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Serotonin (5-HT) produced by enterochromaffin (EC) cells in the digestive tract is crucial for maintaining gut function and homeostasis. Nutritional and non-nutritional stimuli in the gut lumen can modulate the ability of EC cells to produce 5-HT in a temporal- and spatial-specific manner that toning gut physiology and immune response. Of particular interest, the interactions between dietary factors and the gut microbiota exert distinct impacts on gut 5-HT homeostasis and signaling in metabolism and the gut immune response. However, the underlying mechanisms need to be unraveled. This review aims to summarize and discuss the importance of gut 5-HT homeostasis and its regulation in maintaining gut metabolism and immune function in health and disease with special emphasis on different types of nutrients, dietary supplements, processing, and gut microbiota. Cutting-edge discoveries in this area will provide the basis for the development of new nutritional and pharmaceutical strategies for the prevention and treatment of serotonin homeostasis-related gut and systematic disorders and diseases.

The monoamine serotonin, 5-hydroxytryptamine (5-HT), is a remarkable molecule with conserved production in prokaryotes and eukaryotes and a wide range of functions. In the gastrointestinal tract, enterochromaffin cells are the most important source for 5-HT production. Some intestinal bacterial species are also able to produce 5-HT. Besides its role as a neurotransmitter, 5-HT acts on immune cells to regulate their activation. Several lines of evidence indicate that intestinal 5-HT signaling is altered in patients with inflammatory bowel disease. In this review, we discuss the current knowledge on the production, secretion, and signaling of 5-HT in the intestine. We present an inventory of intestinal immune and epithelial cells that respond to 5-HT and describe the effects of these signaling processes on intestinal homeostasis. Further, we detail the mechanisms by which 5-HT could affect inflammatory bowel disease course and describe the effects of interventions that target intestinal 5-HT signaling.

Background & Aims: Gastrointestinal (GI) motility is regulated by serotonin (5-hydroxytryptamine [5-HT]), which is primarily produced by enterochromaffin (EC) cells in the GI tract. However, the precise roles of EC cell-derived 5-HT in regulating gastric motility remain a major point of conjecture. Using a novel transgenic mouse line, we investigated the distribution of EC cells and the pathophysiologic roles of 5-HT deficiency in gastric motility in mice and humans., Methods: We developed an inducible, EC cell-specific Tph1 CreERT2/+ mouse, which was used to generate a reporter mouse line, Tph1-tdTom, and an EC cell-depleted line, Tph1-DTA. We examined EC cell distribution, morphology, and subpopulations in reporter mice. GI motility was measured in vivo and ex vivo in EC cell-depleted mice. Additionally, we evaluated 5-HT content in biopsy and plasma specimens from patients with idiopathic gastroparesis (IG)., Results: Tph1-tdTom mice showed EC cells that were heterogeneously distributed throughout the GI tract with the greatest abundance in the antrum and proximal colon. Two subpopulations of EC cells were identified in the gut: self-renewal cells located at the base of the crypt and mature cells observed in the villi. Tph1-DTA mice displayed delayed gastric emptying, total GI transit, and colonic transit. These gut motility alterations were reversed by exogenous provision of 5-HT. Patients with IG had a significant reduction of antral EC cell numbers and 5-HT content, which negatively correlated with gastric emptying rate., Conclusions: The Tph1 CreERT2/+ mouse provides a powerful tool to study the functional roles of EC cells in the GI tract. Our findings suggest a new pathophysiologic mechanism of 5-HT deficiency in IG., (Copyright © 2021 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The gastrointestinal tract is known as the largest endocrine organ that encounters and integrates various immune stimulations and neuronal responses due to constant environmental challenges. Enterochromaffin (EC) cells, which function as chemosensors on the gut epithelium, are known to translate environmental cues into serotonin (5-HT) production, contributing to intestinal physiology. However, how immune signals participate in gut sensation and neuroendocrine response remains unclear. Interleukin-33 (IL-33) acts as an alarmin cytokine by alerting the system of potential environmental stresses. We here demonstrate that IL-33 induced instantaneous peristaltic movement and facilitated Trichuris muris expulsion. We found that IL-33 could be sensed by EC cells, inducing release of 5-HT. IL-33-mediated 5-HT release activated enteric neurons, subsequently promoting gut motility. Mechanistically, IL-33 triggered calcium influx via a non-canonical signaling pathway specifically in EC cells to induce 5-HT secretion. Our data establish an immune-neuroendocrine axis in calibrating rapid 5-HT release for intestinal homeostasis., Competing Interests: Declaration of Interests The authors declare no competing interests., (Published by Elsevier Inc.)

Small intestinal neuroendocrine tumors (SI-NET) are serotonin-secreting well-differentiated neuroendocrine tumors of putative enterochromaffin (EC) cell origin. Recent studies recognize a subset of EC cells that is label-retaining at the +4 position in the crypt and functions as a reserve intestinal stem cell. Importantly, this +4 reserve EC cell subset not only contributes to regeneration of the intestinal epithelium during injury and inflammation but also to basal crypt homeostasis at a constant rate. The latter function suggests that the +4 EC cell subset serves as an active reserve stem cell via a constant rate of dedifferentiation. Characterization of early tumor formation of SI-NET, observed as crypt-based EC cell clusters in many cases of familial SI-NETs, suggests that the +4 active reserve EC cell subset is the cell of origin. This newly discovered active reserve stem cell property of EC cells can account for unique biological mechanisms and processes associated with the genesis and development of SI-NETs. The recognition of this property of the +4 active reserve EC cell subset may provide novel opportunities to explore NETs in the gastrointestinal tract and other organs.

Enterochromaffin (EC) cell is the main cell type that responsible for 5-hydroxytryptamine (5-HT) synthesis, storage and release of the gut. Intestinal 5-HT play a key role in visceral sensation, intestinal motility and permeability, EC cell hyperplasia and increased 5-HT bioavailability in the gut have been found to be involved in the symptoms generation of irritable bowel syndrome and inflammatory bowel disease. EC cells originate from intestinal stem cells, the interaction between proliferation and differentiation signals on intestinal stem cells enable EC cell number to be regulated in a normal level. This review focuses on the impact factors, pathogenesis mechanisms, and therapeutic clues for intestinal EC cells hyperplasia, and showed that EC cell hyperplasia was observed under the condition of physiological stress, intestinal infection or intestinal inflammation, the disordered proliferation and/or differentiation of intestinal stem cells as well as their progenitor cells all contribute to the pathogenesis of intestinal EC cell hyperplasia. The altered intestinal niche, i.e. increased corticotrophin releasing factor (CRF) signal, elevated nerve growth factor (NGF) signal, and Th2-dominant cytokines production, has been found to have close correlation with intestinal EC cell hyperplasia. Currently, CRF receptor antagonist, nuclear factor-κB inhibitor, and NGF receptor neutralizing antibody have been proved useful to attenuate intestinal EC cell hyperplasia, which may provide a promising clue for the therapeutic strategy in EC cell hyperplasia related diseases., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory cells, EC cells sense luminal stimuli and convert them into serotonin (5-hyroxytryptamine, 5-HT) release events. However, the electrophysiology of these cells is poorly understood because they are difficult to culture and to identify. The method presented in this paper outlines primary EC cell cultures optimized for single cell electrophysiology. This protocol utilizes a transgenic cyan fluorescent protein (CFP) reporter to identify mouse EC cells in mixed primary cultures, advancing the approach to obtaining high-quality recordings of whole cell electrophysiology in voltage- and current-clamp modes.

Enterochromaffin (EC) cells constitute the largest population of intestinal epithelial enteroendocrine (EE) cells. EC cells are proposed to be specialized mechanosensory cells that release serotonin in response to epithelial forces, and thereby regulate intestinal fluid secretion. However, it is unknown whether EE and EC cells are directly mechanosensitive, and if so, what the molecular mechanism of their mechanosensitivity is. Consequently, the role of EE and EC cells in gastrointestinal mechanobiology is unclear. Piezo2 mechanosensitive ion channels are important for some specialized epithelial mechanosensors, and they are expressed in mouse and human EC cells. Here, we use EC and EE cell lineage tracing in multiple mouse models to show that Piezo2 is expressed in a subset of murine EE and EC cells, and it is distributed near serotonin vesicles by superresolution microscopy. Mechanical stimulation of a subset of isolated EE cells leads to a rapid inward ionic current, which is diminished by Piezo2 knockdown and channel inhibitors. In these mechanosensitive EE cells force leads to Piezo2-dependent intracellular Ca 2+ increase in isolated cells as well as in EE cells within intestinal organoids, and Piezo2-dependent mechanosensitive serotonin release in EC cells. Conditional knockout of intestinal epithelial Piezo2 results in a significant decrease in mechanically stimulated epithelial secretion. This study shows that a subset of primary EE and EC cells is mechanosensitive, uncovers Piezo2 as their primary mechanotransducer, defines the molecular mechanism of their mechanotransduction and mechanosensitive serotonin release, and establishes the role of epithelial Piezo2 mechanosensitive ion channels in regulation of intestinal physiology., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Enterochromaffin cells were the first endocrine cells of the gastrointestinal tract to be chemically distinguished, almost 150 years ago. It is now known that the chromaffin reaction of these cells was due to their content of the reactive aromatic amine, 5-hydroxytryptamine (5-HT, also known as serotonin). They have commonly been thought to be a special class of gut endocrine cells (enteroendocrine cells) that are distinct from the enteroendocrine cells that contain peptide hormones. The study by Martin et al. in the current issue of this journal reveals that the patterns of expression of nutrient receptors and transporters differ considerably between chromaffin cells of the mouse duodenum and colon. However, even within regions, chromaffin cells differ; in the duodenum there are chromaffin cells that contain both secretin and 5-HT, cholecystokinin and 5-HT, and all three of secretin, cholecystokinin, and 5-HT. Moreover, the ratios of these different cell types differ substantially between species. And, in terms of function, 5-HT has many roles, including in appetite, motility, fluid secretion, release of digestive enzymes and bone metabolism. The paper thus emphasizes the need to define the many different classes of enterochromaffin cells and relate this to their roles., (© 2017 John Wiley & Sons Ltd.)

Key Points: The gastrointestinal epithelial enterochromaffin (EC) cell synthesizes the vast majority of the body's serotonin. As a specialized mechanosensor, the EC cell releases this serotonin in response to mechanical forces. However, the molecular mechanism of EC cell mechanotransduction is unknown. In the present study, we show, for the first time, that the mechanosensitive ion channel Piezo2 is specifically expressed by the human and mouse EC cells. Activation of Piezo2 by mechanical forces results in a characteristic ionic current, the release of serotonin and stimulation of gastrointestinal secretion. Piezo2 inhibition by drugs or molecular knockdown decreases mechanosensitive currents, serotonin release and downstream physiological effects. The results of the present study suggest that the mechanosensitive ion channel Piezo2 is specifically expressed by the EC cells of the human and mouse small bowel and that it is important for EC cell mechanotransduction., Abstract: The enterochromaffin (EC) cell in the gastrointestinal (GI) epithelium is the source of nearly all systemic serotonin (5-hydroxytryptamine; 5-HT), which is an important neurotransmitter and endocrine, autocrine and paracrine hormone. The EC cell is a specialized mechanosensor, and it is well known that it releases 5-HT in response to mechanical forces. However, the EC cell mechanotransduction mechanism is unknown. The present study aimed to determine whether Piezo2 is involved in EC cell mechanosensation. Piezo2 mRNA was expressed in human jejunum and mouse mucosa from all segments of the small bowel. Piezo2 immunoreactivity localized specifically within EC cells of human and mouse small bowel epithelium. The EC cell model released 5-HT in response to stretch, and had Piezo2 mRNA and protein, as well as a mechanically-sensitive inward non-selective cation current characteristic of Piezo2. Both inward currents and 5-HT release were inhibited by Piezo2 small interfering RNA and antagonists (Gd 3+ and D-GsMTx4). Jejunum mucosal pressure increased 5-HT release and short-circuit current via submucosal 5-HT3 and 5-HT4 receptors. Pressure-induced secretion was inhibited by the mechanosensitive ion channel antagonists gadolinium, ruthenium red and D-GsMTx4. We conclude that the EC cells in the human and mouse small bowel GI epithelium selectively express the mechanosensitive ion channel Piezo2, and also that activation of Piezo2 by force leads to inward currents, 5-HT release and an increase in mucosal secretion. Therefore, Piezo2 is critical to EC cell mechanosensitivity and downstream physiological effects., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

Background: Advanced age is associated with a reduction in clinical visceral pain perception. However, the underlying mechanisms remain largely unknown. Previous studies have suggested that an abnormal interplay between mast cells, enterochromaffin (EC) cells, and afferent nerves contribute to nociception in gastrointestinal disorders. The aim of this study was to investigate how aging affects afferent sensitivity and neuro-immune association in the human bowel., Methods: Mechanical and chemical sensitivity of human bowel afferents were examined by ex vivo afferent nerve recordings. Age-related changes in the density of mast cells, EC cells, sensory nerve terminals, and mast cell-nerve micro-anatomical association were investigated by histological and immune staining., Key Results: Human afferents could be broadly classified into subpopulations displaying mechanical and chemical sensitivity, adaptation, chemo-sensitization, and recruitment. Interestingly human bowel afferent nerve sensitivity was attenuated with age. The density of substance P-immunoreactive (SP-IR) nerve varicosities was also reduced with age. In contrast, the density of ileal and colonic mucosal mast cells was increased with age, as was ileal EC cell number. An increased proportion of mast cells was found in close apposition to SP-IR nerves., Conclusions & Inferences: Afferent sensitivity in human bowel was reduced with advancing age. Augmentation of mast cells and EC cell numbers and the mast cell-nerve association suggest a compensatory mechanism for sensory neurodegeneration., (© 2016 The Authors. Neurogastroenterology & Motility Published by John Wiley & Sons Ltd.)

The major source of serotonin (5-HT) in the body is the enterochromaffin (EC) cells lining the intestinal mucosa of the gastrointestinal tract. Despite the fact that EC cells synthesise ∼95% of total body 5-HT, and that this 5-HT has important paracrine and endocrine roles, no studies have investigated the mechanisms of 5-HT release from single primary EC cells. We have developed a rapid primary culture of guinea-pig and human EC cells, allowing analysis of single EC cell function using electrophysiology, electrochemistry, Ca(2+) imaging, immunocytochemistry and 3D modelling. Ca(2+) enters EC cells upon stimulation and triggers quantal 5-HT release via L-type Ca(2+) channels. Real time amperometric techniques reveal that EC cells release 5-HT at rest and this release increases upon stimulation. Surprisingly for an endocrine cell storing 5-HT in large dense core vesicles (LDCVs), EC cells release 70 times less 5-HT per fusion event than catecholamine released from similarly sized LDCVs in endocrine chromaffin cells, and the vesicle release kinetics instead resembles that observed in mammalian synapses. Furthermore, we measured EC cell density along the gastrointestinal tract to create three-dimensional (3D) simulations of 5-HT diffusion using the minimal number of variables required to understand the physiological relevance of single cell 5-HT release in the whole-tissue milieu. These models indicate that local 5-HT levels are likely to be maintained around the activation threshold for mucosal 5-HT receptors and that this is dependent upon stimulation and location within the gastrointestinal tract. This is the first study demonstrating single cell 5-HT release in primary EC cells. The mode of 5-HT release may represent a unique mode of exocytosis amongst endocrine cells and is functionally relevant to gastrointestinal sensory and motor function.

Constipation and fecal impaction are conditions of the bowel whose prevalence increases with age. Limited information is known about how these conditions manifest; however, functional deficits are likely to be due to changes in signaling within the bowel. This study investigated the effects of age on colonic mucosal melatonin (MEL) release and the consequences this had on colonic motility. Electrochemical measurements of MEL overflow demonstrated that both basal and mechanically stimulated MEL release decreased with age. The MEL/serotonin also decreased with increasing age, and the trend was similar to that of MEL overflow, suggestive that age-related changes were primarily due to a reduction in MEL levels. Levels of N-acetylserotonin and the N-acetylserotonin/serotonin ratio were reduced with age, providing an explanation for the reduction in MEL release. Decreases in colonic motility were observed in animals between 3 and 24 months old. Exogenous application of MEL could reverse this deficit in aged colon. In summary, we propose that the age-related decline in MEL release may be due to either decreases or alterations in mechanosensory channels and/or a loss in levels/activity of the N-acetyltransferase enzyme responsible for the synthesis of N-acetylserotonin. Decreases in MEL release may explain the decreases in colonic motility observed in 24 month old animals and could offer a new potential therapeutic treatment for age-related constipation.

The chemomechanosensory function of the gut enterochromaffin (EC) cell enables it to respond to dietary agents and mechanical stretch. We hypothesized that the EC cell, which also sensed alterations in luminal or mucosal oxygen level, was physiologically sensitive to fluctuations in O(2). Given that low oxygen levels induce 5-HT production and secretion through a hypoxia inducible factor 1α (HIF-1α)-dependent pathway, we also hypothesized that increasing O(2) would reduce 5-HT production and secretion. Isolated normal EC cells as well as the well-characterized EC cell model KRJ-I were used to examine HIF signaling (luciferase-assays), hypoxia transcriptional response element (HRE)-mediated transcription (PCR), signaling pathways (Western blot), and 5-HT release (ELISA) during exposure to different oxygen levels. Normal EC cells and KRJ-I cells express HIF-1α, and transient transfection with Renilla luciferase under HRE control identified a hypoxia-mediated pathway in these cells. PCR confirmed activation of HIF-downstream targets, GLUT1, IGF2, and VEGF under reduced O(2) levels (0.5%). Reducing O(2) also elevated 5-HT secretion (2-3.2-fold) as well as protein levels of HIF-1α (1.7-3-fold). Increasing O(2) to 100% inhibited HRE-mediated signaling, transcription, reduced 5-HT secretion, and significantly lowered HIF-1α levels (∼75% of control). NF-κB signaling was also elevated during hypoxia (1.2-1.6-fold), but no significant changes were noted in PKA/cAMP. We concluded that gut EC cells are oxygen responsive, and alterations in O(2) levels differentially activate HIF-1α and tryptophan hydroxylase 1, as well as NF-κB signaling. This results in alterations in 5-HT production and secretion and identifies that the chemomechanosensory role of EC cells extends to oxygen sensing.

The gut contains the bulk of the body's serotonin (5-hydroxytryptamine, 5-HT); nevertheless, the physiological role that enteric 5-HT plays has not been determined. 5-HT is linked to gastrointestinal (GI) motility; increased intraluminal pressure causes enterochromaffin (EC) cells to secrete 5-HT, which stimulates intrinsic primary afferent neurons that initiate peristaltic reflexes. 5-HT is also an enteric neurotransmitter. Surprisingly, deletion of tryptophan hydroxylase-1 (TPH1), upon which 5-HT biosynthesis in EC cells depends, does not alter constitutive GI motility, whereas deletion of TPH2, upon which biosynthesis of neuronal 5-HT depends, slows intestinal transit and accelerates gastric emptying. TPH1 deletion, however, protects mice from experimental inflammation; 5-HT potentiation and TPH2 deletion each make inflammation more severe. Neuronal 5-HT is neuroprotective and recruits stem cells to give rise to new enteric neurons in adult mice. Mucosal 5-HT, therefore, may mobilize inflammatory effectors, which protect the gut from invasion, whereas neuronal 5-HT shields enteric neurons from inflammatory damage.

The mechanisms underlying distension-evoked peristalsis in the colon are incompletely understood. It is well known that, following colonic distension, 5-hydroxytryptamine (5-HT) is released from enterochromaffin (EC) cells in the intestinal mucosa. It is also known that exogenous 5-HT can stimulate peristalsis. These observations have led some investigators to propose that endogenous 5-HT release from EC cells might be involved in the initiation of colonic peristalsis, following distension. However, because no direct evidence exists to support this hypothesis, the aim of this study was to determine directly whether release of 5-HT from EC cells was required for distension-evoked colonic peristalsis. Real-time amperometric recordings of 5-HT release and video imaging of colonic wall movements were performed on isolated segments of guinea pig distal colon, during distension-evoked peristalsis. Amperometric recordings revealed basal and transient release of 5-HT from EC cells before and during the initiation of peristalsis, respectively. However, removal of mucosa (and submucosal plexus) abolished 5-HT release but did not inhibit the initiation of peristalsis nor prevent the propagation of fecal pellets or intraluminal fluid. Maintained colonic distension by fecal pellets induced repetitive peristaltic waves, whose intrinsic frequency was also unaffected by removal of the submucosal plexus and mucosa, although their propagation velocities were slower. In conclusion, the mechanoreceptors and sensory neurons activated by radial distension to initiate peristalsis lie in the myenteric plexus and/or muscularis externa, and their activation does not require the submucosal plexus, release of 5-HT from EC cells, nor the presence of the mucosa. The propagation of peristalsis and propulsion of liquid or solid content along the colon is entrained by activity within the myenteric plexus and/or muscularis externa and does not require sensory feedback from the mucosa, nor neural inputs arising from submucosal ganglia.

Gastric carcinoid tumors comprise 7% of all gastrointestinal carcinoids and have significantly increased in incidence over the past few decades. Seventy to 80% of gastric carcinoids are type I, which usually are clinically asymptomatic and found incidentally at endoscopic evaluation for abdominal pain or anemia. In this review, advances in understanding the pathophysiology of type I gastric carcinoid are highlighted. In addition, various current diagnostic and treatment options are discussed. Although type I carcinoids generally hold a benign course, rigorous investigation is needed to ensure accurate diagnosis and optimal treatment. This includes appropriate diagnostic procedures and imaging and accurate staging of tumor. Tumor size, depth of invasion, presence of metastasis, and the tumor's gastrin dependency dictate treatment options. Appropriate treatments can consist of endoscopic resection, antrectomy, medical management, or frequent follow-up. This article provides a systematic method of evaluating and treating type I gastric carcinoid.

Background: Lumenal glucose initiates changes in gastrointestinal (GI) function, including inhibition of gastric emptying, stimulation of pancreatic exocrine and endocrine secretion, and intestinal fluid secretion. Glucose stimulates the release of GI hormones and 5-hydroxytryptamine (5-HT), and activates intrinsic and extrinsic neuronal pathways to initiate changes in GI function. The precise mechanisms involved in luminal glucose-sensing are not clear; studying gut endocrine cells is difficult due to their sparse and irregular localization within the epithelium., Methods: Here we show a technique to determine activation of gut epithelial cells and the gut-brain pathway in vivo in rats using immunohistochemical detection of the activated, phosphorylated, form of calcium-calmodulin kinase II (pCaMKII)., Key Results: Perfusion of the gut with glucose (60 mg) increased pCaMKII immunoreactivity in 5-HT-expressing enterochromaffin (EC) cells, cytokeratin-18 immunopositive brush cells, but not in enterocytes or cholecystokinin-expressing cells. Lumenal glucose increased pCaMKII in neurons in the myenteric plexus and nodose ganglion, nucleus of the solitary tract, dorsal motor nucleus of the vagus and the arcuate nucleus. pCaMKII expression in neurons, but not in EC cells, was significantly attenuated by pretreatment with the 5-HT(3) R antagonist ondansetron. Deoxynojirimycin, a selective agonist for the putative glucose sensor, sodium-glucose cotransporter-3 (SGLT-3), mimicked the effects of glucose with increased pCaMKII in ECs and neurons; galactose had no effect., Conclusions & Inferences: The data suggest that native EC cells in situ respond to glucose, possibly via SGLT-3, to activate intrinsic and extrinsic neurons and thereby regulate GI function., (© 2011 Blackwell Publishing Ltd.)

Background & Aims: Gastric stem cells are located in the isthmus of the gastric glands and give rise to epithelial progenitors that undergo bipolar migration and differentiation into pit and oxyntic lineages. Although gastric mucus neck cells located below the isthmus express trefoil factor family 2 (TFF2) protein, TFF2 messenger RNA transcripts are concentrated in cells above the neck region in normal corpus mucosa, suggesting that TFF2 transcription is a marker of gastric progenitor cells., Methods: Using a BAC strategy, we generated a transgenic mouse with a tamoxifen-inducible Cre under the control of the TFF2 promoter (TFF2-BAC-Cre(ERT2)) and analyzed the lineage derivation from TFF2 mRNA transcript-expressing (TTE) cells., Results: TTE cells were localized to the isthmus, above and distinct from TFF2 protein-expressing mucus neck cells. Lineage tracing revealed that these cells migrated toward the bottom of the gland within 20 days, giving rise to parietal, mucous neck, and chief cells, but not to enterochromaffin-like-cell. Surface mucus cells were not derived from TTE cells and the progeny of the TTE lineage did not survive beyond 200 days. TTE cells were localized in the isthmus adjacent to doublecortin CaM kinase-like-1(+) putative progenitor cells. Induction of spasmolytic polypeptide-expressing metaplasia with DMP-777-induced acute parietal cell loss revealed that this metaplastic phenotype might arise in part through transdifferentiation of chief cells as opposed to expansion of mucus neck or progenitor cells., Conclusions: TFF2 transcript-expressing cells are progenitors for mucus neck, parietal and zymogenic, but not for pit or enterochromaffin-like cell lineages in the oxyntic gastric mucosa., (Copyright © 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Although vgf gene knockout mice are hypermetabolic, administration of the VGF peptide TLQP-21 itself increased energy consumption. Agonist-antagonist roles are thus suggested for different VGF peptides, and the definition of their tissue heterogeneity is mandatory. We studied the rat stomach using antisera to C- or N-terminal sequences of known or predicted VGF peptides in immunohistochemistry and ELISA. TLQP (rat VGF(556-565)) peptide/s were most abundant (162±11 pmol/g, mean±s.e.m.) and were brightly immunostained in enterochromaffin-like (ECL) cells and somatostatin cells. A peptide co-eluting with TLQP-21 was revealed in HPLC of gastric and hypothalamic extracts, while the extended TLQP-62 form was restricted to the hypothalamus. Novel PGH (rat VGF(422-430)) peptide/s were revealed in ghrelin cells, mostly corresponding to low MW forms (0.8-1.5  kDa), while VGF C-terminus peptides were confined to neurons. VGF mRNA was present in the above gastric endocrine cell types, and was prominent in chief cells, in parallel with low-intensity staining for further cleaved products from the C-terminal region of VGF (HVLL peptides: VGF(605-614)). In swine stomach, a comparable profile of VGF peptides was revealed by immunohistochemistry. When fed and fasted rats were studied, a clear-cut, selective decrease on fasting was observed for TLQP peptides only (162±11 vs 74±5.3  pmol/g, fed versus fasted rats, mean±s.e.m., P<0.00001). In conclusion, specific VGF peptides appear to be widely represented in different gastric endocrine and other mucosal cell populations. The selective modulation of TLQP peptides suggests their involvement in peripheral neuro-endocrine mechanisms related to feeding responses and/or ECL cell regulation.

Background: Neuroendocrine tumors (NETs) of the gastrointestinal (GI) system are increasing in incidence with minimal improvement in prognosis. Although the cell of origin has been identified as the enterochromaffin (EC) cell, its secretory and proliferative regulation has not been defined at a mechanistic level. To date, the BON cell line has been the most widely used in vitro EC cell model despite its pancreatic origin. Using whole-genome mathematical analysis as well as secretory and proliferative studies, we compared the BON cell line to the small intestine (SI) EC cell-derived NET cell line, KRJ-I, to assess individual cell line validity and applicability for the investigation of GI-NET disease., Methods and Results: Principal component analysis and ANOVA of KRJ-I and BON transcriptomes (U133 Plus 2) identified substantially different (<10%) overlap in transcripts with minimal (R(2) = 0.24) correlation in gene expression profiles. RT-PCR detected large variability (>12%) in neuroendocrine (NE) marker transcripts in the BON cell line and the absence of Tph-2, DDC, TGFbetaR2, and M3 transcripts in KRJ-I. The KRJ-I cell line secreted serotonin (5-HT) in response to isoproterenol (EC(50) = 100 nM), noradrenaline (EC(50) = 1.7 nM), and pituitary adenylate cyclase (PACAP, EC(50) = 0.03 nM). Cholecystokinin (IC(50) = 430 nM), somatostatin (IC(50) = 400 nM), acetylcholine (IC(50) = 3.7 nM), and gamma-aminobutyric acid A (GABA(A), IC(50) = 2 nM) all inhibited 5-HT release, while gastrin and bombesin had no effect. 5-HT secretion in the BON cell line was stimulated by isoproterenol (EC(50) = 900 nM), noradrenaline (EC(50) = 20 nM), cholecystokinin (EC(50) = 130 nM), PACAP (EC(50) = 0.12 nM), bombesin (EC(50) = 15 nM), and acetylcholine (EC(50) = 0.2 nM). It was inhibited by somatostatin (IC(50) = 300 nM) but not GABA(A). KRJ-I responded with proliferation to connective tissue growth factor (CTGF, EC(50) = 0.002 ng/ml), transforming growth factor-alpha (TGFalpha, EC(50) = 0.63 ng/ml) and transforming growth factor-beta (TGFbeta, EC(50) = 0.63 ng/ml). Epidermal growth factor (EGF) and somatostatin had no significant effect. BON cell proliferation was stimulated only by EGF and TGFalpha (EC(50) = 15.8 and 10 ng/ml). TGFbeta (IC(50) = 0.16 ng/ml), MZ-4-147 (IC(50) = 0.5 nM), and BIM23A761 (IC(50) = 0.06 nM) all inhibited proliferation. CTGF and somatostatin had no effect., Conclusion: KRJ-I and BON cell lines demonstrate substantial differences in gene level transcripts, inconsistent receptor profile expression, wide variability in NE marker transcript levels, and significantly differential proliferative and secretory responses. Given the EC cell origin of KRJ-I, these results provide evidence that the BON cell line does not represent an EC cell system and is not a valid study model of (carcinoid) EC cell-derived NET., (Copyright 2009 S. Karger AG, Basel.)

Background and Aims: Well-differentiated neuro-endocrine ileal carcinoids are composed of serotonin-producing enterochromaffin (EC) cells. Life expectancy is determined by metastatic spread to the liver because medical treatment options are still very limited. Selective inhibition of angiogenesis or lymphangiogenesis might prevent tumour growth and metastatic spread. We examined the role of the vascular endothelial growth factors (VEGFs) A, B, C, D, and their receptors (VEGFRs) 1, 2, 3 in angiogenesis and lymphangiogenesis of ileal EC cell carcinoids with and without liver metastases., Methods: The expression of various VEGFs and VEGFRs was determined by quantitative real-time RT-PCR in healthy mucosa, primary tumour, lymph node metastases and liver metastases of 25 patients with ileal EC cell carcinoids. Microvessel density (MVD) was determined by CD-31 staining in primary tumours and lymphatic vessel density (LVD) by LYVE-1 staining. VEGF expression levels, MVD, LVD, and patients' survival time were correlated using logistic regression and Kaplan-Meier survival analysis., Results: VEGF-A was highly expressed with no difference between normal mucosa and tumours. VEGF-B and -D as well as VEGFR-1 and -2 expression levels were significantly increased in the tumours when compared to normal mucosa. Patients with liver metastasis, however, had a significantly lower expression of the factors A, B, and C and the receptors 2 and 3. MVD in primary tumours positively correlated with the expression of VEGF ligands and their receptors, except for VEGF-D. LVD did not correlate with any VEGF ligand or receptor. Interestingly, low expression levels of VEGF-B were associated with poor survival., Conclusion: Patients with more aggressive metastatic spreading had relatively decreased expression levels of VEGF ligands and receptors. Thus, anti-angiogenic therapy may not be a suitable target in metastatic ileal EC cell carcinoids., (Copyright 2009 S. Karger AG, Basel.)

5-Hydroxytryptamine (5-HT) is a major transmitter molecule within the gastrointestinal tract. It is contained in enterochromaffin (EC) cells, which form part of the epithelial lining of the gut and in enteric neurones in the submucosal and myenteric plexuses. 5-HT is present in murine mucosal mast cells in the lamina propria and some studies have suggested that human mast cells may also contain 5-HT especially in conditions associated with mastocytosis. The strategic positioning of the enteric and extrinsic sensory innervation in close proximity to these sources of 5-HT, in conjunction with their demonstrated sensitivity to this mediator, suggests the involvement of 5-HT in the transduction of visceral stimuli and reflex responses affecting motor and secretory function. Under physiological conditions, the release of 5-HT from these storage sites may result in the orchestration of reflexes responsible for transit of material along the bowel at a rate that is appropriate for digestion and absorption of nutrients. However, in the pathophysiological state, 5-HT acting together with other inflammatory mediators may cause inappropriate intestinal secretomotor activity and/or initiate sensations such as nausea or discomfort/pain. Current evidence suggests that the bioavailability of 5-HT within the gut wall is altered in a number of post-inflammatory models of gut dysfunction with increased numbers of EC cells and mast cells with increased 5-HT content in proximity to sensory nerve endings, and decreased serotonin reuptake mechanisms. Changes may also occur in the sensory innervation or pathways within the central nervous system. These processes may contribute to pain mechanisms in the irritable bowel syndrome, in which visceral hypersensitivity is a predominant feature and may also contribute to motor dysfunction leading to altered bowel habit.

This mini-review addresses the role of the neuroendocrine substance serotonin (5-hydroxytryptamine, 5-HT) in intestinal luminal sensing and secretion. Intestinal sensory neurones are activated by several mechanisms, in particular following stimulation of 'specialized' cells in the mucosa. These specialized cells are the enteroendocrine cells, which contain a wide variety of neuroendocrine transmitters. One of these enteroendocrine cells is the enterochromaffin (EC) cell, which is present throughout the intestines and contains large amounts of serotonin, predominantly in the duodenum in humans. The EC cells act as mucosal sensory transducers and secrete serotonin in response to various physiological and pathological luminal stimuli. Following release, serotonin participates in several mucosal protecting processes, one being secretion. Serotonin stimulates active ion, mucus and fluid secretion. Epithelial 5-HT(2) receptors and neuronal 5-HT(1P), 5-HT(3) and 5-HT(4) receptors mediate the secretory effect of serotonin. A transmembrane serotonin transporter terminates epithelial serotonergic signalling. The transient receptor potential ion channel family is important for processing intestinal luminal sensory signalling. Accumulating evidence suggests a significant interaction between serotonin and one of the transient receptor potential ion channels, the capsaicin-sensitive transient receptor potential vanilloid type 1. Accordingly, EC cells, serotonergic receptors and transporter(s), and transient receptor potential vanilloid type-1 ion channels are all explored as pharmacological targets for treatment of some intestinal functional disorders.

The functional and morphological organization of stomach vegetative innervation was studied. It is shown, that the sympathetic nerve strengthens vagal stimulation of motor activity of various departments of a stomach. In realization of the phenomenon participate preganglionic serotoninergic fibres transmitting excitation on ganglionic serotoninergic neurons. It is established, that stimulation of a wandering nerve results to degranulation of serotoninergic EC-cells carrying on the surface ganglionic 5-HT3,4-receptors. The irritation of a wandering nerve results in reduction of volume of EC-cells in older age.

Background & Aims: Release of serotonin from mucosal enterochromaffin cells triggered by luminal substances is the key event in the regulation of gut motility and secretion. We were interested to know whether nasal olfactory receptors are also expressed in the human gut mucosa by enterochromaffin cells and whether their ligands and odorants present in spices, fragrances, detergents, and cosmetics cause serotonin release., Methods: Receptor expression was studied by the reverse-transcription polymerase chain reaction method in human mucosal enterochromaffin cells isolated by laser microdissection and in a cell line derived from human enterochromaffin cells. Activation of the cells by odorants was investigated by digital fluorescence imaging using the fluorescent Ca(2+) indicator Fluo-4. Serotonin release was measured in culture supernatants by a serotonin enzyme immunoassay and amperometry using carbon fiber microelectrodes placed on single cells., Results: We found expression of 4 olfactory receptors in microdissected human mucosal enterochromaffin cells and in a cell line derived from human enterochromaffin cells. Ca(2+) imaging studies revealed that odorant ligands of the identified olfactory receptors cause Ca(2+) influx, elevation of intracellular free Ca(2+) levels, and, consequently, serotonin release., Conclusions: Our results show that odorants present in the luminal environment of the gut may stimulate serotonin release via olfactory receptors present in human enterochromaffin cells. Serotonin controls both gut motility and secretion and is implicated in pathologic conditions such as vomiting, diarrhea, and irritable bowel syndrome. Thus, olfactory receptors are potential novel targets for the treatment of gastrointestinal diseases and motility disorders.

In the gut, 5-HT acts as a paracrine signalling molecule released by enterochromaffin cells and as a transmitter released by some descending serotonergic interneurons. It has a prominent role in the regulation of motility, vascular tone, secretion and perception both in normal and under certain pathophysiological conditions, such as the carcinoid syndrome and the irritable bowel syndrome (IBS). Serotonin is known to markedly influence bowel function by activating at least five receptor types (5-HT(1,2,3,4,7)). Among all 5-HT receptors, those belonging to the 5-HT3 (a ionotropic receptor) and 5-HT4 (a metabotropic receptor) type are the most extensively studied in gastroenterology, resulting in commercially available (although not worldwide) serotonergic agents for the treatment of IBS and functional dyspepsia. Recently, 5-HT7 receptors have been found to participate in the accommodation process of the circular muscle during the preparatory phase of ileal peristalsis. Since an exaggerated accommodation of the gut wall may contribute to abdominal distension and bloating, 5-HT7 receptor ligands may offer innovative opportunities for the pharmacological treatment of functional bowel disorders.

Dietary fibre consumption is known to be beneficial to increase stool bulk and frequency. In contrast, it is unclear whether chronic dietary fibre deficiency affects colonic motor functions, especially neuronally mediated muscle contractions. In this study, rats were fed a fibre-free diet or diet containing dietary fibre (cellulose or guar gum) for 27 days. Furthermore, neurogenic and myogenic contractions were evaluated in circular and longitudinal muscle strips of the distal colon. Additionally, the number of enterochromaffin (EC) cells, which play important roles in the initiation of the peristaltic reflex, was also examined by immunohistochemistry for serotonin. Myogenic contractions induced by carbachol or substance P were examined in the presence of tetrodotoxin. Circular muscle was hyposensitive to carbachol, but longitudinal muscle was hypersensitive to substance P in the fibre-free group. Nerve-mediated circular (5-20 Hz) and longitudinal (1-2 Hz) muscle contractions evoked by electrical field stimulation were attenuated in the fibre-free group and the latter response was almost abolished by atropine, suggesting functional changes of cholinergic neurons. EC cell number was decreased in the fibre-free group. In conclusion, changes in neurogenic and myogenic contractions and a decrease in EC cell number observed may affect colonic motility of the fibre-free group.

Mucosal changes in inflammatory bowel disease (IBD) are characterized by ulcerative lesions accompanied by a prominent infiltrate of inflammatory cells including lymphocytes, macrophages, and neutrophils and alterations in 5-hydroxytryptamine (5-HT)-producing enterochromaffin (EC) cells. Mechanisms involved in recruiting and activating these cells are thought to involve a complex interplay of inflammatory mediators. Studies in clinical and experimental IBD have shown the upregulation of various chemokines including monocyte chemoattractant protein (MCP)-1 in mucosal tissues. However, precise information on the roles of this chemokine or the mechanisms by which it takes part in the pathogenesis of IBD are not clear. In this study, we investigated the role of MCP-1 in the development of hapten-induced experimental colitis in mice deficient in MCP-1. Our results showed a significant reduction in the severity of colitis both macroscopically and histologically along with a decrease in mortality in MCP-1-deficient mice compared with wild-type control mice. This was correlated with a downregulation of myeloperoxidase activity, IL-1beta, IL-12p40, and IFN-gamma production, and infiltration of CD3+ T cells and macrophages in the colonic mucosa. In addition, we observed significantly lower numbers of 5-HT-expressing EC cells in the colon of MCP-1-deficient mice compared with those in wild-type mice after dinitrobenzenesulfonic acid. These results provide evidence for a critical role of MCP-1 in the development of colonic inflammation in this model in the context of immune and enteric endocrine cells.

Although the enterochromaffin (EC) cell is one of the primary neuroendocrine regulatory cells of the small intestine, the lack of a purified cell system has precluded characterization of the cell and limited precise physiological evaluation. We developed methodology to obtain a pure population of Mastomys ileal EC cells, evaluated their functional regulation, and defined the transcriptome. Mastomys ilea were everted, end ligated, pronase-collagenase digested, and Nycodenz gradient centrifuged, and EC cells were collected by fluorescence-activated cell sorting (FACS) of acridine orange-labeled cells. Enrichment was confirmed by immunostaining of tryptophan hydroxylase and chromogranin A, specific EC cell markers, serotonin content, EC cell marker gene expression, and electron microscopy. Pituitary adenylate cyclase-activating polypeptide (PACAP), somatostatin, and gastrin receptor expression was determined by real-time RT-PCR. Live post-FACS-sorted cells were cultured, and the effects of forskolin, isoproterenol, acetylcholine, GABAA, PACAP-38, and gastrin on serotonin secretion were measured by ELISA. GeneChip Affymetrix profiling of FACS-sorted cells was undertaken to obtain the EC cell transcriptome. FACS produced a >70-fold enrichment of EC cells with a serotonin content of 240 +/- 22 ng/mg protein. Preparations were 99 +/- 0.7% pure by immunostaining for tryptophan hydroxylase. Vasoactive intestinal peptide/PACAP receptor 1 (VPAC1) and somatostatin receptor 2 were present, whereas PACAP receptor 1 (PAC1) and CCK2 receptors were undetectable. Forskolin, isoproterenol, and PACAP-38 stimulated serotonin secretion at EC50 values of 5 x 10(-10), 4.5 x 10(-10), and 1.2 x 10(-9) M, respectively. Isoproterenol stimulated cAMP levels by approximately 3.5 +/- 0.62-fold vs. unstimulated cells (EC50 of approximately 10(-9) M). Octreotide, acetylcholine, and GABAA inhibited serotonin secretion with IC50 values of 3 x 10(-11), 3 x 10(-10), and 2.9 x 10(-10) M, respectively. Gastrin had no effect on serotonin secretion. The naive EC cell transcriptome revealed highly expressed EC cell marker genes, the absence of marker genes for other small intestinal cell types, and a receptor profile that included cholinergic, adrenergic, dopaminergic, serotoninergic, GABAergic, and prostaglandin receptors. We were able to isolate homogeneous preparations (>99%) of live ileal EC cells and demonstrated regulation of serotonin secretion as well as established the normal EC cell transcriptome. Application of this methodology to normal and diseased human ileum will facilitate the elucidation of the pathophysiology of EC cells.

Context: Neuroendocrine regulation of small intestinal (SI) function is poorly understood because pure neuroendocrine cells are unavailable, whereas the biological basis of SI carcinoid tumors is unknown because neoplastic human enterochromaffin (EC) cells are unavailable., Objective: The objective of this study was to define the secretory regulation and transcriptome of naive and neoplastic SI neuroendocrine cells., Design: EC cells from human ilea were isolated and purified, and a malignant EC cell carcinoid cell line (KRJ-I) was characterized., Methods: Human ilea from right hemicolectomies were pronase/collagenase digested and Nycodenz gradient centrifuged, and EC cells were fluorescence-activated cell sorting (FACS) sorted after acridine orange labeling. Enrichment was defined by immunostaining, gene expression, serotonin (5-HT) content, and real-time RT-PCR. Naive FACS-sorted EC and KRJ-I cells were cultured, and 5-HT secretion was measured after stimulation with forskolin, isoproterenol, acetylcholine, gamma-aminobutyric acid A (GABA(A)), pituitary adenylate cyclase-activating polypeptide (PACAP)-38, and gastrin. Normal and neoplastic EC cell transcriptomes were acquired by Affymetrix profiling (U133A)., Results: FACS produced 100 +/- 0.3% (chromogranin A staining) and 99 +/- 0.7% pure EC cells by immunostaining for tryptophan hydroxylase with greater than 67-fold enrichment and a 5-HT content of 180 +/- 18 ng/mg protein (mucosa, 3.5 +/- 0.9). Forskolin- and isoproterenol-stimulated 5-HT secretion was 10-100 times more potent for naive cells (EC(50), 1.8 x 10(-9) m; 5.1 x 10(-9) m) than neoplastic cells (EC(50), 2.1 x 10(-7) m; 8.1 x 10(-8) m), but the effect of PACAP-38 was similar (EC(50), 1 x 10(-7) m). Isoproterenol stimulated cAMP levels 1.6 +/- 0.1-fold vs. basal (EC(50), 2.7 x 10(-9) m). Acetylcholine inhibited naive EC cell 5-HT secretion more potently than neoplastic (IC(50), 3.2 x 10(-9) vs. 1.6 x 10(-7) m), whereas GABA(A) was more potent in neoplastic cells (IC(50), 3.9 x 10(-10) vs. 4.4 x 10(-9) m). Octreotide inhibited naive, but not neoplastic, basal 5-HT secretion. Gastrin had no effect on 5-HT secretion. Comparison of naive and neoplastic transcriptomes revealed shared neuroendocrine and EC cell-specific marker genes. Real-time PCR confirmed that expression of adrenergic (beta1), somatostatinergic (SST(R)2), and neural (VPAC(1) and GABA(A)) receptors occurred on both cell types, but PACAP type 1 (PAC(1)) and cholecystokinin type 2 (CCK(2)) were undetectable. The putative carcinoid malignancy genes (MTA1 and MAGE-D2) were unique to the neoplastic EC cell transcriptome., Conclusion: These data support novel methodology to purify live human EC cells for functional characterization and transcriptome assessment, which will allow identification of new targets to control the secretion and proliferation of SI carcinoids.

Background: The rationale for bariatric surgery is to reduce food intake by gastric restriction and/or malabsorption by intestinal bypass. Unlike ghrelin, gastrin is released in response to food intake. Here we studied the possible role of gastrin in the reduction of body weight after gastric bypass surgery., Methods: Rats were divided into four experimental groups and were subjected to different treatments: sham operation, gastric bypass, sham operation + gastrin infusion, and gastric bypass + gastrin infusion. The gastric bypass was done by anastomosing the esophagus to the duodenal bulb without bypassing the intestine. Gastrin-17 was infused continuously for 2 months via subcutaneously implanted osmotic minipumps. Body weights were recorded; serum gastrin and ghrelin levels were measured, and the stomachs were analyzed morphologically., Results: Gastric bypass resulted in reducing the body weight, stomach weight, thickness of the oxyntic mucosa, serum gastrin concentration, and activity of the ECL cells. Gastrin infusion prevented mucosal atrophy and ECL cell inactivation, and attenuated the body weight reduction that occurred following gastric bypass. Circulating ghrelin and ghrelin-producing A-like cells in stomachs that had undergone gastric bypass were unchanged with or without gastrin infusion and are thus unlikely to be responsible for the reduced body weight., Conclusion: We suggest that hypogastrinemia and impaired ECL cell function in the oxyntic mucosa of the stomach might be partially responsible for the reduction in body weight that occurs after gastric bypass.

Inhibitors of gastric acid secretion are efficient drugs in the treatment of acid-related diseases. However, by reducing gastric acidity, hypergastrinemia develops. Gastrin regulates its target cell, the enterochromaffin (ECL) cell, both functionally and tropicaly. Long-term hypergastrinemia in whatever species studied, has been shown to induce tumors originating from the ECL cell. In man, at least 10 years of hypergastrinemia, accompanied by high or reduced gastric acidity is necessary to induce ECL cell carcinoids. There are reports indicating development of ECL cell carcinoids after long-term treatment with proton pump inhibitors. Moreover, the ECL cell may give rise to gastric carcinomas of diffuse type, which have increased during the last decades. Furthermore, most of the carcinomas developing in patients with long-lasting hypergastrinemia are of ECL cell origin. Therefore, long-lasting iatrogenic hypergastrinemia induced by potent inhibitors of acid secretion may be expected to increase the occurrence of gastric carcinomas in the future.

Gustatory and anticipatory cephalic stimuli that are detected during a meal yield nutritional information and aid in the efficient digestion of food. It is possible that animals can detect the amount of dietary food and its quality via cephalic relay to initiate proper digestion in the alimentary tract. The abdominal vagus conveys primary afferent nutritional information from the digestive system to the brain and modulates gastrointestinal motor and secretory activity that ensures the food digestion through local and central reflexes. Almost all the 5-HT in the body exists in the enterochromaffin (EC) cells in the alimentary tract, but the physiological significance of the mucosal 5-HT is not well elucidated. In the present paper, we reviewed recent advances in studies on gut nutrient perception and proposed the hypothesis that 5-HT derived from EC cells acts as a general transmitter of gut nutrient sensing by the abdominal vagus.

Aim and Methods: Enterochromaffin (EC) cells, interspersed throughout the gastrointestinal mucosa, provide most of the serotonin of the body and control intestinal motility, secretion and absorption. We purified EC cells from the rat ileum by a combination of elutriation and density gradient centrifugation in order to characterize the function of this important cell type., Results: Immunostaining showed that there were 84% serotonin-positive cells in the highly enriched EC fraction as compared with 12% in unfractionated cells, yielding a approximately sevenfold enrichment. Serotonin measurements in the cell suspensions indicated a seven to 14-fold enrichment. Presence of alpha- and beta-adrenoreceptor isoforms, muscarinic M3 and gama-aminobutyric acid (GABA)-A receptors was confirmed by RT-PCR and cytochemistry. Increased expression of VMAT-1 and GABA-A mRNA was also shown by quantitative TaqMan PCR using EC cell RNA. Serotonin release in isolated EC cells was stimulated by noradrenaline, and to a smaller extent, by carbachol, while GABA addition was without effect., Conclusion: Our data provide a basis for a new approach to characterize receptors on this unique cell type.

Objective: Parietal cell antibodies (PCAs) are found in 20% of type 1 diabetic patients, denoting autoimmune gastritis and pernicious anemia, which may predispose to enterochromaffin-like (ECL) cell hyper/dysplasia and gastric carcinoid tumors. We evaluated whether chromogranin A (CgA), 5-hydroxyindole acetic acid (5-HIAA), and neuron-specific enolase (NSE) contribute to screening for ECL cell hyper/dysplasia., Research Design and Methods: Sera from 93 type 1 diabetic patients (53 men and 40 women, 31 PCA(+) and 62 PCA(-), aged 45 +/- 13 years) were analyzed for PCAs by indirect immunofluorescence and for CgA, NSE, and gastrin by radioimmunoassay. Urinary 5-HIAA was tested by high-performance liquid chromatography. Corpus atrophy and ECL cell proliferation were assessed in gastric biopsies., Results: PCA(+) patients had higher gastrin (P < 0.0001) and CgA levels (P = 0.003) and were more prone to autoimmune gastritis (odds ratio [OR] 17, P < 0.0001) and ECL cell hyper/dysplasia (OR = 23, P = 0.005) than PCA(-) subjects. ECL cell hyper/dysplasia was present in seven PCA(+) patients who showed higher CgA levels (P < 0.0001) than subjects without ECL cell hyper/dysplasia, but NSE and 5-HIAA levels were similar. CgA levels correlated with gastrinemia (r = 0.50, P < 0.0001), PCA titer (r = 0.42, P = 0.001), and 5-HIAA levels (r = 0.38, P = 0.012). Logistic regression identified the CgA level (beta = 0.01, P = 0.027) as an independent risk factor for ECL cell hyper/dysplasia when PCA, CgA, 5-HIAA, NSE, gastrin, sex, and age were tested. Multivariate linear regression demonstrated that CgA level was determined by ECL cell density (r = 0.59, P < 0.0001) and gastrin level (r = 0.67, P = 0.02). One PCA(+) patient with elevated gastrin, CgA, and 5-HIAA levels had a gastric carcinoid tumor., Conclusions: PCA(+) patients, particularly those with high gastrin and CgA levels, risk developing ECL cell hyper/dysplasia. The determination of CgA, but not NSE and 5-HIAA, may complement histology in evaluating ECL cell mass.

The enteric nervous system in combination with inputs from parasympathetic and sympathetic nerves regulate the contractile, secretory and vasomotor activity of the gastrointestinal track via neural reflexes. Sensory elements which may be present in specialized neurones, enteroendocrine cells or mast cells detect changes in force, chemical composition or even foreign antigens. Sensory elements signal the enteric nervous system to correct these changes by altering contractile activity, secretion and blood flow. Advances have been made in understanding the sensory mechanisms that are involved in 5-hydroxytryptamine (5-HT) release from enterochromaffin cells (EC) or a model for EC cells. These advances relate to roles for ATP and its metabolites ADP and adenosine in mechanotransduction and a role for a sodium glucose cotransporter, a SGLT-like protein, in chemotransduction.

The peptide hormone gastrin is the key regulator of gastric acid secretion. Gastrin exerts its effects as acid secretagogue through functional activation of gastric enterochromaffin-like (ECL) cells, which control acid secretion through biosynthesis and release of histamine. In ECL cells, concerted activation of histidine decarboxylase (HDC), vesicular monoamine transporter 2 (VMAT2), and chromogranin A (CgA) genes by gastrin is a prerequisite for proper acid control. To elucidate the molecular pathways underlying gastrin-dependent control of ECL cell genes, we recently analyzed the signaling cascades, regulatory promoter elements, and transcription factors mediating the transcriptional effects of gastrin. Our studies identified the Raf>MEK1>ERK 1/-2 kinase module as the common signaling pathway mediating gastrin-dependent ECL cell gene transcription. In contrast to this uniform signaling cascade, pronounced heterogeneity was detected between cis- and trans-activating regulatory factors conferring gastrin responsiveness. The molecular diversity of transcription factors and regulatory enhancer elements transmitting gastrin-triggered gene transcription offers the molecular basis for synergistic, but differential, regulation of HDC, VMAT2, and CgA genes during a secretory challenge of ECL cells by gastrin and possibly other acid secretagogues.

Disorders of gastrointestinal function are common and significantly reduce quality-of-life, as well as negatively impacting healthcare costs. Consequently, there is much interest in understanding the pathogenesis of these disorders. Increasing, albeit as yet limited, evidence has implicated alterations in 5-hydroxytryptamine (5-HT) release, and the subsequent interaction of 5-HT with specific 5-HT receptor subtypes, in the altered gut function of patients with irritable bowel syndrome (IBS) and other functional bowel diseases. Alterations to enterochromaffin cells and/or 5-HT signaling can result in gastrointestinal dysmotility, visceral hypersensitivity and secretomotor abnormalities in the gut. Evidence is beginning to link disturbed 5-HT physiology with the pathophysiology of diarrhea and constipation in IBS, and with slow-transit constipation. This review discusses the current evidence on the pathobiology of these systems.

This manuscript provides a gene-chip examination of gastric ECL cell proliferation in an animal model of neuroendocrine tumour disease. Data that were used to identify molecular targets were then utilised to develop novel therapeutic strategies as appropriate adjuncts to surgery in human disease. Alterations in growth-mediated cell signaling (the AP-1 pathway) and in the cell cycle were identified in ECL cell tumours in the animal model and confirmed in human tumour tissue. The growth-inhibitory somatostatin receptor subtype 2 was identified as a potential clinical target. An investigation of patients with neuroendocrine tumours treated using SSTR2 targeted radiotherapy [111In]pentetreotide producing encouraging preliminary results. Fifty-six per cent of patients with evaluable hormone markers demonstrated stable levels or a significant decrease in one or more measured markers. This data demonstrate that gene pathways recognised to be altered in an animal model of a human disease can be used to identify therapeutic agents. This approach was successfully used to discover novel strategies that can be both effective and appropriate adjuncts to surgery for patients with neuroendocrine tumour disease.

During the last 10 years many attempts have been made to estimate the number of enterochromaffin-like (ECL) cells in various animal studies. This is the first presentation of an unbiased stereological estimator of the total number of histamine-positive ECL cells per rat and linked to estimators of related parameters: total volume of the oxyntic mucosa, total oxyntic mucosal surface area, total oxyntic serosal surface area, surface amplification factor, average thickness of the oxyntic mucosa, total and mean volume of the ECL cells, total number of oxyntic glands and pits, mean number of ECL cells per gland, and mean number of ECL cells and glands per oxyntic serosal surface area. This study is the first application of the smooth fractionator and includes a description of all sources of sampling variance in the smooth fractionator design with newly developed predictors.

Melatonin, a pineal hormone, because of its wide activity spectrum, is a subject of much current interest for biologists and physicians. It has been demonstrated that pineal gland is not an exclusive source of melatonin synthesis. Melatonin synthesis has been found in different sites of the organism, and a major source of extrapineal melatonin is the gastrointestinal tract. The role of melatonin in gastrointestinal functions is considered in the present review.

Activation of G(q) protein-coupled receptors usually causes a biphasic increase in intracellular calcium concentration ([Ca(2+)](i)) that is crucial for secretion in nonexcitable cells. In gastric enterochromaffin-like (ECL) cells, stimulation with gastrin leads to a prompt biphasic calcium response followed by histamine secretion. This study investigates the underlying signaling events in this neuroendocrine cell type. In ECL cells, RT-PCR suggested the presence of inositol 1,4,5-trisphosphate receptor (IP(3)R) subtypes 1-3. The IP(3)R antagonist 2-aminoethoxydiphenyl borate abolished both gastrin-induced elevation of [Ca(2+)](i) and histamine release. Thapsigargin increased [Ca(2+)](i), however, without inducing histamine secretion. In thapsigargin-pretreated cells, gastrin increased [Ca(2+)](i) through calcium influx across the plasma membrane. Both nimodipine and SKF-96365 inhibited gastrin-induced histamine release. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate induced histamine secretion, an effect that was prevented by nimodipine. In summary, gastrin-stimulated histamine release depends on IP(3)R activation and plasmalemmal calcium entry. Gastrin-induced calcium influx was mediated by dihydropyridine-sensitive calcium channels that appear to be L-type channels activated through a pathway involving activation of PKC.

N alpha-methylhistamine (N alpha-MH) is one of unusual metabolite of histamine that was found in Helicobacter pylori-infected stomach and is believed to interact with specific histamine H1, H2 and H3-receptors to stimulate gastric acid secretion and gastrin release from isolated G-cells but the effects of N alpha-MH on gastric mucosal integrity have been little studied. This study was designed; 1) to compare the effect of intraperitoneal (i.p.), intracerebroventricular (i.c.v.) and gastric topical (intragastric i.g.) application of exogenous N alpha-MH with that of standard histamine on gastric secretion in rats equipped with gastric fistula (series A) and 2) to compare the effect of i.c.v. administration of histamine and N alpha-MH with that of peripheral (i.p. and i.g.) application of these amines on gastric lesions induced by 100% ethanol (series B) in rats with or without capsaicin-induced deactivation of sensory nerves. The area of gastric lesions was determined planimetrically, gastric blood flow (GBF) was assessed by H2-gas clearance method and venous blood was collected for determination of plasma gastrin levels by RIA. N alpha-MH and histamine (0.1-10 mg/kg i.p. or i.g.) dose-dependently increased gastric acid output (series A); whereas i.c.v. administration of histamine or N alpha-MH inhibited dose-dependently this secretion; the dose attenuating gastric acid output by 50% (ED50) being 4 and 6 microg/kg i.c.v. Both, N alpha-MH and histamine (2 mg/kg i.p. and i.g.) attenuated significantly the area of gastric lesions induced by 100% ethanol (series B) while producing significant rise in the GBF and plasma immunoreactive gastrin increments. Central application of N alpha-MH and histamine (0.01-5 microg/kg i.c.v.) inhibited ethanol-induced gastric damage whereas higher doses ranging from 10-100 microg/kg of histamine and N alpha-MH were significantly less effective. Capsaicin-induced deactivation of sensory nerves by itself augmented significantly ethanol damage and attenuated significantly the protective and hyperemic effects of histamine and its methylated analog applied i.p. but failed to affect significantly those caused by i.c.v. administration of these amines. We concluded that: 1) central histamine and N alpha-MH inhibits gastric acid secretion and exhibits gastroprotective activity against ethanol in similar manner to that afforded by parenteral and topical histamine and N-alphaMH, 2) central N-alphaMH- and histamine-induced protection involve the enhancement in gastric microcirculation unrelated to neuropeptides released from capsaicin-sensitive afferent nerves, and 3) the major difference between central and peripheral histamine and its methylated analog is the influence on gastric acid secretion which does not appear to play any major role in gastroprotective activity of these agents.

Gastric enterochromaffin-like cells produce histamine in response to the antral hormone gastrin and accumulate the biogenic amine in secretory organelles via vesicular monoamine transporter subtype 2. The putative effects of gastrin on vesicular monoamine transporter subtype 2 expression and promoter activity are poorly understood. In the present study we used highly enriched rat enterochromaffin-like cells (purity, >90%) and rat pheochromocytoma cells stably transfected with a gastrin/cholecystokinin B receptor to investigate the expression and transcriptional regulation of vesicular monoamine transporter subtype 2. Stimulation of vesicular monoamine transporter subtype 2 mRNA and protein expression was observed in isolated enterochromaffin-like cells after 3- to 7-h incubation with gastrin (10(-7) M), forskolin (10(-5) M), or ionomycin (10(-5) M). Deletion analysis of the rat vesicular monoamine transporter subtype 2 promoter defined the minimal promoter sequence necessary for full basal activity as a -121 bp segment upstream of exon 1 containing two Sp1 sites (-97 to -88 bp and -68 to -59 bp) and a cAMP-responsive element (-44 to -35 bp). Gastrin (10(-7) M) stimulated extracellular signal related kinase1/2 phosphorylation, activated Sp1 and cAMP-responsive element-binding protein, and further induced activity of the complete rat vesicular monoamine transporter subtype 2 promoter (-800 bp) in gastrin/cholecystokinin B receptor cells. The -121-bp fragment was able to confer full gastrin responsiveness, and site-directed mutagenesis of the Sp1 and cAMP-responsive element motifs demonstrated their crucial importance for basal and inducible activities. Comparison of promoter activity of histidine decarboxylase, chromogranin A, or vesicular monoamine transporter subtype 2 in transfected cell lines revealed significant differences in basal and gastrin-stimulated activities. Our current study provides the first evidence that gastrin directly stimulates the expression and promoter activity of vesicular monoamine transporter subtype 2. Sp1 and cAMP-responsive element-binding protein recognition motifs located within 121 bp upstream of exon 1 appear to be indispensable for full basal and inducible promoter activities. Diverging effects of gastrin on histidine decarboxylase, chromogranin A, and vesicular monoamine transporter subtype 2 promoter may account for the coordinated synthesis and storage of histamine in this neuroendocrine cell type.

Background: Enteroendocrine cell-derived peptides modulate postresectional small bowel adaptation, which may be attenuated by transplantation. We investigated whether autotransplantation modulates the number and distribution of ileal enteroendocrine cells in pigs with proximal small bowel resection., Materials and Methods: Fifteen pigs were assigned into either small intestinal transection or 75% proximal small intestinal resection with or without autotransplantation of the remaining ileum. After 14 weeks the number and subtype distribution of enteroendocrine cells, crypt cell proliferation, and mucosal histology were analyzed from the proximal and distal ends of the remaining ileum., Results: When compared to resected controls, autotransplantation of the ileum decreased the absolute (P < 0.05 in proximal ileum) and proportional (P < 0.05 in distal ileum) crypt enteroendocrine cell number. In addition, autotransplantation reduced somatostatin and glicentin expressing cell counts and abolished the proximodistal gradient of the enteroendocrine cell number. When compared to transected controls, villus height, crypt depth, number of proliferating crypt cells, and crypt cell proliferation index increased after the proximal resection (P < 0.05 in all except in crypt depth and proliferation index of the distal ileum) but remained virtually unchanged after autotransplantation of the ileal remnant., Conclusions: Autotransplantation decreases the crypt enteroendocrine cell number and alters their proximodistal and subtype distribution in the remaining ileum in pigs with proximal small bowel resection. These alterations are associated with attenuated adaptive response of the autotransplanted ileum., (Copyright 2000 Academic Press.)

Secretin, gastrin and cholecystokinin were the first discovered gut hormones. Today we recognize more than 30 gut hormone genes and a multitude of bioactive peptides, which make the gut the largest endocrine organ in the body. Due to structural homologies gut peptide hormones/growth factors have been divided into separate families. It has been emphasized that those peptides are widely distributed, but have a specific expression in different cell types. The intestine can also be regarded as a sensory organ operating via neurons, endocrine cells and immune cells with gut peptides as signalling substances. Expression studies of peptide receptors in gut neuroendocrine tumours in combination with tailored peptide analogs have been helpful in developing new diagnostic and therapeutic strategies. New fields of research will relate to gut peptides associated with deficiency diseases and as potential growth factors in malignancies. Enterochromaffin cells, interspersed throughout the entire gastrointestinal mucosa, form the largest endocrine cell system. The physiological role of hormonal messengers, peptide receptors and amine transporters is currently under investigation as well as their potential involvement in disease, e.g. the secretory diarrhea associated with midgut carcinoid tumours.

Background & Aims: Regenerating (Reg) protein has a trophic effect on gastric mucosal cells. We have shown that Reg gene expression is increased in enterochromaffin-like (ECL) cells during the healing of damaged gastric mucosa around mucosal erosion. This study was designed to explore the stimulants of Reg expression during the healing of gastric mucosal damage., Methods: Time course changes of the expression of genes for various proinflammatory cytokines and Reg were investigated after induction of gastric mucosal lesions in rats. The direct effect of proinflammatory cytokines on Reg gene expression and Reg protein production were investigated in vitro using counterflow elutriation-enriched rat ECL cells. CXC receptor 2 (CXCR-2) expression was investigated in ECL cells by reverse-transcription polymerase chain reaction. Reg gene expression was also investigated in rats treated by the neutralizing antibody of cytokine-induced neutrophil chemoattractant (CINC-2 beta)., Results: During healing, the gene expression of several proinflammatory cytokines and Reg was markedly augmented. Among the proinflammatory cytokines, CINC-2 beta is the only cytokine in which augmented expression preceded the increase of Reg gene expression. In rats treated with CINC-2 beta neutralizing antibody, the augmentation of Reg gene expression was significantly inhibited. When ECL cells were incubated with these proinflammatory cytokines, CINC-2 beta dose-dependently increased Reg messenger RNA and Reg protein in ECL cells. CXCR-2 was identified in isolated ECL cells., Conclusions: CINC-2 beta, expressed in damaged gastric mucosa, stimulates the production of Reg protein in ECL cells via CXCR-2 and may be involved in the accelerated healing of injured gastric mucosa.

The theoretical basis for and clinical experience with using oral serotonin type 3 (5-HT3)-receptor antagonists for preventing chemotherapy-induced emesis are discussed. Evidence supports the idea that antineoplastic drugs and irradiation can initiate emesis by releasing serotonin from enterochromaffin cells in the gut mucosa, which activates peripheral vagal afferent nerves. In view of the GI site of serotonin release and vagal afferent activation, the proximity of neuronal 5-HT3 receptors, and the pharmacologic properties of 5-HT3-receptor antagonists, the oral use of these agents is rational. Oral granisetron 2 mg once daily or 1 mg twice daily has been evaluated in more than 4500 patients receiving highly or moderately emetogenic chemotherapy. Rates of total control of emesis ranged from 44% to 60%, and complete-response rates ranged from 70% to 94%. Oral ondansetron 8 mg three times daily has proven effective in patients receiving antineoplastics with moderate or moderately high emetogenic potential. Two double-blind studies demonstrated the efficacy of a single 24-mg oral dose of ondansetron administered approximately 30 minutes before cisplatin-based chemotherapy. Patients randomized to oral ondansetron had higher total-control and complete-response rates than patients receiving intravenous granisetron or ondansetron. Oral dolasetron 100 or 200 mg once daily also prevented emesis. Oral administration of 5-HT3-receptor antagonists for the prevention of acute emesis associated with chemotherapy is rational and appears to be effective.