Gastric Serotonin Biosynthesis and Its Functional Role in L-Arginine-Induced Gastric Proton Secretion.

Among mammals, serotonin is predominantly found in the gastrointestinal tract, where it has been shown to participate in pathway-regulating satiation. For the stomach, vascular serotonin release induced by gastric distension is thought to chiefly contribute to satiation after food intake. However, little information is available on the capability of gastric cells to synthesize, release and respond to serotonin by functional changes of mechanisms regulating gastric acid secretion. We investigated whether human gastric cells are capable of serotonin synthesis and release. First, HGT-1 cells, derived from a human adenocarcinoma of the stomach, and human stomach specimens were immunostained positive for serotonin. In HGT-1 cells, incubation with the tryptophan hydroxylase inhibitor p-chlorophenylalanine reduced the mean serotonin-induced fluorescence signal intensity by 27%. Serotonin release of 147 ± 18%, compared to control HGT-1 cells (set to 100%) was demonstrated after treatment with 30 mM of the satiating amino acid L-Arg. Granisetron, a 5-HT3 receptor antagonist, reduced this L-Arg-induced serotonin release, as well as L-Arg-induced proton secretion. Similarly to the in vitro experiment, human antrum samples released serotonin upon incubation with 10 mM L-Arg. Overall, our data suggest that human parietal cells in culture, as well as from the gastric antrum, synthesize serotonin and release it after treatment with L-Arg via an HTR3-related mechanism. Moreover, we suggest not only gastric distension but also gastric acid secretion to result in peripheral serotonin release.

Bitter Sensing TAS2R50 Mediates the trans-Resveratrol-Induced Anti-inflammatory Effect on Interleukin 6 Release in HGF-1 Cells in Culture.

Recent data have shown anti-inflammatory effects for trans-resveratrol (RSV) and rosmarinic acid (RA) in various immune-competent cell models through reduction of lipopolysaccharide (LPS)-induced interleukin 6 (IL-6) release. Because both compounds have been reported to taste bitter, we hypothesized an involvement of human bitter taste sensing receptors (TAS2Rs) on IL-6 release in LPS-treated human gingival fibroblasts (HGF-1). First, the bitter taste intensity of RSV and RA was compared in a sensory trial with 10 untrained panelists, of whom 90% rated a 50 ppm of RSV in water solution more bitter than 50 ppm of RA. A mean 19 ± 6% reduction of the RSV-induced bitter taste intensity was achieved by co-administration of 50 ppm of the bitter-masking, TAS2R43 antagonist homoeriodictyol (HED). Mechanistic experiments in a stably CRISPR-Cas9-edited TAS2R43ko gastric cell model revealed involvement of TAS2R43 in the HED-evoked effect on RSV-induced proton secretion, whereas the cellular response to RSV did not depend upon TAS2R43. Next, the IL-6 modulatory effect of 100 µM RSV was studied in LPS-treated immune-competent HGF-1 cells. After 6 h of treatment, RSV reduced the LPS-induced IL-6 gene expression and protein release by -46.2 ± 12.7 and -73.9 ± 2.99%, respectively. This RSV-evoked effect was abolished by co-administration of HED. Because real-time quantitative polymerase chain reaction analyses revealed a regulation of TAS2R50 in RSV with or without HED-treated HGF-1 cells, an siRNA knockdown approach of TAS2R50 was applied to verify TAS2R50 involvement in the RSV-induced reduction of the LPS-evoked IL-6 release in HGT-1 cells.

Impact of free Nε-carboxymethyllysine, its precursor glyoxal and AGE-modified BSA on serotonin release from human parietal cells in culture.

Advanced glycation end products (AGEs) are frequently encountered in a western diet, in addition to their formation in vivo. N-Epsilon-carboxymethyllysine (CML), one of the chemically diverse compounds formed in the reaction between reducing carbohydrates and amines, is often used as a marker of advanced glycation, and has been shown to stimulate serotonin release from cells representing the central (SH-SY5Y cells) and the peripheral (Caco-2 cells) serotonin system in vitro. Here, we investigated the effect of glyoxal, free CML, and protein-linked AGE-BSA on serotonin release from human gastric tumour cells, which originate from an adenocarcinoma of the stomach and have recently been shown to be capable of serotonin synthesis and release. Microarray experiments showed both CML and glyoxal to alter genes associated with serotonin receptors. Furthermore, treatment with glyoxal resulted in a small change in RAGE expression while CML did not alter its expression. On a functional level, treatment with 500 µM CML increased extracellular serotonin content by 341 ± 241%, while treatment with 1 mg mL-1 AGE-BSA led to a reduction by 49 ± 11% compared to non-treated cells. The CML-induced serotonin release was reduced by the HTR3 antagonist granisetron. Incubation with the RAGE antagonist FPS-ZM1 abolished the effect of AGE-BSA on serotonin release, while no impact on CML-induced serotonin release was observed. Furthermore, treatment with 5 mM CML stimulated proton secretion as a functional outcome measure, assessed using a pH sensitive dye. Taken together, these results indicate a likely HTR3-mediated, RAGE-independent effect of free CML on serotonin release and a RAGE-dependent mechanism for the protein linked AGE-BSA.

Noncaloric Sweeteners Induce Peripheral Serotonin Secretion via the T1R3-Dependent Pathway in Human Gastric Parietal Tumor Cells (HGT-1).

The role of sweet taste in energy intake and satiety regulation is still controversial. Noncaloric artificial sweeteners (NCSs) are thought to help reduce energy intake, although little is known about their impact on the satiating neurotransmitter serotonin (5-HT). In the gastrointestinal (GI) tract, 5-HT regulates gastric acid secretion and gastric motility, both part of the complex network of mechanisms regulating food intake and satiety. This study demonstrated a stimulating impact compared to controls (100%) on 5-HT release in human gastric tumor cells (HGT-1) by the NCSs cyclamate (50 mM, 157% ± 6.3%), acesulfame potassium (Ace K, 50 mM, 197% ± 8.6%), saccharin (50 mM, 147% ± 6.7%), sucralose (50 mM, 194% ± 11%), and neohesperidin dihydrochalcone (NHDC, 1 mM, 201% ± 13%). Although these effects were not associated with the sweet taste intensity of the NCSs tested, involvement of the sweet receptor subunit T1R3 in the NCS-evoked response was demonstrated by mRNA expression of TAS1R3, co-incubation experiments using the T1R3 receptor antagonist lactisole, and a TAS1R3 siRNA knockdown approach. Analysis of the downstream signaling revealed activation of the cAMP/ERK/Ca2+ cascade. Co-treatment experiments with 10 mM glucose enhanced the 5-HT release induced by cyclamate, Ace K, saccharin, and sucralose, thereby supporting the enhancing effect of glucose on a NCS-mediated response. Overall, the results obtained identify NCSs as potent inducers of 5-HT release via T1R3 in human gastric parietal cells in culture and warrant in vivo studies to demonstrate their efficacy.

The flavanone homoeriodictyol increases SGLT-1-mediated glucose uptake but decreases serotonin release in differentiated Caco-2 cells.

Flavanoids and related polyphenols, among them hesperitin, have been shown to modulate cellular glucose transport by targeting SGLT-1 and GLUT-2 transport proteins. We aimed to investigate whether homoeriodictyol, which is structurally related to hesperitin, affects glucose uptake in differentiated Caco-2 cells as a model for the intestinal barrier. The results revealed that, in contrast to other polyphenols, the flavanon homoeriodictyol promotes glucose uptake by 29.0 ± 3.83% at a concentration of 100 µM. The glucose uptake stimulating effect was sensitive to phloridzin, but not to phloretin, indicating an involvement of the sodium-coupled glucose transporter SGLT-1, but not of sodium-independent glucose transporters (GLUT). In addition, in contrast to the increased extracellular serotonin levels by stimulation with 500 mM D-(+)-glucose, treatment with 100 µM homoeriodictyol decreased serotonin release by -48.8 ± 7.57% in Caco-2 cells via a phloridzin-sensitive signaling pathway. Extracellular serotonin levels were also reduced by -57.1 ± 5.43% after application of 0.01 µM homoeriodictyol to human neural SH-SY5Y cells. In conclusion, we demonstrate that homoeriodictyol affects both the glucose metabolism and the serotonin system in Caco-2 cells via a SGLT-1-meditated pathway. Furthermore, the results presented here support the usage of Caco-2 cells as a model for peripheral serotonin release. Further investigations may address the value of homoeriodictyol in the treatment of anorexia and malnutrition through the targeting of SGLT-1.

Express photolithographic DNA microarray synthesis with optimized chemistry and high-efficiency photolabile groups.

BACKGROUND: DNA microarrays are a core element of modern genomics research and medical diagnostics, allowing the simple and simultaneous determination of the relative abundances of hundreds of thousands to millions of genomic DNA or RNA sequences in a sample. Photolithographic in situ synthesis, using light projection from a digitally-controlled array of micromirrors, has been successful at both commercial and laboratory scales. The advantages of this synthesis method are its ability to reliably produce high-quality custom microarrays with a very high spatial density of DNA features using a compact device with few moving parts. The phosphoramidite chemistry used in photolithographic synthesis is similar to that used in conventional solid-phase synthesis of oligonucleotides, but some unique differences require an independent optimization of the synthesis chemistry to achieve fast and low-cost synthesis without compromising microarray quality. RESULTS: High microarray quality could be maintained while reducing coupling time to a few seconds using DCI activator. Five coupling activators were compared, which resulted in microarray hybridization signals following the order ETT > Activator 42 > DCI â‰« BTT â‰« pyridinium chloride, but only the use of DCI led to both high signal and highly uniform feature intensities. The photodeprotection time was also reduced to a few seconds by replacing the NPPOC photolabile group with the new thiophenyl-NPPOC group. Other chemical parameters, such as oxidation and washing steps were also optimized. CONCLUSIONS: Highly optimized and microarray-specific phosphoramidite chemistry, along with the use of the very photosensitive thiophenyl-NPPOC protecting group allow for the synthesis of high-complexity DNA arrays using coupling times of 15 s and deprotection times of 9 s. The resulting overall cycle time (coupling to coupling) of about 50 s, results in a three-fold reduction in synthesis time.

Nonivamide, a capsaicin analog, increases dopamine and serotonin release in SH-SY5Y cells via a TRPV1-independent pathway.

SCOPE: Dietary intake of capsaicin has been shown to reduce body weight by increasing energy expenditure, and to enhance alertness and mood by stimulating the brain's reward system. Binding of capsaicin to the vanilloid receptor 1 (transient receptor potential cation channel subfamily V member 1 (TRPV1)) is one of the major cellular mechanisms responsible for these effects. However, strong TRPV1 agonists like capsaicin elicit a sharp, burning pain that limits their dietary intake. The present study aimed to investigate the effect of the less pungent capsaicin-analog nonivamide on dopamine and serotonin release in neural SH-SY5Y cells. METHODS AND RESULTS: Nonivamide (1 µM) stimulated the Ca(2+) -dependent release of serotonin (272 ± 115%) and dopamine (646 ± 48%) in SH-SY5Y cells compared to nontreated cells (100%) to a similar extent as capsaicin. qRT-PCR analysis of 1 µM nonivamide-treated SH-SY5Y cells revealed gene regulation of the receptors dopamine D1 and D2, serotonin HTR1A, 1B and 2A, cannabinoid 1, and TRPV1. Co-incubation experiments of SH-SY5Y cells with the TRPV1 inhibitors trans-tert-butylcyclohexanol and capsazepine demonstrated that capsaicin, but not nonivamide, induces serotonin and dopamine release through TRPV1 activation. CONCLUSION: The results indicate a TRPV1-independent signaling pathway for nonivamide that might allow dietary administration of higher doses of nonivamide compared to capsaicin.

N(ε)-Carboxymethyllysine (CML), a Maillard reaction product, stimulates serotonin release and activates the receptor for advanced glycation end products (RAGE) in SH-SY5Y cells.

Maillard reaction products, which are formed in highly thermally treated foods, are commonly consumed in a Western diet. In this study, we investigated the impact of N(ε)-carboxymethyllysine (CML), a well-characterized product of the Maillard reaction, on the gene regulation of the human neuroblastoma cell line SH-SY5Y. Pathway analysis of data generated from customized DNA microarrays revealed 3 h incubation with 50 µM and 500 µM CML to affect serotonin receptor expression. Further experiments employing qRT-PCR showed an up-regulation of serotonin receptors 2A, 1A and 1B after 0.25 h and 3 h. In addition, 500 µM CML increased serotonin release, thus showing effects of CML not only at a genetic, but also at a functional level. Intracellular calcium mobilization, which mediates serotonin release, was increased by CML at concentrations of 0.05-500 µM. Since calcium mobilization has been linked to the activation of the receptor for advanced glycation end products (RAGE), we further investigated the effects of CML on RAGE expression. RAGE was found to be up-regulated after incubation with 500 µM CML for 0.25 h. Co-incubation with the calcium blocker neomycin for 0.25 h blocked the up-regulation of RAGE and the serotonin receptors 2A, 1A and 1B. These results indicate a possible link between a CML-induced calcium-mediated serotonin release and RAGE.