Nano-analysis Reveals High Fraction of Serotonin Release during Exocytosis from a Gut Epithelium Model Cell.

Electrochemical methods were used to explore the exocytotic nature of serotonin (5-HT) release in human carcinoid BON cells, an in vitro human enterochromaffin cell model, to understand the mechanisms operating the release of gut-derived 5-HT in the intestinal mucosal epithelium. We show that the fractional vesicular 5-HT release in BON cells is 80 % compared to previous work in pancreatic beta cells (34 %). The fractional release increased from 80 % in control BON cells to 87 % with 5-HT preincubation and nearly 100 % with the combination of 5-HT and the 5-HT4 autoreceptor agonist, cisapride. Thus, partial release is the primary mechanism of exocytosis in BON cells, resulting in a variable amount of the vesicular content being released. Factors that control secretion of 5-HT from enterochromaffin cells or BON cells are important as partial release provides a mechanism for development of effective therapeutic strategies to treat gastrointestinal diseases.

Serotonin 3 receptor signaling regulates 5-fluorouracil-mediated apoptosis indirectly via TNF-α production by enhancing serotonin release from enterochromaffin cells.

Antagonists of the 5-hydroxytryptamine (serotonin) 3 receptor (5-HT3R) have anti-inflammatory and anti-apoptotic activities, but the detailed, underlying mechanisms are not well understood. We focused on anti-apoptotic activities via 5-HT3R signaling to clarify the underlying mechanisms. Mice were administered 5-fluorouracil (5-FU), which induced apoptosis in intestinal epithelial cells. Coadministration with 5-HT3R antagonists or agonists tended to decrease or increase the number of apoptotic cells, respectively. In serotonin 3A receptor (5-HT3AR) null (HTR3A-/-) mice, the number of apoptotic cells induced by 5-FU was decreased compared with that in wild-type (WT) mice. Bone marrow (BM) transplantation was performed to determine if BM-derived immune cells regulated 5-FU-induced apoptosis, but they were found to be unrelated to this process. Data from 5-HT3AR/enhanced green fluorescent protein reporter mice revealed that 50% of enterochromaffin (EC) cells expressed 5-HT3AR, but the number of apoptotic cells induced by 5-FU in the intestinal crypt organoids of HTR3A-/- mice was not altered compared with WT mice. In contrast, plasma 5-HT concentrations in WT mice but not in HTR3A-/- mice administered 5-FU were increased significantly. In conclusion, 5-HT3R signaling may enhance 5-HT release, possibly from EC cells intravascularly, or paracrine, resulting in increases in plasma 5-HT concentration, which in turn, enhances apoptotic activities induced by 5-FU.-Mikawa, S., Kondo, M., Kaji, N., Mihara, T., Yoshitake, R., Nakagawa, T., Takamoto, M., Nishimura, R., Shimada, S., Ozaki, H., Hori, M. Serotonin 3 receptor signaling regulates 5-fluorouracil-mediated apoptosis indirectly via TNF-α production by enhancing serotonin release from enterochromaffin cells.

Histamine causes influx via T-type voltage-gated calcium channels in an enterochromaffin tumor cell line: potential therapeutic target in adverse food reactions.

The P-STS human ileal neuroendocrine tumor cells, as a model for gut enterochromaffin cells, are strongly and synergistically activated by histamine plus acetylcholine (ACh), presumably via histamine 4 receptors, and weakly activated by histamine alone. Sensing these signals, enterochromaffin cells could participate in intestinal intolerance or allergic reactions to food constituents associated with elevated histamine levels. In this study we aimed to analyze the underlying molecular mechanisms. Inhibition by mepyramine and mibefradil indicated that histamine alone caused a rise in intracellular calcium concentration ([Ca2+]i) via histamine 1 receptors involving T-type voltage-gated calcium channels (VGCCs). Sensitivity to histamine was enhanced by pretreatment with the inflammatory cytokine tumor necrosis factor-α (TNF-α). In accordance with the relief it offers some inflammatory bowel disease patients, otilonium bromide, a gut-impermeable inhibitor of T-type (and L-type) VGCCs and muscarinic ACh receptors, efficiently inhibited the [Ca2+]i responses induced by histamine plus ACh or by histamine alone in P-STS cells. It will take clinical studies to show whether otilonium bromide has promise for the treatment of adverse food reactions. The cells did not react to the nutrient constituents glutamate, capsaicin, cinnamaldehyde, or amylase-trypsin inhibitors and the transient receptor potential channel vanilloid 4 agonist GSK-1016790A. The bacterial product butyrate evoked a rise in [Ca2+]i only when added together with ACh. Lipopolysaccharide had no effect on [Ca2+]i despite the presence of Toll-like receptor 4 protein. Our results indicate that inflammatory conditions with elevated levels of TNF-α might enhance histamine-induced serotonin release from intestinal neuroendocrine cells. NEW & NOTEWORTHY We show that histamine synergistically enhances the intracellular calcium response to the physiological agonist acetylcholine in human ileal enterochromaffin tumor cells. This synergistic activation and cell activation by histamine alone largely depend on T-type voltage-gated calcium channels and are inhibited by the antispasmodic otilonium bromide. The cells showed no response to wheat amylase-trypsin inhibitors, suggesting that enterochromaffin cells are not directly involved in nongluten wheat sensitivity.

Problems Associated with Deprescribing of Proton Pump Inhibitors.

Proton pump inhibitors (PPIs) are recommended as a first-line treatment for gastroesophageal reflux disease (GERD) and other acid related disorders. In recent years, concerns have been raised about the increasing prevalence of patients on long-term PPI therapy and inappropriate PPI use. It is well known that short-term PPI therapy is generally well tolerated and safe; however, their extensive long-term use is a major global issue. One of these long-standing concerns is PPI-induced gastrin elevation secondary to hypoacidity. Hypergastrinemia is believed to play a role in rebound hyperacidity when PPIs are discontinued resulting in induced dyspeptic symptoms that might result in the reinstitution of therapy. Gastrin exerts tropic effects in the stomach, especially on enterochromaffin-like (ECL) cells, and concerns have also been raised regarding the potential progression to dysplasia or tumor formation following long-term therapy. It is well known that a substantial number of patients on long-term PPI therapy can discontinue PPIs without recurrence of symptoms in deprescribing trials. What is unknown is how sustainable deprescribing should be undertaken in practice and how effective it is in terms of reducing long-term outcomes like adverse drug events, morbidity and mortality. Moreover, there is no clear consensus on when and how deprescribing strategies should be attempted in practice. This review sought to summarize the harms and benefits of long-term PPI therapy with special focus on gastrin elevation and its relation to deprescribing studies and future interventions that may improve PPI use.

FFA2 activation combined with ulcerogenic COX inhibition induces duodenal mucosal injury via the 5-HT pathway in rats.

Serotonin (5-HT), predominantly synthesized and released by enterochromaffin cells, is implicated in gastrointestinal symptoms such as emesis, abdominal pain, and diarrhea. Because luminal short-chain fatty acids (SCFAs) release 5-HT from enterochromaffin cells, which express the SCFA receptor free fatty acid receptor 2 (FFA2) in rat duodenum, we examined the effects of the selective FFA2 agonist phenylacetamide-1 (PA1) on duodenal 5-HT release with consequent bicarbonate secretion [duodenal bicarbonate secretion (DBS)] and on indomethacin (IND)-induced enteropathy. Intestinal injury was induced by IND (10 mg/kg sc) with or without PA1. We measured DBS in vivo in a duodenal loop perfused with PA1 while measuring 5-HT released in the portal vein. Duodenal blood flow was measured by laser-Doppler flowmetry. IND induced small intestinal ulcers with duodenal sparing. PA1 given with IND (IND + PA1) dose dependently induced duodenal erosions. IND + PA1-induced duodenal lesions were inhibited by the FFA2 antagonist GLPG-0974, ondansetron, or omeprazole but not by RS-23597 or atropine. Luminal perfusion of PA1 augmented DBS accompanied by increased portal blood 5-HT concentrations with approximately eight times more release at 0.1 mM than at 1 µM, with the effects inhibited by coperfusion of GLPG-0974. Luminal PA1 at 1 µM increased, but at 0.1 mM diminished, duodenal blood flow. Cosuperfusion of PA1 (0.1 mM) decreased acid-induced hyperemia, further reduced by IND pretreatment but restored by ondansetron. These results suggest that, although FFA2 activation enhances duodenal mucosal defenses, FFA2 overactivation during ulcerogenic cyclooxygenase inhibition may increase the vulnerability of the duodenal mucosa to gastric acid via excessive 5-HT release and 5-HT3 receptor activation, implicated in foregut-related symptoms such as emesis and epigastralgia.NEW & NOTEWORTHY Luminal free fatty acid receptor 2 agonists stimulate enterochromaffin cells and release serotonin, which enhances mucosal defenses in rat duodenum. However, overdriving serotonin release with high luminal concentrations of free fatty acid 2 ligands such as short-chain fatty acids injures the mucosa by decreasing mucosal blood flow. These results are likely implicated in serotonin-related dyspeptic symptom generation because of small intestinal bacterial overgrowth, which is hypothesized to generate excess SCFAs in the foregut, overdriving serotonin release from enterochromaffin cells.

Methotrexate causes acute hyperplasia of enterochromaffin cells containing substance P in the intestinal mucosa of rats.

This study aimed to investigate the acute and chronic effect of methotrexate on the intestinal substance P metabolism after a single administration to rats. Methotrexate caused a significant increase in the number of substance P-containing cells in the ileal mucosa both at 24 and 96 h. Most of enterochromaffin cells expressing l-tryptophan hydroxylase contained substance P. The expression of Tac1 mRNA was increased by methotrexate at 24 h, but not at 96 h. Thus, methotrexate causes acute hyperplasia of enterochromaffin cells in the intestinal mucosa of rats with a transient increase in the production of substance P.

Blocking peripheral serotonin synthesis by telotristat etiprate (LX1032/LX1606) reduces severity of both chemical- and infection-induced intestinal inflammation.

Mucosal inflammation is accompanied by an alteration in 5-HT. Intestinal 5-HT synthesis is catalyzed by tryptophan hydroxylase 1 (Tph1) and we have shown that mice deficient in this rate-limiting enzyme have reduced severity of intestinal inflammation in models of chemical-induced experimental colitis. Here, we investigated the effect of blocking peripheral 5-HT synthesis in generation of intestinal inflammation by a using peripheral Tph inhibitor, telotristat etiprate (LX1606), in models of intestinal inflammation. LX1606 was given orally either prophylactically or therapeutically to mice with dextran sulfate sodium (DSS)-induced colitis or with infection with Trichuris muris. Severity of intestinal inflammation was measured by assessment of disease activity scores, histological damage, and MPO and inflammatory cytokine levels. LX1606 significantly reduced intestinal 5-HT levels and delayed onset and severity of DSS-induced acute and chronic colitis. This was associated with decreased MPO and proinflammatory cytokine levels compared with vehicle-treated controls. In the infection-induced inflammation model, treatment with LX1606 enhanced worm expulsion as well as increased IL-10 production and goblet cell numbers. LX1606-treated mice had significantly lower MPO and IL-1ß levels compared with controls postinfection. Our results demonstrate that peripheral 5-HT plays an important role in intestinal inflammation and in the generation of immune responses. Pharmacological reduction of peripheral 5-HT may serve as a potential strategy for modulating various intestinal inflammatory disorders.

Serum serotonin reduced the expression of hepatic transporter Mrp2 and P-gp via regulating nuclear receptor CAR in PI-IBS rats.

Hepatic transporters and drug metabolizing enzymes (DMEs) play important roles in the pharmacological effects and (or) side-effects of many drugs, and are regulated by several mediators, including neurotransmitters. This work aimed to investigate whether serum levels of 5-hydroxytryptamine (5-HT) affected the expression of hepatic transporters or DMEs. The expression of hepatic transporters was assessed using the Western-blot technique in a 2,4,6-trinitrobenzenesulfonic-acid-induced rat model of post-infectious irritable bowel syndrome (PI-IBS), in which serum levels of 5-HT were significantly elevated. To further clarify the underlying mechanism, the 5-HT precursor 5-hydroxytryptophan (5-HTP) and the 5-HT depleting agent parachlorophenylalanine (pCPA) were applied to adjust serum levels of 5-HT. Serum levels of 5-HT were measured using LC-MS/MS; the expression of hepatic transporters, DMEs, and nuclear receptors were examined by Western-blot technique. Our results showed that in PI-IBS rats the expression of multidrug resistance protein 2 (Mrp2) was significantly decreased, while colonic enterochromaffin cell density and serum levels of 5-HT were all significantly increased. Moreover, 5-HTP treatment significantly increased serum levels of 5-HT and decreased the expression of Mrp2 and glycoprotein P (P-gp), whereas treatment with pCPA markedly decreased serum levels of 5-HT and increased the expression of Mrp2 and P-gp. Our results indicated that serum 5-HT regulates the expression of Mrp2 and P-gp, and the underlying mechanism may be related to the altered expression of the nuclear receptor constitutive androstane receptor (CAR).

The receptor TGR5 mediates the prokinetic actions of intestinal bile acids and is required for normal defecation in mice.

BACKGROUND & AIMS: Abnormal delivery of bile acids (BAs) to the colon as a result of disease or therapy causes constipation or diarrhea by unknown mechanisms. The G protein-coupled BA receptor TGR5 (or GPBAR1) is expressed by enteric neurons and endocrine cells, which regulate motility and secretion. METHODS: We analyzed gastrointestinal and colon transit, as well as defecation frequency and water content, in wild-type, knockout, and transgenic mice (trg5-wt, tgr5-ko, and tgr5-tg, respectively). We analyzed colon tissues for contractility, peristalsis, and transmitter release. RESULTS: Deoxycholic acid inhibited contractility of colonic longitudinal muscle from tgr5-wt but not tgr5-ko mice. Application of deoxycholic acid, lithocholic acid, or oleanolic acid (a selective agonist of TGR5) to the mucosa of tgr5-wt mice caused oral contraction and caudal relaxation, indicating peristalsis. BAs stimulated release of the peristaltic transmitters 5-hydroxytryptamine and calcitonin gene-related peptide; antagonists of these transmitters suppressed BA-induced peristalsis, consistent with localization of TGR5 to enterochromaffin cells and intrinsic primary afferent neurons. tgr5-ko mice did not undergo peristalsis or transmitter release in response to BAs. Mechanically induced peristalsis and transmitter release were not affected by deletion of tgr5. Whole-gut transit was 1.4-fold slower in tgr5-ko than tgr5-wt or tgr5-tg mice, whereas colonic transit was 2.2-fold faster in tgr5-tg mice. Defecation frequency was reduced 2.6-fold in tgr5-ko and increased 1.4-fold in tgr5-tg mice compared with tgr5-wt mice. Water content in stool was lower (37%) in tgr5-ko than tgr5-tg (58%) or tgr5-wt mice (62%). CONCLUSIONS: The receptor TGR5 mediates the effects of BAs on colonic motility, and deficiency of TGR5 causes constipation in mice. These findings might mediate the long-known laxative properties of BAs, and TGR5 might be a therapeutic target for digestive diseases.

Activation of colonic mucosal 5-HT(4) receptors accelerates propulsive motility and inhibits visceral hypersensitivity.

BACKGROUND & AIMS: 5-hydroxytryptamine receptor (5-HT(4)R) agonists promote gastrointestinal motility and attenuate visceral pain, but concerns about adverse reactions have restricted their availability. We tested the hypotheses that 5-HT(4) receptors are expressed in the colonic epithelium and that 5-HT(4)R agonists can act intraluminally to increase motility and reduce visceral hypersensitivity. METHODS: Mucosal expression of the 5-HT(4)R was evaluated by reverse-transcriptase polymerase chain reaction and immunohistochemical analysis of tissues from 5-HT(4)R(BAC)-enhanced green fluorescent protein mice. Amperometry, histology, and short-circuit current measurements were used to study 5-HT, mucus, and Cl(-) secretion, respectively. Propulsive motility was measured in guinea pig distal colon, and visceromotor responses were recorded in a rat model of colonic hypersensitivity. 5-HT(4)R compounds included cisapride, tegaserod, naronapride, SB204070, and GR113808. RESULTS: Mucosal 5-HT(4) receptors were present in the small and large intestines. In the distal colon, 5-HT(4) receptors were expressed by most epithelial cells, including enterochromaffin and goblet cells. Stimulation of 5-HT(4)Rs evoked mucosal 5-HT release, goblet cell degranulation, and Cl(-) secretion. Luminal administration of 5-HT(4)R agonists accelerated propulsive motility; a 5-HT(4)R antagonist blocked this effect. Bath application of 5-HT(4)R agonists did not affect motility. Oral or intracolonic administration of 5-HT(4)R agonists attenuated visceral hypersensitivity. Intracolonic administration was more potent than oral administration, and was inhibited by a 5-HT(4)R antagonist. CONCLUSIONS: Mucosal 5-HT(4) receptor activation can mediate the prokinetic and antinociceptive actions of 5-HT(4)R agonists. Colon-targeted, intraluminal delivery of 5-HT(4)R agonists might be used to promote motility and alleviate visceral pain, while restricting systemic bioavailability and resulting adverse side effects.

Serotonin availability in rat colon is reduced during a Western diet model of obesity.

Constipation and slowed transit are associated with diet-induced obesity, although the mechanisms by which this occurs are unclear. Enterochromaffin (EC) cells within the intestinal epithelium respond to mechanical stimulation with the release of serotonin [5-hydroxytryptamine (5-HT)], which promotes transit. Thus our aim was to characterize 5-HT availability in the rat colon of a physiologically relevant model of diet-induced obesity. EC cell numbers were determined immunohistochemically in chow-fed (CF) and Western diet-fed (WD) rats, while electrochemical methods were used to measure mechanically evoked (peak) and steady-state (SS) 5-HT levels. Fluoxetine was used to block the 5-HT reuptake transporter (SERT), and the levels of mRNA for tryptophan hydroxylase 1 and SERT were determined by quantitative PCR, and SERT protein was determined by Western blot. In WD rats, there was a significant decrease in the total number of EC cells per crypt (0.86 ± 0.06 and 0.71 ± 0.05 in CF and WD, respectively), which was supported by a reduction in the levels of 5-HT in WD rats (2.9 ± 1.0 and 10.5 ± 2.6 µM at SS and peak, respectively) compared with CF rats (7.3 ± 0.4 and 18.4 ± 3.4 µM at SS and peak, respectively). SERT-dependent uptake of 5-HT was unchanged, which was supported by a lack of change in SERT protein levels. In WD rats, there was no change in tryptophan hydroxylase 1 mRNA but an increase in SERT mRNA. In conclusion, our data show that foods typical of a WD are associated with decreased 5-HT availability in rat colon. Decreased 5-HT availability is driven primarily by a reduction in the numbers and/or 5-HT content of EC cells, which are likely to be associated with decreased intestinal motility in vivo.

Gastrin upregulates the prosurvival factor secretory clusterin in adenocarcinoma cells and in oxyntic mucosa of hypergastrinemic rats.

We show that the gastric hormone gastrin induces the expression of the prosurvival secretory clusterin (sCLU) in rat adenocarcinoma cells. Clusterin mRNA was still upregulated in the presence of the protein synthesis inhibitor cycloheximide, although at a lower level. This indicates that gastrin induces clusterin transcription independently of de novo protein synthesis but requires de novo protein synthesis of signal transduction pathway components to achieve maximal expression level. Luciferase reporter assay indicates that the AP-1 transcription factor complex is involved in gastrin-mediated activation of the clusterin promoter. Gastrin-induced clusterin expression and subsequent secretion is dependent on sustained treatment, because removal of gastrin after 1-2 h abolished the response. Neutralization of secreted clusterin by a specific antibody abolished the antiapoptotic effect of gastrin on serum starvation-induced apoptosis, suggesting that extracellular clusterin is involved in gastrin-mediated inhibition of apoptosis. The clusterin response to gastrin was validated in vivo in hypergastrinemic rats, showing increased clusterin expression in the oxyntic mucosa, as well as higher levels of clusterin in plasma. In normal rat oxyntic mucosa, clusterin protein was strongly expressed in chromogranin A-immunoreactive neuroendocrine cells, of which the main cell type was the histidine decarboxylase-immunoreactive enterochromaffin-like (ECL) cell. The association of clusterin with neuroendocrine differentiation was further confirmed in human gastric ECL carcinoids. Interestingly, in hypergastrinemic rats, clusterin-immunoreactive cells formed distinct groups of diverse cells at the base of many glands. Our results suggest that clusterin may contribute to gastrin's growth-promoting effect on the oxyntic mucosa.

The role of mechanical forces and adenosine in the regulation of intestinal enterochromaffin cell serotonin secretion.

Enterochromaffin (EC) cells of the diffuse neuroendocrine cell system secrete serotonin (5-HT) with activation of gut motility, secretion, and pain. These cells express adenosine (ADORA) receptors and are considered to function as mechanosensors. Physiological pathways mediating mechanosensitivity and adenosine responsiveness remain to be fully elucidated, as do their roles in inflammatory bowel disease (IBD) and neoplasia. Pure (98-99%) FACS-sorted normal and IBD human EC cells and neoplastic EC cells (KRJ-I) were studied. IBD-EC cells and KRJ-I overexpressed ADORA2B. NECA, a general ADORA receptor agonist, stimulated, whereas the A2B receptor antagonist MRS1754 inhibited, 5-HT release (EC50 = 1.8 × 10-6 M; IC50 = 3.7 × 10-8 M), which was associated with corresponding alterations in intracellular cAMP levels and pCREB (Ser133). Mechanical stimulation using a rhythmic flex model induced transcription and activation of Tph1 (tryptophan hydroxylase) and VMAT1 (vesicular monoamine transporter 1) and the release of 5-HT, which could be inhibited by MRS1754 and amplified by NECA. Secretion was also inhibited by H-89 (PKA inhibitor) while Tph1 and VMAT1 transcription was regulated by PKA/MAPK and PI3K-mediated signaling. Normal and IBD-EC cells also responded to NECA and mechanical stimulation with PKA activation, cAMP production, and 5-HT release, effects reversible by MRS1754. EC cells express stimulatory ADORA2B, and rhythmic stretch induces A2B activation, PKA/MAPK/IP3-dependent transcription, and PKA-dependent secretion of 5-HT synthesis and secretion. Receptor expression is amplified in IBD and neoplasia, and 5-HT release is increased. Determination of factors that regulate EC cell function are necessary for understanding its role as a mechanosensory cell and to facilitate the development of agents that can selectively target cell function in EC cell-associated disease.

An in vitro screening method for probiotics with antidepressant-like effect using the enterochromaffin cell model.

Certain probiotics can regulate the host's neurobehavioral function through the microbiota-gut-brain axis. However, screening these probiotics is mainly carried out in animal models, and is costly and inefficient. Herein, a putative enterochromaffin cell line (RIN14B) was used as an in vitro pre-screening model; 30 bacterial strains were tested for bacteria-stimulated tryptophan hydroxylase 1 gene (Tph1) expression and 5-hydroxytryptophan/5-hydroxytryptamine secretion. All strains were further validated for their neurobehavioral effects in chronic stress-induced depressive mice. Using partial least squares (PLS) modeling of in vitro and in vivo datasets, we found that the level of Tph1 mRNA in RIN14B significantly correlated with the performance of a forced swim test and sucrose preference test, and serum corticosterone level in chronically stressed mice. Four strains were identified as the best candidates among 30 strains using principal component analysis on all in vivo measures, and unsurprisingly, three of them could enhance Tph1 expression in RIN14B, which further proved that the RIN14B-based screening method (especially the detection of bacteria-stimulated Tph1 mRNA) has good predictive validity and screening efficiency for the strain's antidepressant-like capacity. Collectively, this study provides a novel in vitro method for screening probiotics (or other related bioproducts) with antidepressant-like potential.

The outer membrane protein Amuc_1100 of Akkermansia muciniphila promotes intestinal 5-HT biosynthesis and extracellular availability through TLR2 signalling.

Akkermansia muciniphila is a probiotic inhabiting host intestinal mucus layers and displays evident easing or therapeutic effects on host enteritis and metabolic disorders such as obesity and diabetes. The outer membrane protein Amuc_1100 of A. muciniphila is likely to play a crucial role during the interaction with the host. 5-HT is a neurotransmitter and a key signal molecule regulating the gastrointestinal tract functions and other organs, which is involved in diverse physiological and pathological processes. This study demonstrated that Amuc_1100 could promote the expression of the 5-HT synthesis rate-limiting enzyme Tph1 in RIN-14B cells and reduce the expression of the serotonin reuptake transporter (SERT) in Caco-2 cells through direct interaction with TLR2, thereby improving 5-HT biosynthesis and extracellular availability. Using antibiotic-treated mice as animal models, we found that after gavage with A. muciniphila or Amuc_1100, Tph1 expression increased and SERT expression decreased in colon tissues. The 5-HT concentrations in colon tissues and blood were markedly elevated simultaneously. We also found that A. muciniphila or Amuc_1100 improved the gastrointestinal motility function and restored gut microbiota abundance and species diversity in antibiotic-treated mice. These results suggest that A. muciniphila can regulate the host intestinal 5-HT system via its outer membrane protein Amuc_1100 and TLR2. This mechanism represented an important approach through which A. muciniphila interacts with the host and further influences 5-HT-related physiological functions. These results advance the understanding of interplay mechanisms between the gut microbiota and the host, which could be the basis for new intervention strategies for related diseases.

Identification and molecular study on the interaction of Schisandrin C with human 5-HT3A receptor.

Schisandrin C (Sch C) is one of the main components of Schisandra chinensis (Schisandra). Since the olden times, Schisandra has been used as a traditional herbal medicine in Asia. Recent studies have shown that Schisandra is effective against irritable bowel syndrome (IBS) in an animal model and affects IBS through the 5-HT3A pathway in the IBS rat model. However, there lacks fundamental research on the interaction of specific components of Schisandra with the 5-HT3A receptor for the treatment of IBS. We hypothesized that a component of Schisandra binds to the 5-HT3A receptor and identified Sch C via a screening work using two electrode-voltage clamps (TEVC). Thus, we aimed to elucidate the neuropharmacological actions between Sch C and the 5-HT3A receptor at molecular and cellular levels. Co-treatment of Sch C with 5-HT inhibited I5-HT in a reversible, concentrate-dependent, like-competition, and voltage-independent manner, and IC50 values of Sch C. Besides, the main binding positions of Sch C were identified through 3D modeling and point mutation were V225A and V288Y on 5-HT3A receptor. Thus, we suggest the potential of Sch C in treating IBS in a manner that suppresses excessive neuronal serotonin signaling in the synapse of sensory neurons and enterochromaffin (EC) cells. In conclusion, the results demonstrate the mechanism of interaction between Sch C and 5-HT3A receptor and reveal Sch C as a novel antagonist.

KRJ-I and BON cell lines: defining an appropriate enterochromaffin cell neuroendocrine tumor model.

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.

Effects of serotonin on acid secretion in isolated rat stomach: the role of 5-HT3 receptors.

Serotonin (5-hydroxytryptamin; 5-HT) content has been measured using high-performance liquid chromatography with electrochemical detection (HPLC-ECD). The distributions of 5-HT-containing cells and 5-HT3 receptors have been determined with specific antibodies against 5-HT and 5-HT3 receptors, respectively. The effect of serotonin on acid secretion has been studied using an isolated rat stomach model. It has been shown that 5-HT concentrations in the fundus, mucosal layers of the corpus, remaining layer of the corpus and antrum are approximately 152, 498, 1494 and 972 nmol/mg protein, respectively. The distribution of 5-HT-containing cells is concentrated in the enteric plexus and enterochromaffin (EC) cells in the deep mucosal layer. Immunoreactivity to 5-HT3 receptors is localized in numerous neurons of the myenteric and submucosal plexus and concentrated in the neuronal plasma membrane, submucosa, endocrine cells and lamina propria. In the present study, the effect of 5-HT on gastric acid secretion was investigated on an everted preparation of isolated rat stomach. 5-HT at 1-100 microM reduced acid secretion stimulated by oxotremorine while 10 microM 5-HT did not modify the basal secretion of gastric acid. We further showed that 10 microM 5-HT reduced acid secretion and pepsin output stimulated by oxotremorine, histamine and pentagastrin; among the 5-HT receptors agonists tested, 2-methyl-5-HT (1-10 microM), a 5-HT3 receptor agonist, inhibited oxotremorine-, histamine- and pentagastrin-stimulated acid secretions, and this inhibitory effect was blocked by 1 microM MDL 72222, a specific 5-HT3 receptor antagonist. These results suggest that 5-HT is released from serotoninergic neurons, their processes and EC cells. The effect of 5-HT mediated by 5-HT3 receptors involves distinct neuronal and non-neuronal pathways which modulate gastric acid secretion.

Sugar Responses of Human Enterochromaffin Cells Depend on Gut Region, Sex, and Body Mass.

Gut-derived serotonin (5-HT) is released from enterochromaffin (EC) cells in response to nutrient cues, and acts to slow gastric emptying and modulate gastric motility. Rodent studies also evidence a role for gut-derived 5-HT in the control of hepatic glucose production, lipolysis and thermogenesis, and in mediating diet-induced obesity. EC cell number and 5-HT content is increased in the small intestine of obese rodents and human, however, it is unknown whether EC cells respond directly to glucose in humans, and whether their capacity to release 5-HT is perturbed in obesity. We therefore investigated 5-HT release from human duodenal and colonic EC cells in response to glucose, sucrose, fructose and α-glucoside (αMG) in relation to body mass index (BMI). EC cells released 5-HT only in response to 100 and 300 mM glucose (duodenum) and 300 mM glucose (colon), independently of osmolarity. Duodenal, but not colonic, EC cells also released 5-HT in response to sucrose and αMG, but did not respond to fructose. 5-HT content was similar in all EC cells in males, and colonic EC cells in females, but 3 to 4-fold higher in duodenal EC cells from overweight females (p < 0.05 compared to lean, obese). Glucose-evoked 5-HT release was 3-fold higher in the duodenum of overweight females (p < 0.05, compared to obese), but absent here in overweight males. Our data demonstrate that primary human EC cells respond directly to dietary glucose cues, with regional differences in selectivity for other sugars. Augmented glucose-evoked 5-HT release from duodenal EC is a feature of overweight females, and may be an early determinant of obesity.

Clostridium ramosum regulates enterochromaffin cell development and serotonin release.

Peripheral serotonin (5-hydroxytryptamine: 5-HT) synthesized in the intestine by enterochromaffin cells (ECs), plays an important role in the regulation of peristaltic of the gut, epithelial secretion and promotes the development and maintenance of the enteric neurons. Recent studies showed that the indigenous gut microbiota modulates 5-HT signalling and that ECs use sensory receptors to detect dietary and microbiota-derived signals from the lumen to subsequently transduce the information to the nervous system. We hypothesized that Clostridium ramosum by increasing gut 5-HT availability consequently contributes to high-fat diet-induced obesity. Using germ-free mice and mice monoassociated with C. ramosum, intestinal cell lines and mouse organoids, we demonstrated that bacterial cell components stimulate host 5-HT secretion and program the differentiation of colonic intestinal stem progenitors toward the secretory 5-HT-producing lineage. An elevated 5-HT level regulates the expression of major proteins involved in intestinal fatty acid absorption in vitro, suggesting that the presence of C. ramosum in the gut promotes 5-HT secretion and thereby could facilitates intestinal lipid absorption and the development of obesity.