Primary structure, tissue distribution, and biological activity of chicken motilin receptor

Motilin is a peptide hormone involved in gastrointestinal motility. GPR38, initially cloned as an orphan receptor, is now considered a specific receptor for motilin. Previously, molecular characterization of the motilin receptor had only been performed in mammalian and fish species. In this study, we cloned cDNA for chicken motilin receptor from the duodenum and characterized its primary structure, tissue distribution, and biological activity. The cDNA encoded 349 amino acids showing significant overall sequence identity to mammalian motilin receptors. Chicken motilin increased intracellular Ca2+ concentration in human embryonic kidney (HEK) 293 cells transiently expressing the recombinant chicken motilin receptor. Comparison of the cDNA sequence with the genomic sequence of chicken motilin receptor revealed that the chicken motilin receptor gene consists of two exons separated by an intron. Real-time PCR analysis showed that chicken motilin receptor mRNA is expressed in a wide range of tissues in 21-day-old chickens, with markedly high levels in the proventriculus, duodenum, and oviduct. The expression levels of the mRNA in the proventriculus and duodenum were highest just before hatching and rapidly decreased during post-hatch development. These results suggest that chicken motilin receptor is largely involved in gastrointestinal functions at pre- and post-hatch periods through an intracellular signaling pathway accompanied by an increase in Ca2+ levels.     

Effects of Motilin and Mitemcinal (GM-611) on Gastrointestinal Contractile Activity in Rhesus Monkeys In Vivo and In Vitro

Neither the presence of motilin receptors nor their action has been investigated in monkeys. The object of this study was to determine the effects of motilin and mitemcinal (GM-611), an erythromycin derivative, on the gastrointestinal tract in rhesus monkeys in vivo and in vitro. In in vivo investigations in conscious monkeys, both motilin and mitemcinal induced migrating motor complex-like contractions in the interdigestive state and also accelerated gastric emptying. In in vitro investigations, the presence of motilin receptors in the gastrointestinal tract was demonstrated by receptor binding assays. Motilin and mitemcinal contracted isolated duodenum strips in a concentration-dependent manner. In conclusion, rhesus monkeys may be useful for studying the physiological and pharmacological roles of the motilin agonistic mechanism because they show reactivity to motilin both in vivo and in vitro.     

胃动素受体的表达与疾病的关系

胃动素受体是一种G蛋白偶联受体,分布于胃肠道,对胃肠运动功能的调节起到重要的作用,目前胃动素受体激动剂逐步应用于临床疾病的治疗.本文就胃动素受体的分布及其与疾病的关系作一概述.     

Ghrelin and motilin are cosecreted from a prominent endocrine cell population in the small intestine

CONTEXT: Ghrelin is a novel hormone produced mainly in the gastric body. Hitherto, mapping studies of ghrelin cells covering the entire gastrointestinal (GI) tract in humans have been lacking. Furthermore, the phenotype of extragastric ghrelin cells is not known. OBJECTIVE: The objective of the study was to perform a detailed mapping with specimens from all parts of the GI tract, and colocalization studies to phenotype ghrelin cells along the tract. In addition, mapping of ghrelin cells was performed in porcine GI tract, and the plasma profiles of ghrelin and motilin in blood from the porcine intestine were measured. DESIGN: Biopsies from patients were obtained during gastroscopy or surgery. Ghrelin cell density and phenotyping was assessed with immunocytochemistry, in situ hybridization, and immunogold electron microscopy. Plasma ghrelin and motilin levels were measured in pigs, fitted with cannulas in the mesenteric vein. RESULTS: The upper small intestine is unexpectedly rich in ghrelin cells, and these cells contribute to circulating ghrelin. Ghrelin and motilin are coproduced in the same cells in the duodenum and jejunum of both species, and ghrelin and motilin are stored in all secretory granules of such cells in humans, indicating cosecretion. The plasma profiles of ghrelin and motilin in pig were parallel, and a correlation between ghrelin and motilin (r(2) = 0.22; P < 0.001) was evident in intestinal blood. CONCLUSIONS: The upper small intestine is an important source of ghrelin. The likely cosecretion of intestinal ghrelin and motilin suggests concerted actions of the two hormones. These data may have implications for understanding gut motility and clinical implications for dysmotility and bariatric surgery.     

Identification of genes for the ghrelin and motilin receptors and a novel related gene in fish, and stimulation of intestinal motility in zebrafish (Danio rerio) by ghrelin and motilin

In mammals ghrelin has a diverse range of effects including stimulation of gut motility but although present in teleost fish its effects on motility have not been investigated. The present study used bioinformatics to search for fish paralogues of the ghrelin receptor and the closely related motilin receptor, and investigated the effects of ghrelin and motilin on gut motility in zebrafish, Danio rerio. Fish paralogues of the human ghrelin and motilin receptor genes were identified, including those from the zebrafish. In addition, a third gene was identified in three species of pufferfish (the only fish genome completely sequenced), which is distinct from the ghrelin and motilin receptors but more closely aligned to these receptors relative to other G-protein coupled receptors. Immunohistochemistry demonstrated strong ghrelin receptor-like reactivity in the muscle of the zebrafish intestine. In isolated intestinal bulb and mid/distal intestine preparations, ghrelin, motilin, and the motilin receptor agonist erythromycin all evoked contraction; these responses ranged between 9% and 51% of the contractions evoked by carbachol (10(-6) M). There were some variations in the concentrations found to be active in the different tissues, e.g., whereas motilin and rat ghrelin caused contraction of the intestinal bulb circular muscle at concentrations as low as 10(-8) M, human ghrelin (10(-8) to 10(-6) M) was without activity. Neither ghrelin (10(-7) M) nor erythromycin (10(-5) M) affected the contractions evoked by electrical field stimulation. The results suggest that both ghrelin and motilin can regulate intestinal motility in zebrafish and most likely other teleosts, and are discussed in relation to the evolution of these regulatory peptides.     

Motilin: Spectrum and mode of gastrointestinal actions

Conclusion Motilin is found in the enterochromaffin cells of the human intestine. It can be released by duodenal acidification or fat intake. Motilin effects appear to be confined exclusively to the gastrointestinal tract. Before, however, hormonal status can be assigned to a candidate hormone, it must be shown that the peptide in question is released in amount and kind sufficient to account for the physiologic event under consideration (81). Synthetic motilin is capable of increasing lower esophageal sphincter pressure and gastric pepsin secretion while it decreases gastric mucosal protein biosynthesis and inhibits gastric emptying. These effects can be achieved by exogenous motilin infusions at doses that produce plasma motilin levels comparable to those after endogenous motilin release. So, motilin has taken first steps out of the candidate hormone reserve towards the respectability of recognition as a gastrointestinal hormone.     

Molecular characterization of structure and tissue distribution of chicken neurotensin receptor

Neurotensin, a tridecapeptide, is distributed in a wide range of tissues and exhibits multiple functions through its receptors. Hitherto molecular characterization of the neurotensin receptor has been reported in mammalian, amphibian, and fish species but not in avian species. In this study, we cloned the cDNA encoding chicken neurotensin receptor from the duodenum and characterized its primary structure, biological activity and distribution in the gastrointestinal tract. The cDNA encoded a protein consisting of 399 amino acids that had significant overall sequence homology to other vertebrate neurotensin receptor 1 with higher extent in the seven transmembrane domains. Chicken neurotensin increased intracellular Ca²⁺ concentrations in human embryonic kidney 293 cells transiently expressing the chicken neurotensin receptor 1. Real-time PCR analysis showed that chicken neurotensin receptor 1 mRNA is expressed throughout the gastrointestinal tract with markedly higher level in the colon/rectum. These results indicate that the chicken neurotensin receptor 1 is involved in gastrointestinal functions through an intracellular signaling pathway accompanied by an increase in Ca²⁺ levels.     

Somatostatin, cortistatin and their receptors in tumours

Somatostatin (SS) and its synthetic analogs have a role in the treatment of neuroendocrine tumours both in terms of symptoms control and antiproliferative activities. These effects are mediated by five SS receptors, widely expressed in both human neuroendocrine and non-neuroendocrine tumours, which were demonstrated to be diagnostically and therapeutically valuable targets. Cortistatin (CST), a brain cortex peptide, partially homologous to SS and having similar functions is also expressed in peripheral tissues and tumours. CST binds all SS receptors, and, differently from SS, also the ghrelin receptor GHSR1a and the CST specific receptor MrgX2. The expression profile of CST is mostly restricted to neuroendocrine tumours (gastrointestinal, pancreas, lung, parathyroid, thyroid, adrenal). In these tumours, CST probably acts via the SS or ghrelin receptor, the MrgX2 receptor being absent. Thus, in comparison to SS analogs, CST synthetic analogs may represent additional diagnostic/therapeutic tools in those tumours expressing the receptors for SS, for ghrelin or for both peptides.     

Progastrin-releasing peptide and gastrin-releasing peptide receptor mRNA expression in non-tumor tissues of the human gastrointestinal tract

AIM: To investigate the expression of gastrin-releasing peptide (GRP) and GRP-receptor mRNA in non-tumor tissues of the human esophagus, gastrointestinal tract, pancreas and gallbladder using molecular biology techniques. METHODS: Poly A mRNA was isolated from total RNA extracts using an automated nucleic acid extractor and, subsequently, converted into single-stranded cDNA (sscDNA). PCR amplifications were carried out using genespecific GRP and GRP-receptor primers. The specificity of the PCR amplicons was further confirmed by Southern blot analyses using gene-specific GRP and GRP-receptor hybridization probes. RESULTS: Expression of GRP and GRP-receptor mRNA was detected at various levels in nearly all segments of the non-tumor specimens analysed, except the gallbladder. In most of the biopsy specimens, coexpression of both GRP and GRP-receptor mRNA appeared to take place. However, expression of GRP mRNA was more prominent than was GRP-receptor mRNA. CONCLUSION: GRP and GRP-receptor mRNAs are expressed throughout the gastrointestinal tract and provides information for the future mapping and determination of its physiological importance in normal and tumor cells.     

Ontogeny and tissue-specific regulation of ghrelin mRNA expression suggest that ghrelin is primarily involved in the control of extraendocrine functions in the rat

Ghrelin is a 28-amino-acid gastric peptide that potently stimulates growth hormone (GH) secretion in vivo and in vitro. Ghrelin-expressing cells have been found in the oxyntic region of the stomach and in the arcuate nucleus of the hypothalamus. The aim of this work was to investigate the regional distribution and developmental changes in ghrelin mRNA levels in the pituitary, hypothalamus and gastrointestinal (GI) tract of the rat using a semiquantitative RT-PCR assay. We also describe the effects of ghrelin immunoneutralization in late gestation and those resulting from induction of an isolated GH deficiency in adult rats. Ghrelin mRNA was already expressed in the fetus by embryonic day 12 (E12), by E17 most of ghrelin mRNA was in the trunk. At E17, in situ hybridization did not reveal a clear expression of ghrelin mRNA in fetal stomach but showed high ghrelin mRNA levels in the placenta. In the pituitary gland, levels of ghrelin mRNA were high after birth but declined significantly with puberty, whereas in the hypothalamus they were barely detectable at birth and remained very low at all subsequent time points tested. In the GI tract, ghrelin mRNA levels were high from birth to 270 days of life. Immunoneutralization of ghrelin at E16 had no effect on survival or development. Rats showed normal somatotropic function, ghrelin expression and onset of puberty. In young adult rats, passive immunization against GHRH did not affect ghrelin mRNA levels in the pituitary, hypothalamus and stomach. Only a 72-hour fasting period induced a significant increase in ghrelin mRNA levels in the stomach, but not in the pituitary and hypothalamus. These results strongly indicate that ghrelin is an important GI hormone expressed early in life and primarily sensitive to nutritional status.     

Ontogeny of ghrelin mRNA expression and identification of ghrelin-immunopositive cells in the gastrointestinal tract of the Peking duck, Anas platyrhychos

Ghrelin is an acylated peptide and an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), and stimulates growth hormone release and food intake in mammals. Peking duck is a very fast growing species of poultry. Although the sequence and structure of ghrelin have recently been determined, the expression of ghrelin in Peking duck has not been studied. Here, we investigated the tissue expression and distribution of ghrelin by RT-PCR and immunohistochemistry, respectively, in Peking duck at different stages of development. Ghrelin mRNA expression was mainly detected in the proventriculus and proventriculus-gizzard junction. It was first expressed, but weakly, on embryonic day 14 (E14); the expression increased by embryonic day 21 (E21), and was maintained at high levels between post-hatching-day 1 (P1) and post-hatching-day 60 (P60). Weak expression of ghrelin mRNA was also found in the gizzard and duodenum. In the gastrointestinal tract of growing Peking duck in P60, the largest number of ghrelin-ip cells was detected in the epithelium of the compound tubular glands in the proventriculus and the next largest number was in the proventriculus-gizzard junction. Very few ghrelin-ip cells were located in the epithelium of the simple tubular glands adjacent to the gizzard. No ghrelin-ip cells were observed elsewhere in the gastrointestinal tract. Ghrelin-ip cells were found in embryos as early as day E21; at the same time, the compound tubular glands in the proventriculus had formed. The numbers of ghrelin-ip cells on P1 were similar to those of E21 embryos. However, on P60, high numbers of strongly stained ghrelin-ip cells were found to be scattered in the epithelium of the compound tubular glands in the proventriculus. The density of ghrelin-ip cells (cells/mm²) in the proventriculus on P60 was significantly greater than those of P1 and E21 embryos. These results demonstrate that ghrelin is expressed in the Peking duck gastrointestinal tract, especially in the proventriculus, from mid-late-stage embryos to growing period and suggested an involvement of ghrelin in the development and biology of the gastrointestinal tract of the Peking duck.     

The endogenous growth hormone secretagogue (ghrelin) is synthesized and secreted by chondrocytes

Ghrelin, the endogenous ligand for the GH secretagogue receptor (GHS-R), is a recently isolated hormone, prevalently expressed in stomach but also in other tissues such as hypothalamus and placenta. This novel acylated peptide acts at a central level to stimulate GH secretion and, notably, to regulate food intake. However, the existence of further, as yet unknown, effects or presence of ghrelin in peripheral tissues cannot be ruled out. In this report, we provide clear evidence for the expression of ghrelin peptide and mRNA in human, mouse, and rat chondrocytes. Immunoreactive ghrelin was identified by immunohistochemistry in rat cartilage, being localized prevalently in proliferative and maturative zone of the epiphyseal growth plate, and in mouse and human chondrocytic cell lines. Moreover, ghrelin mRNA was detected by RT-PCR and confirmed by Southern analysis in rat cartilage as well as in mouse and human chondrocytes cell lines. Ghrelin mRNA expression has been studied in rat along early life development showing a stable profile of expression throughout. Although ghrelin expression in chondrocytes suggests the presence of an unexpected autocrine/paracrine pathway, we failed to identify the functional GH secretagogue receptor type 1A by RT-PCR. On the other hand, binding analysis with 125I ghrelin suggests the presence of specific receptors different from the 1A isotype. Scatchard analysis revealed the presence of two receptors with respectively high and low affinity. Finally, ghrelin, in vitro, was able to significantly stimulate cAMP production and inhibits chondrocytes metabolic activity both in human and murine chondrocytes. In addition, ghrelin is able to actively decrease both spontaneous or insulin-induced long chain fatty acid uptake in human and mouse chondrocytes. This study is the first to provide evidence for the presence of this novel peptide in chondrocytes and suggests novel potential roles for this newly recognized component of the GH axis in cartilage metabolism.     

Novel expression and functional role of ghrelin in rat testis.

Ghrelin, the endogenous ligand for the GH-secretagogue receptor (GHS-R), is a recently cloned peptide, primarily expressed in the stomach and hypothalamus, that acts at central levels to elicit GH release and, notably, to regulate food intake. However, the possibility of additional, as yet unknown, peripheral effects of ghrelin cannot be ruled out. In the present communication, we provide evidence for the novel expression of ghrelin and its functional receptor in rat testis. Testicular ghrelin gene expression was demonstrated throughout postnatal development, and ghrelin protein was detected in Leydig cells from adult testis specimens. Accordingly, ghrelin mRNA signal became undetectable in rat testis following selective Leydig cell elimination. In addition, testicular expression of the gene encoding the cognate ghrelin receptor was observed from the infantile period to adulthood, with the GHS-R mRNA being persistently expressed after selective withdrawal of mature Leydig cells. From a functional standpoint, ghrelin, in a dose-dependent manner, induced an average 30% inhibition of human CG- and cAMP-stimulated T secretion in vitro. This inhibitory effect was associated with significant decreases in human CG-stimulated expression levels of the mRNAs encoding steroid acute regulatory protein, and P450 cholesterol side-chain cleavage, 3beta-hydroxy steroid dehydrogenase, and 17beta-hydroxy steroid dehydrogenase type III enzymes. Overall, our data are the first to provide evidence for a possible direct action of ghrelin in the control of testicular function. Furthermore, the present results underscore an unexpected role of ghrelin as signal with ability to potentially modulate not only growth and body weight homeostasis but also reproductive function, a phenomenon also demonstrated recently for the adipocyte-derived hormone, leptin.     

Ghrelin and unacylated ghrelin stimulate human osteoblast growth via mitogen-activated protein kinase (MAPK)/phosphoinositide 3-kinase (PI3K) pathways in the absence of GHS-R1a

Recent studies demonstrate widespread expression of ghrelin among tissues and have uncovered its pleiotropic nature. We have examined gene expression of ghrelin and its two receptor splice variants, growth hormone secretagogue receptors (GHS-R) 1a and 1b, in human bone biopsies and in the human pre-osteoblastic SV-HFO cell line during differentiation. Additionally, we examined proliferative effects of ghrelin and unacylated ghrelin (UAG) in differentiating and non-differentiating cells. We detected GHS-R1b mRNA in human bone and osteoblasts but not ghrelin's cognate receptor GHS-R1a, using two different real-time PCR assays and both total RNA and mRNA. In osteoblasts GHS-R1b mRNA expression remained low during the first 14 days of culture, but increased 300% in differentiating cells by day 21. Both human bone biopsies and osteoblasts expressed ghrelin mRNA, and osteoblasts were found to secrete ghrelin. Overall, ghrelin gene expression was greater in differentiating than non-differentiating osteoblasts, but was not increased during culture in either group. Ghrelin and UAG induced thymidine uptake dose-dependently, peaking at 1 and 10 nM respectively, at day 6 of culture in both non-differentiating and differentiating osteoblasts. The proliferative response to ghrelin and UAG declined with culture time and state of differentiation. The proliferative effects of ghrelin and UAG were suppressed by inhibitors of extracellular-signal-regulated kinase (ERK) and phosphoinositide-3 kinase, and both peptides rapidly induced ERK phosphorylation. Overall, our data suggest new roles for ghrelin and UAG in modulating human osteoblast proliferation via a novel signal transduction pathway.     

Ghrelin mRNA and GH secretagogue receptor mRNA in human GH-producing pituitary adenomas is affected by mutations in the alpha subunit of G protein

OBJECTIVE: Ghrelin and its receptor, growth hormone secretagogue (GHS) receptor (GHSR), are expressed in the normal pituitary gland and various types of pituitary adenoma. Somatic mutations in the subunit of Gs alpha protein (gsp), which led to a constitutive activation of adenylyl cyclase, are reported in GH-producing pituitary adenomas. We analysed the relationship between ghrelin mRNA and GHSR mRNA expression levels in gsp mutation-positive and -negative GH-producing pituitary adenomas. PATIENTS: Pituitary adenoma tissue was obtained at surgery from 20 patients with acromegaly. METHODS: The expression levels of human ghrelin mRNA and GHSR mRNA were quantified using a competitive RT-PCR method. To detect the gsp mutations, amplified Gs alpha subunit cDNA fragments were sequenced directly using RT-PCR method. RESULTS: There was no significant difference in the expression of ghrelin mRNA between mutation-positive and -negative adenomas. The expression of GHSR mRNA was significantly lower in gsp mutation-positive than -negative adenomas. There was a significant negative correlation between the levels of ghrelin mRNA and GHSR mRNA expression in mutation-negative adenomas; no such correlation was found in mutation-positive adenomas. CONCLUSION: These results suggest that GHSR mRNA expression is downregulated by ghrelin in gsp mutation-negative GH-producing pituitary adenomas, and that changes in intracellular signalling pathways in gsp mutation-positive GH-producing pituitary adenomas affect the expression of G protein-coupled receptors such as GHSR. The absence of negative correlation between ghrelin and GHSR expression might be induced by lowered GHSR expression in gsp mutation-positive GH-producing adenomas.     

Ghrelin-producing endocrine tumors of the stomach and intestine

Ghrelin is a novel gastrointestinal hormone produced by about 20% of the rat and human gastric neuroendocrine cell population, which possesses strong GH-releasing activity, but also plays other central and peripheral roles, including influence on food intake, gastric motility, and acid secretion. The aim of the present study was to determine whether gastrointestinal endocrine hyperplastic and neoplastic lesions produce ghrelin, at both protein (immunohistochemistry) and mRNA (in situ hybridization and/or RT-PCR) levels, and express the GH secretagogue receptor mRNA by RT-PCR. Sixteen gastric and 20 intestinal carcinoids as well as normal gastrointestinal mucosa and atrophic gastritis-associated neuroendocrine cell hyperplasia were studied. The majority (12 of 16, 75%) of gastric carcinoids and only 5 of 18 (27%) of intestinal endocrine tumors were immunoreactive for ghrelin. In situ hybridization confirmed the immunohistochemical data, but also showed ghrelin mRNA in 1 gastric and 8 intestinal additional tumors. RT-PCR showed ghrelin mRNA in 14 of 14 cases, indicating a low level of ghrelin gene expression in all gastrointestinal endocrine tumors tested. Gastric neuroendocrine hyperplastic cells were also strongly positive for ghrelin. GH secretagogue receptor mRNA was absent in 3 gastric, but present in 7 of 11 intestinal carcinoids studied by RT-PCR. These findings demonstrate that most gastric carcinoids (and related neuroendocrine cell hyperplasias) and some intestinal carcinoids produce ghrelin. These hyperplastic/neoplastic conditions could represent the clinical model to clarify the existence and impact of ghrelin hypersecretion on endocrine and nonendocrine functions.     

Release of motilin by oral and intravenous nutrients in man.

Motilin is a hormonal peptide found in the duodenum and jejunum which potently influences gastrointestinal tract motility. Its role in human physiology is not yet established. After a standard hospital lunch the plasma concentration of motilin showed a small, transient, but significant rise in 28 healthy subjects. Individual food components either stimulated (oral fat) or suppressed release (oral glucose). Plasma motilin levels were, in addition, altered to an equal extent by intravenous nutrients, with glucose and amino acids suppressing release, and intravenous fat causing a significant rise in plasma concentration. These results demonstrate a consistent response to food stimuli, whether oral or intravenous. The release mechanism appears to be complicated and after a balanced meal, containing food components which both stimulate and suppress release, there is only a small net change.