominal surgery. Motilin thus remains a hormone seeking a physiological function in some species and a pathological role in all species.     

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'.     

The isolation and characterization of rabbit motilin precursor cDNA.

The cDNA sequence of rabbit motilin precursor has been determined. The predicted amino acid sequence indicates that the precursor consists of 133 amino acids and includes a 25 amino acid signal peptide followed by the 22 amino acid motilin sequence and an 86 amino acid motilin associated peptide (MAP). As in the human and porcine precursors, two lysine residues follow motilin in the rabbit sequence. Rabbit motilin shares 64% amino acid sequence identity with human and porcine motilin, and all amino acid substitutions represent conservative changes. Amino acid sequence alignments of the rabbit, human and porcine MAP sequences suggest three functional/structural motifs corresponding to a putative endoproteinase recognition site, a putative PEST site and a potential posttranslational processing recognition element.     

Sequence of an intestinal cDNA encoding human motilin precursor

A cDNA clone encoding the human motilin precursor was isolated from an intestinal library using synthetic oligonucleotide probes. The predicted amino acid sequence indicates that the motilin precursor consists of 115 amino acids and includes a 25-residue N-terminal signal peptide followed by the 22-amino-acid motilin sequence and a long, 68-residue C-terminal peptide. The amino acid sequence of human motilin predicted from the cDNA sequence is identical to its porcine counterpart, which has been determined by protein sequencing. Proteolytic processing of promotilin to motilin occurs at the sequence, Lys-Lys, this being the first reported instance of processing occurring at a pair of Lys residues. In other precursors it occurs at Lys-Arg, Arg-Arg, Arg, or very rarely Lys.     

Characterization of complementary deoxyribonucleic acid for precursor of porcine motilin

Cloned cDNAs encoding the precursor protein for motilin and a novel peptide, motilin-associated peptide, were isolated from a library derived from porcine intestinal mucosa mRNA. Nucleotide sequence analysis predicts a precursor protein of 119 amino acids including a signal peptide in direct linkage with the 22 amino acid sequence for motilin, and a 70 amino acid peptide of unknown function. The putative bioactive moieties are separated by Lys-Lys, dibasic residues that serve as substrates for cleavage by proteolytic maturation enzymes in many polyprotein precursors. While there is an abundant literature detailing a spectrum of tissues and cell types which express motilin like immunoreactivity, analysis of mRNA derived from many of these tissues suggests that the mRNA for the mucosal motilin precursor is only transcribed in this tissue. The nature of the immunoreactive material in the central nervous system and other peripheral tissues remains to be determined.     

北极狐GHR基因cDNA的克隆及序列分析

本文根据狗(AF133835)的GHR基因cDNA编码全序列设计了三对引物,利用RT-PCR方法克隆出北极狐GHR基因编码区全长cDNA序列(GenBank accession No.EU304325)。结果表明,北极狐GHR的ORF为1917bp,编码638个氨基酸的前体蛋白,由18个氨基酸的信号肽和620个氨基酸的成熟肽组成。通过同源性比较发现北极狐与狗的同源性最高,达到98%。另外,利用邻接法(NJ法)构建的分子系统进化树聚类结果表明,北极狐与狗先聚为一类,该聚类结果与传统的物种进化关系基本一致。另外,通过氨基酸对位序列比较发现,北极狐GHR在氨基酸序列上存在明显的特异性,如45和451位分别为A和E,而其它物种均分别为T(大鼠为K)和A(牛羊为V,鼠为T)。     

Pancreatic polypeptide is not involved in the regulation of the migrating motor complex in man

The role of pancreatic polypeptide (PP) and motilin in the regulation of the migrating motor complex (MMC) was studied in normal subjects. Both plasma motilin and PP levels changed cyclically in the fasted state and were highest in the late phase II period preceding the activity front in the duodenum. A continental breakfast invariably disrupted the MMC and induced a fed pattern of motility. After the meal plasma motilin levels decreased whereas PP levels rose significantly. Infusion of pure porcine motilin during the fasted state induced an activity front and a rise in plasma PP levels. Infusion of bovine PP in doses producing plasma PP levels above the postprandial values neither induced an activity front nor prevented its occurrence. During PP infusion, however, plasma motilin levels were low, although the activity front was not inhibited. PP seems to have no clear role in the regulation of the motor component of the MMC of man. The role of motilin in the production of the activity front of the MMC is discussed.     

Molecular identification and characterization of the dog motilin receptor

Motilin, a 22-amino acid peptide hormone secreted by endocrine cells of the intestinal mucosa, plays an important role in the regulation of gastrointestinal motility. The actions of motilin agonists have been extensively investigated in dogs due to physiological similarities between the dog and human alimentary tracts. The amino acid sequence of the dog motilin receptor, however, was previously unknown. We have cloned a cDNA from dog stomach corresponding to the motilin receptor. The deduced protein shared 71% and 72% sequence identity with the human and rabbit motilin receptors, respectively. Expression of the dog motilin receptor in CHO cells promoted the typical cellular responses to the agonists, motilin and erythromycin. The rank order of potency determined for these agonists was similar to that found for the human motilin receptor, with motilin being more potent than erythromycin. Immunohistochemistry of the dog stomach revealed that the motilin receptor was localized in neuronal cell bodies and fibers. This is the first study detailing the cloning, expression, and functional characterization of the dog motilin receptor. Determination of the full sequence and functional properties of the dog motilin receptor will provide useful information enabling us to interpret previous and future studies of motilin agonists in dogs.     

Motilin receptors in the human antrum

Motilin is an intestinal peptide that stimulates contraction of gut smooth muscle. The motilin receptor has not been cloned yet, but motilin-receptor agonists appear to be potent prokinetic agents for the treatment of dysmotility disorders. The aim of this study was to determine neural or muscular localization of motilin receptors in human upper gastrointestinal tract and to investigate their pharmacological characteristics. The binding of (125)I-labeled motilin to tissue membranes prepared from human stomach and duodenum was studied; rabbit tissues were used for comparison. Solutions enriched in neural synaptosomes or in smooth muscle plasma membranes were obtained. Various motilin analogs were used to displace the motilin radioligand from the various tissue membranes. The highest concentration of human motilin receptors was found in the antrum, predominantly in the neural preparation. Human motilin receptors were sensitive to the NH(2)-terminal portion of the motilin molecule, but comparison with rabbit showed that both species had specific affinities for various motilin analogs [i.e., Mot-(1-9), Mot-(1-12), Mot-(1-12) (CH(2)NH)(10-11), and erythromycin]. Motilin receptors obtained from synaptosomes or muscular plasma membranes of human antrum expressed different affinity for two motilin-receptor agonists, Mot-(1-12) and Mot-(1-12) (CH(2)NH)(10-11), suggesting that they correspond to specific receptor subtypes. We conclude that human motilin receptors are located predominantly in nerves of the antral wall, are functionally (and probably structurally) different from those found in other species such as the rabbit, and express specific functional (and probably structural) characteristics dependent on their localization on antral nerves or muscles, suggesting the existence of specific receptor subtypes, potentially of significant physiological or pharmacological relevance.     

Peripheral motilin administration stimulates feeding in fasted rats

Although the physiologic function of the gastrointestinal hormone motilin remains uncertain, plasma levels of this peptide vary with migrating myoelectric complexes (MMCs) in the small intestine. In the fed state, both MMCs and plasma motilin are suppressed. During fasting, cyclical peaks of motilin in plasma occur at the same time as Phase III of the MMC cycle occurs in the duodenum. This dependence of motilin concentrations in plasma on the feeding state of the animal prompted an investigation of the effects of motilin on feeding behavior. Intraperitoneal injection of motilin into fasted, but not fed, rats stimulated eating in a dose dependent manner. A significant stimulation of feeding was seen at doses of 5 and 10 μg/kg. Sated rats did not eat whether injected with motilin or vehicle. The feeding response to motilin was blocked by prior injection of the rats with naloxone, naltrexone, or pentagastrin. The dose response suppression of food intake by naloxone was similar in fasted animals treated with motilin or vehicle. Motilin may function as a hunger hormone during periods of fasting.     

Lack of CCK-like pancreatic stimulation by intraduodenal amino acids in the anesthetized cat

We have attempted to demonstrate a pancreatic secretory response to intraduodenal amino acids in the anesthetized cat. In four cats stimulated with supramaximal doses of secretin, protein concentrations in pancreatic juice were measured after intraduodenal bolus injection of various amino acids, IV CCK, or electrical stimulation of the vagus nerve. In addition, the duodenum was perfused with phenylalanine (50 mM) for 30 min in two cats, and the vagus nerve stimulated electrically for 15 min in one. In no case did amino acids produce pancreatic protein secretion, whereas CCK and vagal stimulation always did so. We conclude that this insensitivity to amino acids in the cat is a species difference from the dog and man.     

Erythromycin and its derivatives with motilin-like biological activities inhibit the specific binding of 125I-motilin to duodenal muscle

Erythromycin, one of the macrolide antibiotics, and its derivatives had been found to mimic actions of exogenous motilin, a gastrointestinal peptide hormone. We found that some of the macrolide compounds inhibited the specific binding of 125I-motilin to rabbit duodenum muscle at 15 C in a dose-dependent fashion. The inhibitory activity of several macrolides examined did not relate to their antibacterial activity but to their motilin-like activity. A 50% inhibition by EM536, a non-antibacterial erythromycin derivative with the highest motilin-like activity, was obtained at 3-40 nM and little higher than that of non-radioactive motilin (5-6 nM) under the present conditions. The results suggest that erythromycin and its derivatives mimic physiological actions of motilin by acting as agonists for a motilin receptor.     

Molecular identification of GHS-R and GPR38 in Suncus murinus

We previously identified ghrelin and motilin genes in Suncus murinus (suncus), and also revealed that motilin induces phase III-like strong contractions in the suncus stomach in vivo, as observed in humans and dogs. Moreover, repeated migrating motor complexes were found in the gastrointestinal tract of suncus at regular 120-min intervals. We therefore proposed suncus as a small laboratory animal model for the study of gastrointestinal motility. In the present study, we identified growth hormone secretagogue receptor (GHS-R) and motilin receptor (GPR38) genes in the suncus. We also examined their tissue distribution throughout the body. The amino acids of suncus GHS-R and GPR38 showed high homology with those of other mammals and shared 42% amino acid identity. RT-PCR showed that both the receptors were expressed in the hypothalamus, medulla oblongata, pituitary gland and the nodose ganglion in the central nervous system. In addition, GHS-R mRNA expressions were detected throughout the stomach and intestine, whereas GPR38 was expressed in the gastric muscle layer, lower intestine, lungs, heart, and pituitary gland. These results suggest that ghrelin and motilin affect gut motility and energy metabolism via specific receptors expressed in the gastrointestinal tract and/or in the central nervous system of suncus.     

Interdigestive intestinal motility in dogs with chronic exclusion of bile from the digestive tract

To elucidate the role of bile delivery into the duodenum on the regulation of plasma motilin and on the interdigestive migrating complex, three dogs were operated upon to ligate the main bile duct and divert the biliary flow into the urinary bladder via a Foley catheter. After the operation, despite the chronic diversion of bile from the digestive tract, all animals maintained an excellent health status and exhibited recurrent periods of phase III motor activity migrating from the duodenum to the ileum, which were associated with cyclic increases in plasma motilin. Following the infusion of pooled dog bile (1 mL/min for 10 min) into the duodenum, a premature phase III and a concomitant rise in plasma motilin were observed. These results suggest, that although bile delivery into the duodenum can induce motilin increase in plasma and period of phase III activity in the gut, this phenomenon does not constitute an essential stimulus for the release of motilin and for the induction of the phase III of the interdigestive migrating complex.     

Characterization and distribution of bombesin-like peptides in the rat brain and gastrointestinal tract

Extracts of rat brain and gastrointestinal tract, analyzed by reverse-phase high-performance liquid chromatography and radioimmunoassay, contained two bombesin-like immunoreactivity peaks with similar retention times as porcine gastrin-releasing peptide (GRP) and its COOH-terminal decapeptide, neuromedin C or GRP(18-27). However, the GRP-like peptide peak did not elute with exactly the same retention time as porcine GRP. The highest concentration of bombesin-like immunoreactivity was found in extracts of antrum, whereas the lowest was found in whole brain. Neuromedin C was present at lower concentrations than the GRP in antrum, duodenum, and ileum, while similar amounts of each were found in brain.     

Identification of a novel male reproduction-related gene and its regulated expression patterns in the prawn, Macrobrachium rosenbergii

To identify male-specific genes that could be involved in male development, we screened a subtracted male reproductive tract library and isolated a novel gene named Mar-Mrr (M. rosenbergii male reproduction-related gene). The Mar-Mrr cDNA sequence consists of 683 nucleotides with a 333 nucleotide open reading frame, encoding putative 110 amino acids (11.7473 kDa) precursor protein and a signal peptide consisting of 24 amino acids. Significant developmentally dependent accumulation of the mRNA was observed in the male reproductive tract, specifically in epithelial cells of vas deferens and terminal ampullae.     

Cloning and characterization of penicillin V acylase from Streptomyces mobaraensis

We report on the molecular cloning and characterization of penicillin V acylase (PVA) from an actinomycete, Streptomyces mobaraensis (Sm-PVA), which was originally isolated as an acylase that efficiently hydrolyzes the amide bond of various N-fatty-acyl-l-amino acids and N-fatty-acyl-peptides as well as capsaicin (8-methyl-N-vanillyl-6-nonenamide). In addition, the purified Sm-PVA hydrolyzed penicillin V with the highest activity (k(cat)) among the PVAs so far reported, penicillin G, and 2-nitro-5-phenoxyacetamide benzoic acid. The BLAST search revealed that the Sm-PVA precursor is composed of a polypeptide that is characteristic of enzymes belonging to the beta-lactam acylase family with four distinct segments; a signal sequence (43 amino acids), an alpha subunit (173 amino acids), a linker peptide (28 amino acids), and a beta subunit (570 amino acids). The mature, active Sm-PVA is a heterodimeric protein with alpha and beta subunits, in contrast to PVAs isolated from Bacillus sphaericus and B. subtilis, which have a homotetrameric structure. The amino acid sequence of Sm-PVA showed identities to PVA from S. lavendulae, N-acylhomoserine lactone-degrading acylase from Streptomyces sp., cyclic lipopeptide acylase from Streptomyces sp., and aculeacin A acylase from Actinoplanes utahensis with 68, 67, 67, and 41% identities, respectively.     

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.