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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.

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 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.

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.)

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.

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.

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.

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: Enterochromaffin-like (ECL) cells are histamine-containing endocrine cells in the gastric mucosa. Previous studies have shown that the proinflammatory cytokine interleukin (IL)-1beta present during chronic gastritis inhibits histamine synthesis in ECL cells and leads to sustained functional impairment. This study investigated the effects of IL-1beta on ECL cell apoptosis and the related signal-transduction mechanisms., Methods: ECL cells were isolated by pronase digestion and a combination of elutriation, gradient centrifugation, and 48-hour culture (purity >/=90%). Apoptosis was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling reaction and cell death detection enzyme-linked immunosorbent assay., Results: IL-1beta (100 pg/mL) increased the rate of programmed cell death 2-3 fold in ECL cells after 24 hours of incubation (total of 12%-14%). This effect was completely inhibited by the NF-kappaB inhibitor, proteasome inhibitor I, and the nitric oxide synthase inhibitor (iNOS) N(G)-monomethyl-L-arginine (10(-4) mol/L), but not by the caspase 3 inhibitor, Asp-Glu-Val-Asp-CHO. Western blot analysis, reverse-transcription polymerase chain reaction (PCR), and in situ PCR showed that IL-1beta induced gene expression (after 2-4 hours) and protein synthesis (6-18 hours) of the iNOS isoform in ECL cells. Bax protein expression was increased in response to IL-1beta. In contrast, bcl-2 gene expression was increased in response to basic fibroblast growth factor, which has been shown to counteract IL-1beta- induced apoptosis., Conclusions: These data suggest that IL-1beta induces programmed cell death in isolated rat ECL cells via activation of NF-kappaB, iNOS, and the Bax protein.

Although serotonin (5-HT) release from enterochromaffin (EC) cells is considered to be regulated by multiple receptor-mediated mechanisms, little is known about the signal transduction. Here, we introduce the methods to isolate the mouse ileal crypts, which consist of various types of cells including EC cells, and to analyze the intracellular calcium dynamics. Ileal tissue was inserted with a plastic straw and the smooth muscle layers were peeled off. The mucosa were digested with collagenase and then suspended by moderate pipetting. Ileal crypts were separated by stepwise filtrations through 2 different nylon-meshes. The isolated crypt contained 0-3 EC cells as identified by immunostaining using anti-5-HT antibody followed by confocal microscopy. Isolated crypts were attached to a coverglass by an adhesive material (Cell-Tak) and loaded with fura-2/AM. Intracellular calcium dynamics in EC cells were obtained by digital video-imaging analysis of fura-2 fluorescence followed by the identification of EC cells with immunostaining of 5-HT granules. By these methods, it was suggested that norepinephrine elicited a transient elevation of intracellular calcium concentration in EC cells via alpha 2-adrenoceptors. These methods could be also useful to analyze the signal transduction system in intestinal endocrine cells that contain various intestinal hormones such as gastrin, cholecystokinin or secretin.

Objective: To investigate the relationship between Helicobacter pylori (Hp) infection and pathophysiology of abnormal gastric acid secretion and mucosal proliferation by studying the effect of Hp lipopolysaccharide (LPS) on the function of enterochromaffin-like (ECL) cells in vitro., Methods: Pure isolated ECL cell preparation with a purity of +/- 95% was developed by proteinase digestion, elutriation and density gradient centrifugation with viability of > 95% as determined by trypan blue exclusion. After a short duration of culture basal and stimulated histamine secretion and DNA synthesis of ECL cells were measured by enzyme immunoassay and bromodeoxyuridine (BrdU) uptake, respectively., Results: Hp LPS stimulated basal and gastrin-stimulated histamine secretion (EC(50)4 x 10(-11) mol/L and EC(50) 10(-10) mol/L respectively). These effects were completely inhibited by somatostatin (10(-10) mol/L) but not by gastrin receptor antagonist L(365 260). Hp LPS did not stimulate basal DNA synthesis but significantly increased gastrin stimulated BrdU uptake with EC(50) of 10(-10) mol/L. Escherichia coil LPS had no significant effect on both histamine secretion and DNA synthesis., Conclusion: Hp stimulates both ECL cell proliferation and secretion in vitro. An interaction between Hp LPS and ECL cells may contribute to the reported abnormalities in acid secretion and gastric pathophysiology noted in Hp infection.

Background/aim: To investigate whether endogenous prostaglandins participate in the regulation of the gastrointestinal endocrine cell system., Methods: Sprague-Dawley rats were treated with 1 mg/kg indomethacin subcutaneously or indomethacin subcutaneously and 500 microg/kg oral prostaglandin E2 or solvents for 2 months. Endocrine cells were visualized by using immunohistochemistry and by the Sevier-Munger silver stain on specimens from the gastroduodenal mucosa, and their total volume was estimated, using standard stereological methods. Plasma and gastrointestinal tissue concentrations of regulatory peptides were analyzed by radioimmunoassay., Results: Fundic mucosa. The total volume of cells stained with the Sevier-Munger silver stain (enterochromaffin-like) was increased by indomethacin, but reduced by the administration of prostaglandin E2 (P < 0.05 vs. indomethacin). Indomethacin increased the total volume of somatostatin-immunoreactive. Similarly, rats given indomethacin and prostaglandin E2 had higher values than controls. Indomethacin increased the tissue concentration of somatostatin in the gastric fundus whereas prostaglandin E2 prevented such changes (P < 0.05 vs. indomethacin). Antral mucosa. The total volume of serotonin-immunoreactive cells was reduced by indomethacin, but increased by prostaglandin E2 (P < 0.05 vs. controls and indomethacin, respectively). Duodenal mucosa. The total volume of somatostatin-immunoreactive cells was reduced in the rats given indomethacin and prostaglandin E2 (P < 0.05 vs. controls and indomethacin). Indomethacin reduced and simultaneous administration of prostaglandin E2 increased the total volume of CCK-immunoreactive cells (P < 0.05 vs. controls and indomethacin). Indomethacin reduced the total volume of serotonin-immunoreactive cells whereas the simultaneous administration of PGE2 comparatively increased their total volumes (P < 0.05 vs. indomethacin), although they were still lower than the control values. The total volume of GIP-immunoreactive cells was slightly increased in the rats given both indomethacin and indomethacin + prostaglandin E2. The tissue concentration of somatostatin in the duodenum was reduced in rats given indometacin and prostaglandin E2 (P < 0.05 vs. controls and indomethacin)., Conclusion: Endogenous prostaglandins, particularly prostaglandin E2, regulate CCK-, enterochromaffin-like-, somatostatin-, GIP- and enterochromaffin cells in the gastroduodenal mucosa of the rat.

One hundred cases of neurogenic appendicopathy were histochemically studied for schwann cells and enterochromaffin cells. The early phase, labelled as neuro-appendicopathy (29 cases) showed minimum to moderate number of extraepithelial enterochromaffin cells without neurogenous hyperplasia. In 53 cases, there was intra and submucosal neural hyperplasia with increase in the extraepithelial enterochromaffin cells, representing the active phase. The late phase known as obliterative neurogenic appendicopathy, showed extraepithelial enterochromaffin cells and schwann cell proliferation of variable grades (18 cases). The origin of extraepithelial enterochromaffin cells is related to proliferating nerve plexus, rather than epithelial enterochromaffin cells.

Background and Purpose: Female inbred cotton rats develop adenocarcinomas in the oxyntic mucosa. Since a female preponderance is typical for enterochromaffin-like (ECL) cell tumors, we examined such tumors for ECL cells. Gastrin plays a decisive role in ECL cell tumorigenesis, so blood gastrin concentration and gastric mucosal pH were measured., Methods: The stomachs from six female cotton rats (6 to 8 months old) were studied histologically, and at euthanasia, gastric mucosal pH was determined. Euthanasia was performed on 15 other female cotton rats of similar age for determination of blood gastrin values by radioimmunoassay (RIA) and gastric mucosal pH. Rats were classified macroscopically to have normal or thick oxyntic mucosa, with or without tumor., Results: Among the six cotton rats studied histologically, two 6-month-old rats had normal and two others had thick gastric mucosa, whereas two 8-month-old rats had thick mucosa with tumors. The ECL cells were markedly hyperplastic in all rats with thick mucosa, and ECL cells were found in the neoplastic parenchyma. All cotton rats with normal-appearing gastric mucosa had pH <2.5, whereas 14 rats with thick mucosa had pH >3.1 and hypergastrinemia., Conclusions: Gastrin may play a major role in ECL cell hyperplasia and, perhaps, in adenocarcinoma genesis.

Background & Aims: Gastric carcinoids (types I and II) involve the transformation of naive enterochromaffin-like (ECL) cells to the neoplastic state and are associated primarily with hypergastrinemia. In this study, we evaluated the effects of two related neuropeptides, pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP), on ECL cell proliferation and characterized the receptor subtype(s) and signal transduction pathways that mediate this effect., Methods: Purified rat ECL cells were analyzed in culture for DNA synthesis as measured by 24-hour 5-bromo-2-deoxyuridine (BrdU) uptake. Reverse-transcription polymerase chain reaction (RT-PCR) with gene-specific oligonucleotide primers was performed to characterize the PACAP/VIP receptor subtype(s)., Results: PACAP/VIP neuropeptide-stimulated BrdU uptake was significantly greater (3.4-3.8-fold greater than control) than that at the maximal dose of gastrin (2.2-fold greater than control). PACAP-stimulated ECL cell proliferation (EC50, approximately 3 x 10(-)14 mol/L) was approximately 100-fold more potent than VIP (EC50, approximately 3x 10(-)12 mol/L). The stimulated BrdU uptake by both PACAP and VIP was competitively inhibited by PACAP-receptor antagonist (IC50, 10(-)9 mol/L, 3 x 10(-)9 mol/L, respectively) and VIP-receptor antagonist (IC50, 3 x 10(-)7 mol/L, 5 x 10(-)7 mol/L, respectively). RT-PCR identified the presence of the PACAP-specific but not PACAP/VIP receptor subtypes. The PACAP-stimulated BrdU uptake was inhibited (70%-80%) by inhibitors of adenosine 3',5'-cyclic monophosphate, phosphatidylinositol 3 kinase, and protein tyrosine kinase as well as mitogen-activated protein kinase., Conclusions: PACAP/VIP-related peptides are more potent modulators of ECL cell proliferation than gastrin, and their effect is mediated by a PACAP-specific receptor whose activation is transduced by multiple intracellular messenger systems.

Although serotonin (5-HT) release from enterochromaffin (EC) cells is considered to be regulated by multiple receptor-mediated mechanisms, little is known about the signal transduction in EC cells. We investigated the effects of adrenoceptor stimulation on 5-HT release from ileal tissue and intracellular calcium dynamics of epithelial cells in isolated ileal crypts in mice. Ileal tissues placed in organ bath were perfused with a buffered solution. Released 5-HT was measured using HPLC-ECD. Ileal crypts were isolated by collagenase digestion followed by moderate pipetting. Intracellular calcium dynamics were analyzed by digital video-imaging system using fura-2. NE, but not isoprenaline (Iso), induced 5-HT release from mouse ileal tissue. NE-induced 5-HT release was antagonized by yohimbine and rauwolscine, but not by prazosin and bunazosin. NE, but not Iso, also elicited a transient elevation of intracellular calcium in some EC cells. The effect of NE (1 microM) was slightly suppressed by prazosin and bunazosin, but was remarkably suppressed by yohimbine and rauwolscine. UK 14,304 and Clonidine at 10 microM significantly induced an increase in intracellular calcium concentration. NE-induced intracellular calcium dynamics was not significantly affected by timolol, Ro20-1724, rolipram and 8-bromo-cAMP. These results suggest that NE-induced 5-HT release from ileal EC cells is mediated predominantly via alpha 2-, but not beta-adrenoceptors, by a mechanism dependent on elevation of intracellular calcium concentration.

Background & Aims: Helicobacter pylori alterations in gastric acid output and mucosal proliferation may involve the enterochromaffin-like (ECL) cell. To test whether H. pylori affects ECL cell histamine secretion and proliferation, the effect of lipopolysaccharide (LPS) on ECL cell function in vitro was investigated., Methods: Using a rat ECL cell preparation of high purity (+/-95%), basal and stimulated histamine secretion and DNA synthesis were measured by enzyme immunoassay and bromodeoxyuridine (BrdU) uptake, respectively., Results: Escherichia coli LPS (10(-12) to 10(-6) mol/L) had no effect on basal and stimulated histamine secretion at concentrations of > 10(-6) mol/L. H. pylori LPS stimulated basal and gastrin-stimulated histamine secretion. These effects were completely inhibited by somatostatin (10(-10) mol/L) but not by the gastrin receptor antagonist L365,260 at 10(-6) mol/L. E. coli LPS had a weak stimulatory effect on ECL cell BrdU uptake at 10(-6) mol/L but had no effect on gastrin-stimulated BrdU uptake. H. pylori LPS did not stimulate basal synthesis but significantly increased (1.5-fold) gastrin-stimulated BrdU uptake., Conclusions: H. pylori influences both ECL cell proliferation and secretion in vitro. An interaction between H. pylori LPS and ECL cells may contribute to the reported abnormalities in acid secretion and gastric pathobiology noted in H. pylori infections.

1 The so-called enterochromaffin-like (ECL) cells constitute 65-75% of the endocrine cells in the acid-producing part of the rat stomach. They produce and secrete histamine and pancreastatin, a chromogranin A (CGA)-derived peptide, in response to gastrin, Cholecystokinin (CCK)B/gastrin receptor blockade is known to suppress their activity. 2 We have examined the time course of the deactivation of the ECL cells following treatment with the selective CCKB receptor antagonists RP73870 and YM022. The drugs were given by continuous subcutaneous infusion for a time span of 1 h to 3 weeks and the serum gastrin concentration and various ECL cell parameters were measured (oxyntic mucosal histidine decarboxylase (HDC) activity, histamine and pancreastatin concentrations, HDC mRNA and CGA mRNA levels, and circulating pancreastatin concentration). 3 The two antagonists caused a prompt and dramatic decline in the oxyntic mucosal HDC activity and HDC mRNA level. The HDC activity started to decline after 1-2 h, was reduced by 60-70% after 6 h and was maximally suppressed (80-90%) after 24-48 h. The HDC mRNA level was reduced after 12 h and was at about 20% of the pretreatment level after 2-4 days of infusion. The ECL cell histamine concentration was lowered by about 50% after 7-10 days. 4 RP73870 and YM022 lowered the serum pancreastatin concentration and the oxyntic mucosal CGA mRNA level. The serum pancreastatin concentration was reduced by 40% after 6 h and the reduction was maximal after 2-3 days. A decline in the oxyntic mucosal CGA mRNA level was noted after 12 h with a maximal reduction after 2-4 days of infusion. The ECL cell pancreastatin concentration was reduced by 30-40% after 3 weeks. 5 The infusion of RP73870 and YM022 induced hypergastrinaemia. The serum gastrin concentration started to rise after 2-4 h, there was a 2 fold increase after 6 h and maximal increase (3-4 fold) after 2-3 days of treatment. 6 In conclusion, CCKB/gastrin receptor blockade promptly deactivates the ECL cells. Deactivation, manifested in a greatly reduced HDC activity, was apparent after 1-2 h of the infusion. The serum pancreastatin concentration and the oxyntic mucosal HDC mRNA and CGA mRNA levels were greatly reduced after 1-2 days. The ECL cell concentrations of histamine and pancreastatin declined quite slowly by comparison.

Background & Aims: Histamine-producing enterochromaffin-like (ECL) cells play an integrative role in the regulation of acid secretion. Decreased mucosal histamine concentrations and increased levels of interleukin (IL) 1 beta, IL-6, and IL-8 have been detected in the gastric mucosa inflamed with Helicobacter pylori. The aim of this study was to investigate the response of isolated ECL cells to these cytokines., Methods: Enriched rat gastric ECL cells (85%-95%) were cultured for 2-4 days., Results: Polymerase chain reaction showed IL-1 and IL-6, but not IL-8 receptors, in ECL cell complementary DNA. Positive receptor staining with biotinylated IL-1 beta corresponded to ECL cell enrichment (92%). IL-6 and IL-8 had no effect on histamine secretion. IL-1 beta (2 U/mL) stimulated basal histamine secretion and nitric oxide production within 60 minutes and cyclic guanosine monophosphate production within 20 minutes. Pretreatment for 20 minutes with IL-1 beta (2 U/mL) attenuated gastrin-stimulated histamine secretion by 40%-50%, reversed by the IL-1 receptor antagonist (10 U/ mL). Pretreatment for 20 minutes with IL-1 beta (2 U/mL) completely inhibited gastrin-stimulated (1 nmol/L) histidine decarboxylase activity. IL-1 beta (2 U/mL, 60 minutes) increased lactate dehydrogenase release to 25% of cell content. Cells pretreated with IL-1 beta did not respond to gastrin after a further 48-hour culture and showed decreased histamine content., Conclusions: ECL cells appear to express IL-1 receptors. IL-1 beta causes sustained functional impairment of ECL cells in vitro.

The effect of vagotomy on the development of ECL cell tumours was analyzed during drug-induced hypergastrinemia in Mastomys natalensis, a rodent prone to develop ECL cell tumours. Untreated animals were compared with animals receiving the histamine2-receptor blocker loxtidine (LOX) and with animals subjected to unilateral subdiaphragmatic vagotomy prior to loxtidine treatment (VAG+LOX). Loxtidine (2g/l) was administered in drinking water for 48 weeks to allow multiple ECL cell carcinoids to develop. Plasma gastrin levels were increased in LOX animals (94 +/- 31 pmol/l) and in VAG+LOX animals (181 +/- 59 pmol/l) compared to controls (45 +/- 4 pmol/l). Corpus weight and oxyntic mucosal thickness was almost doubled in all loxtidine-treated animals and the density of mucosal endocrine cells was increased by 65% in the LOX group and by 135% in VAG+LOX animals. No significant differences in mucosal thickness and endocrine cell density were seen when denervated and intact parts of the stomach were compared. In the VAG+LOX animals endocrine cell neoplasia was seen in 60% and dysplasia in 40% of animals compared to 40% neoplasia, 45% dysplasia and 15% hyperplasia in LOX animals. The frequency of neoplastic and dysplastic lesions did not differ between denervated and intact parts of the stomach. Untreated animals showed no neoplastic or dysplastic lesions. It is concluded that unilateral vagotomy has no protective effect on the development of ECL-cell tumours in Mastomys during hypergastrinemia, as opposed to previous studies in the rat.

Background: The interaction between adrenergic nerves and enterochromaffin (EC) cells was studied in health and disease using animal models and patients with the midgut carcinoid syndrome., Methods: The methods included morphological techniques (fluorescence microscopy/cytofluorimetry, electronmicroscopy), experimental models (in vivo/in vitro nerve stimulation, pharmacological analyses, axonal transport, tumor transplantation, tumor cell cultures) and clinical tests (pentagastrin provocation, octreotide scintigraphy)., Results: From vagal nerve stimulation studies it was clear that activation of adrenergic fibers could release serotonin (5-HT) from EC cells, which led to the mapping of a vagal adrenergic pathway. Ultrastructurally a direct innervation of EC cells was demonstrated. It was confirmed in vitro that adrenoceptors controlled the release of 5-HT; it was maintained in neoplasia as studied in the tumor models. The tumor cells shared several functional and morphological characteristics with adrenergic neurons and exerted trophic actions on neurons grown in co-culture., Conclusions: Pentagastrin provocation of 5-HT release in carcinoid patients may be mediated via release of catecholamines from the adrenals in turn activating adrenoceptors on tumor cells. Pretreatment with a somatostatin analog can reduce these reactions and thus minimize the risk for carcinoid crisis during surgery.

Little progress has been made in the understanding of the pathobiology of gastric neoplasia over the past 4 decades. This reflects the paucity of information available regarding the biology of gastric mucosal cell proliferation. More recently it has become apparent that growth factor regulation of cell proliferation is of considerable relevance in initiating mucosal mitogenesis. We have recently identified the histamine secreting enterochromaffin-like (ECL) cell as a pivotal cellular regulator of gastric acid secretion. In addition to its critical role in initiating acid secretion, we have proposed that the ECL cell may produce agents responsible for the regulation of mucosal cell proliferation. We have therefore hypothesized that such a function may be subserved by production of transforming growth factor alpha (TGFalpha). TGFalpha is known to play a significant role both in normal physiology and in the transformation of naive cells into a neoplastic form. We therefore proposed that increased levels of gastrin induced by low acid states might stimulate TGFalpha secretion and that this agent might be capable of regulating ECL cell DNA synthesis and cell proliferation. We used the mastomys rodent to generate an in vivo hypergastrinemia model using long-term histamine-2 receptor blockade (loxtidine 1 mg/kg/day). In order to evaluate the cell-specific effects, we developed a pure isolated ECL cell system from the mastomys stomach. This utilized pronase digestion (1.0 mg/ml) and EDTA exposure (1 mM) of the mucosa followed by particle size separation with countercurrent elutriation and density purification on a Nycodenz step gradient. ECL cells were obtained with a purity of 90-95%. Histamine secretion from ECL cells was measured by radioimmunoassay (RIA). TGFalpha content was measured by RIA, and TGFalpha expression was measured by RNAse probe protection assay. DNA synthesis was quantified by measuring bromo-deoxyuridine (BrdU) incorporation into cultured cells. TGFalpha levels were increased in fundic mucosa after 16 weeks of hypergastrinemia from 4.3 +/- 0.6 to 32.6 +/- 2.6 fmole/mg protein, P < 0.05). TGFalpha message was identified in the ECL cells by RNAse probe protection assay, and was fourfold amplified in ECL cell tumors after 16 weeks of exposure to hypergastrinemia. Gastrin stimulated (10 nM) histamine secretion in isolated naive ECL cells was inhibited by TGFalpha (IC50 5 x 10 (-9) M). DNA synthesis was stimulated by gastrin (EC50 2 X 10 M) and TGFalpha (EC50 5 x 10(-9) M). These data are consistent with the proposal that elevated gastrin levels are associated with ECL cell TGFalpha production and that TGFalpha stimulates ECL cell DNA synthesis.

We consider the multiple interacting factors which lead to the proliferation of human gastric enterochromaffin-like (ECL) cells, and control of this proliferation by somatostatin and its analogues. Somatostatin receptors occur in human fundic mucosa, and octreotide can be considered a useful therapeutic tool to control gastrin-driven ECL cell growth in humans.

Background: The enterochromaffin-like (ECL) cells in the rat oxyntic mucosa produce histamine and contain cytoplasmic granules, microvesicles, and secretory vesicles. The cells respond to gastrin by the release of histamine (associated with loss of secretory vesicles), which is thought to mediate the gastrin-induced stimulation of the parietal cells. Gastric acid secretion is stimulated also by vagal excitation, which can be induced, for instance, by pylorus ligation. The present study addresses the question whether the ECL cells are involved in the acid response to pylorus ligation., Methods: Rats were subjected to pylorus ligation and killed 4 or 16 h later. Other rats were subjected to sham operation (laparotomy). Some of the rats received human Leu15-gastrin-17 (5 nmol/kg/h) by intravenous infusion for 30 or 60 min before being killed. The serum gastrin concentration, the oxyntic mucosal histidine decarboxylase (HDC) activity, HDC mRNA concentration, histamine concentration, and gastric acid output were measured. Specimens from the oxyntic mucosa were processed for transmission electron microscopy. Electron micrographs of ECL cells were analyzed planimetrically., Results: The gastric acid output was high, but the serum gastrin concentration was not affected by the pylorus ligation. The HDC activity and the level of HDC mRNA in the oxyntic mucosa were reduced, but the histamine concentration was unchanged. The secretory vesicles and granules of the ECL cells were unaffected, whereas the number and volume density of the microvesicles were reduced. Gastrin administration to sham-operated and pylorus-ligated rats lowered the oxyntic mucosal histamine concentration and increased the HDC activity in both groups., Conclusions: ECL cells in the rat stomach do not mediate the gastric acid response to pylorus ligation, and ECL cells in the pylorus-ligated stomach retain their ability to respond to gastrin with activation.

The idea presented here is that, in gastric mucosa, two independent regulatory systems use the same transmitter: histamine molecules. The IgE/mast cell system is dispersed throughout the body, while the other regulates the gastric acid secretion. IgE molecules in gastric mucosa are attached to the mast cells. Mast cells release histamine molecules after the antigen has been recognized by IgE. These molecules normally act on vascular H1 receptors to promote extravasation and chemotaxy. Gastrin molecules are released from antral G cells to stimulate gastric acid secretion. Their influence on parietal cells is indirectly augmented by gastrin governed release of histamine molecules from enterochromaffin-like cells. These histamine molecules normally act on H2 receptors of parietal cells to promote gastric acid secretion. Chronic infection of gastric mucosa (i.e. with Helicobacter pylori), autoimmune disorders or repetitive mucosal exposure to the same antigen, can develop chronic inflammation of gastric mucosa. Gastric acid secretion is diminished with secondary hypergastrinemia and increased release of histamine from enterochromaffin-like cells in an attempt to stimulate the few remaining parietal cells. Hypothetically, increased concentrations of released histamine in gastric mucosa might activate the vascular H1 receptors with extravasation and aggravated inflammation. This can further decrease the number of active parietal cells, reduce gastric acid secretion and potentiate hypergastrinemia. In this hypothetical setting, H1 blockers might reduce the damage by abolishing the vascular reactions. The prolonged antigen load on gastric mucosa can promote production of specific IgE antibodies. Further exposures to the same antigen degranulate sensitized mucosal mast cells. Liberated histamine can produce extravasation through the vascular H1 receptor and, hypothetically, local hyperacidity through the parietal cell H2 receptors. The result would be hyperacidity and hypogastrinemia with possible ulcer disease. Some individuals are more predisposed to IgE production or have increased numbers of mast cells that might explain why only some people develop ulcer disease after H. pylori infection.