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.

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.

In vitro combinatory effects of the Alternaria mycotoxins alternariol and altertoxin II and potentially involved miRNAs.

Alternariol (AOH) and altertoxin II (ATX II) are mycotoxins formed by Alternaria spp. Since they are expected to co-occur in Alternaria-infested food and feed, we addressed the question of combinatory effects. In addition, potentially involved regulatory microRNAs were surveyed in an exploratory approach. Cytotoxicity measurements in constant ratio combinations of 1:10 or 1:1 (ATX II: AOH) mainly revealed additive effects in HepG2, HT29 and HCEC-1CT cells. Yet, in specific high doses antagonism was found. Microarray analysis of miRNA expression profiles in HepG2 cells indicated different patterns of miRNA regulation by AOH and ATX II, including several miRNA species for which no distinct functions are currently known. Among others, miR-4654, miR-4715_3p and miR-6720_3p were up-regulated by AOH and miR-5583_5p was down-regulated by ATX II. Additionally, miR-1323, involved in hindering DNA repair mechanisms, was decreased by ATX II. Digital droplet PCR (ddPCR) analysis of selected miRNAs indicated regulation of miR-29a by AOH, which might play a role in AOH-induced apoptosis. miR-192 and miR-224 regulation was associated with antagonistic cytotoxic effects of AOH and ATX II combinations. Our study represents the first evaluation on combinatory effects of AOH and ATX II.

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.

Multi-parametric approach to identify coffee components that regulate mechanisms of gastric acid secretion.

SCOPE: Chlorogenic acid (CA), caffeine (CAFF), pyrogallol (PYR), catechol (CAT), (ß)N-alkanoyl-hydroxytryptamides (C5HT) and N-methylpyridinium (N-MP) were evaluated for their influence on mechanisms of gastric acid secretion as single compounds and in biomimetic mixtures. METHODS AND RESULTS: Compounds were tested in coffee representative concentrations. Human gastric cancer cells (HGT-1) were used to study the proton secretory activity by Ussing chamber experiments and FACS analysis. For activation of EGFr, Akt1, ERK1/2, ATF-2 and cAMP levels, we performed pathway screening assays. Time-dependent expression of related genes were determined by real-time PCR. Part of the data was used for neural network modeling to identify the most relevant compounds. N-MP increased the expression of the anti-secretory somatostatin receptor by 114%, whereas C5HT decreased its expression by 52%. N-MP down-regulated the pro-secretory CHRM3 receptor by 36% and the H⁺,K⁺-ATPase by 36%. CAFF stimulated the secretory activity in the functional assays, whereas N-MP and CA decreased proton secretion. After applying a pathway analysis, we were able to discriminate between CAFF, CA, CAT, C5HT, PYR and histamine-activating EGFr signaling and N-MP-associated ERK1/2 signaling. CONCLUSION: By applying a multi-parametric approach, N-MP was shown to effectively down-regulate mechanisms of gastric acid secretion in human parietal gastric cells.