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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.)

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.)

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.

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.

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.

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.

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.

Background & Aims: A nongastrin acid-stimulating peptide (NGASP) has been found in ulcerogenic pancreatic tumor syndrome without hypergastrinemia. The mechanism of gastric acid hypersecretion by NGASP was investigated in rats., Methods: In vivo, gastric acid secretion and in vitro histamine release from enterochromaffin-like (ECL) cells in responses to tumor extract (TE) and synthetic human gastrin-17 I or pentagastrin (PG) were studied. Whether the 2 secretagogues potentiate each other was determined., Results: TE dose-dependently stimulated histamine release, which was not blocked by a cholecystokinin (CCK)-B receptor antagonist. When TE was incubated with trypsin, the activity was abolished but was not affected by antibody. However, when rats were pretreated with antigastrin serum or CCK-B receptor antagonist, the acid secretion by TE was virtually abolished. The dose response of acid secretion to TE in the rats receiving PG in a threshold dose was significantly greater than that achieved by TE alone. Similarly, the dose response to PG combined with a threshold dose of TE was significantly greater than that produced by PG alone., Conclusions: NGASP stimulates histamine release from ECL cells, but the release is not mediated via CCK-B/gastrin receptor. NGASP and gastrin may potentiate each other to produce acid hypersecretion in ulcerogenic pancreatic tumor syndrome.

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.

Background/aims: Gastric enterochromaffinlike (ECL) cells play an important role in peripheral regulation of acid secretion. This study investigated the somatostatin receptor subtype on ECL cells., Methods: ECL cells were isolated from rat fundic mucosa to a purity of 90%-95% by combining enzymatic digestion, elutriation, density gradient centrifugation, and culture., Results: Polymerase chain reaction performed with templates from an ECL cell complementary DNA library and primers specific to each of the five known somatostatin receptor subtypes showed that the somatostatin receptor type 2 was significantly enriched in ECL complementary DNA. Single cell videoimaging of highly purified ECL cells in culture showed that only the somatostatin receptor type 2 selective agonist, DC 32-87, inhibited the gastrin-induced calcium signal at 10(-11) mol/L. The type 3 and type 4 selective agonists, DC 25-12 and DC 32-92, and also somatostatin 14 required 100-1000 times higher concentrations (10(-8) mol/L). The somatostatin receptor type 2 analogue also inhibited gastrin-stimulated histamine release with a 50% inhibitory concentration (IC50) value of 2 x 10(-12) mol/L, whereas somatostatin 14 and the type 3 and 4 analogues showed IC50 values of 1 to 5 x 10(-9) mol/L., Conclusions: The predominant somatostatin receptors on rat gastric ECL cells are of the somatostatin receptor 2 subtype; they inhibit histamine secretion by interfering with the gastrin-induced calcium signal.