Raquel Martín-Venegas

Arachidonic acid cascade and epithelial barrier function during Caco-2 cell differentiation

The small intestinal epithelium is a highly dynamic system continuously renewed by a process involving cell proliferation and differentiation. The intestinal epithelium constitutes a permeability barrier regulating the vectorial transport of ions, water, and solutes. Morphological changes during cell differentiation, as well as changes in the activity of brush-border enzymes and the expression of transport proteins, are well established. However, little is known about the arachidonic acid (AA) cascade underlying epithelial cell differentiation or its role in the development of epithelial barrier function. The main purpose of this study was to examine the activity of the high-molecular-weight phospholipases A2 (PLA2) and cyclooxygenase (COX) pathway during differentiation, with particular emphasis on paracellular permeability. PLA2 activity, AA release, COX-2 expression, prostaglandin E2 (PGE2) production, and paracellular permeability were studied in preconfluent, confluent, and differentiated Caco-2 cell cultures. Our results show that Caco-2 differentiation induces a decrease in both calcium-independent PLA2 activity and COX-2 expression and, consequently, a decrease in AA release and PGE2 synthesis in parallel with a reduction in paracellular permeability. Moreover, the addition of PGE2 to differentiated cells, at concentrations similar to those detected in nondifferentiated cultures, induces the disruption of epithelial barrier function. These results suggest that AA release by calcium-independent PLA2, COX-2 expression, and subsequent PGE2 release are important for the maintenance of paracellular permeability in differentiated Caco-2 cells.

PGE2 promotes Ca2+-mediated epithelial barrier disruption through EP1 and EP4 receptors in Caco-2 cell monolayers

We recently demonstrated that PGE(2) induces the disruption of the intestinal epithelial barrier function. In the present study, our objectives were to study the role of PGE(2) receptors (EP(1)-EP(4)) and the signaling pathways involved in this event. Paracellular permeability (PP) was assessed in differentiated Caco-2 cell cultures from d-mannitol fluxes and transepithelial electrical resistance (TER) in the presence of different PGE(2) receptor agonists (carbacyclin, sulprostone, butaprost, ONO-AE1-259, ONO-AE-248, GR63799, and ONO-AE1-329) and antagonists (ONO-8711, SC-19220, AH-6809, ONO-AE3-240, ONO-AE3-208, and AH-23848). The results indicate that EP(1) and EP(4) but not EP(2) and EP(3) might be involved in PP regulation. These effects were mediated through PLC-inositol trisphosphate (IP(3))-Ca(2+) and cAMP-PKA signaling pathways, respectively. We also observed an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) strengthened by cAMP formation indicating a cross talk interaction of these two pathways. Moreover, the participation of a conventional PKC isoform was shown. The results also indicate that the increase in PP may be correlated with the redistribution of occludin, zona occludens 1 (ZO-1), and the perijunctional actin ring together with an increase in myosin light chain kinase activity. Although the disruption of epithelial barrier function observed in inflammatory bowel disease (IBD) patients has been traditionally attributed to cytokines, the present study focused on the role of PGE(2) in PP regulation, as mucosal levels of this eicosanoid are also increased in these inflammatory processes.

β-Galactomannan and Saccharomyces cerevisiae var. boulardii Modulate the Immune Response against Salmonella enterica Serovar Typhimurium in Porcine Intestinal Epithelial and Dendritic Cells

ABSTRACT Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that causes inflammation, necrosis, and diarrhea in pigs, as well as being an important source of food-borne diseases in humans. Probiotics and prebiotics are promising alternatives to antibiotics to control and prevent intestinal infections. The present work investigated a recently developed β-galactomannan (βGM) prebiotic compared to the proven probiotic Saccharomyces cerevisiae var. boulardii on porcine ileum intestinal epithelial cells (IECs) of the IPI-2I line and monocyte-derived dendritic cells (DCs) cocultured in vitro with Salmonella. We observed that both S. cerevisiae var. boulardii and βGM inhibited the association of Salmonella with IECs in vitro. Our data indicated that βGM has a higher ability than S. cerevisiae var. boulardii to inhibit Salmonella-induced proinflammatory mRNA (cytokines tumor necrosis factor alpha [TNF-α], interleukin-1α [IL-1α], IL-6, and granulocyte-macrophage colony-stimulating factor [GM-CSF] and chemokines CCL2, CCL20, and CXCL8) and at protein levels (IL-6 and CXCL8). Additionally, βGM and S. cerevisiae var. boulardii induced some effects on DCs that were not observed on IECs: βGM and S. cerevisiae var. boulardii showed slight upregulation of mRNA for TNF-α, GM-CSF, and CCR7 receptor on porcine monocyte-derived dendritic cells (DCs). Indeed, the addition of βGM or S. cerevisiae var. boulardii on DCs cocultured with Salmonella showed higher gene expression (mRNA) for TNF-α, GM-CSF, and CXCL8 compared to that of the control with Salmonella. In conclusion, the addition of βGM inhibits Salmonella-induced proinflammatory profiles in IECs but may promote DC activation, although associated molecular mechanisms remain to be elucidated.

Rapid simultaneous analysis of cyclooxygenase, lipoxygenase and cytochrome P-450 metabolites of arachidonic and linoleic acids using high performance liquid chromatography/mass spectrometry in tandem mode.

Eicosanoids are oxidized arachidonate-derived lipid products generated by cyclooxygenase, lipoxygenase and cytochrome P-450 pathways. They are involved in diverse processes in health and disease and they are highly bioactive. Gas chromatography and enzyme immunoassays were used to quantify these mediators in the past. However, the recent availability of high-sensitivity liquid chromatography-mass spectrometry has provided a new approach for quantification that minimizes the sample size and the required preparation. This paper describes a rapid and simple technique for the simultaneous quantitative analysis of prostaglandin (PG) E(2) and PGJ(2); leukotrienes (LT) B(4) and D(4); 5-, 12-, 15- and 20-hydroxyeicosatetraenoic acids (HETEs); 13-hydroxyoctadecadienoic acid (13-HODE); 5,6-, 8,9-, 11,12- and 14,15-epoxyeicosatrienoic acids (EETs); and 11,12- and 14,15-dihydroxieicosatrienoic acids (DHETs) in cell culture supernatants and urine. We simultaneously analyzed 14 arachidonic acid metabolites representative from the three pathways, together with 13-HODE, a linoleic-derived product. Solid phase extraction was used for the sample preparation. The recoveries obtained ranged from 25% to 100%, depending on the metabolites. The LC/MS/MS method used the gradient on a C(18) column and electrospray ionization in negative ion detection mode. The method was optimized for sensitivity and for separation within 20 min. The linear ranges of the calibration curves were 0.1-200 ng/ml for PGE(2), PGJ(2), LTB(4), 5-HETE, 12-HETE, 15-HETE, 13-HODE, 11,12-EET, 11,12-DHET and 14,15-DHET, and 1-200ng/ml for LTD(4), 20-HETE, 5,6-EET, 8,9-EET and 14,15-EET. The advantages of this method include minimal sample preparation, high sensitivity and elimination of the problem associated with thermal instability in gas chromatography analysis.

Effect of pH on l- and d-methionine uptake across the apical membrane of Caco-2 cells

The transport systems involved in intestinal methionine (Met) absorption are described as Na(+)-dependent and Na(+)-independent mechanisms. However, since recent studies have suggested the importance of the H(+) gradient as a driving force for intestinal nutrient absorption, the aim of the present work was to test whether Met transport across the apical membrane of Caco-2 cells is affected by extracellular pH. The results show that l- and d-Met uptake was increased by lowering extracellular pH from 7.4 to 5.5, in both the presence and absence of Na(+). Cis-inhibition experiments revealed that inhibition of l-Met transport by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) or l-lysine (l-Lys) was higher at a pH of 5.5. Moreover, the BCH-insensitive component was not affected by pH, whereas the l-Lys-insensitive component was increased by lowering extracellular pH, thus suggesting the participation of system L. The contribution of another mechanism, sensitive to both BCH and l-Lys, was also considered. The inhibition obtained with taurine (Tau) was also higher at a pH of 5.5, thus suggesting the involvement of system B(0,+) on pH-stimulated component. As for d-Met uptake, the results showed higher inhibition with l-Lys and Tau at a pH of 5.5 and no effect on the l-Lys- or Tau-insensitive component. In conclusion, Met transport across the apical membrane of Caco-2 cells is increased by low extracellular pH as the result of the stimulation of two transport systems functionally identified with systems L and B(0,+) for l-Met and with system B(0,+) for d-Met.

Role of arachidonic acid metabolites on the control of non-differentiated intestinal epithelial cell growth

Increasingly evidence indicates that enzymes, receptors and metabolites of the arachidonic acid (AA) cascade play a role in intestinal epithelial cell proliferation and colorectal tumorigenesis. However, the information available does not provide a complete picture and contains a number of discrepancies. For this reason it might be appropriate a thorough study into the impacts of the AA cascade on intestinal epithelial cell growth. Our data show that non-differentiated Caco-2 cells cultured with 10% fetal bovine serum (FBS) synthesize appreciable amounts of prostaglandin E2 (PGE2), leukotriene B4 (LTB4) and 5-, 12 and 15-hydroxyeicosatetraenoic acid (HETE) but not LTD4, 20-HETE and epoxyeicosatrienoic acids. We also found that inhibitors of PGE2, LTB4 and 5-, 12-, 15-HETE synthesis as well as receptor antagonists of PGE2 and LTB4 blocked Caco-2 cell growth and DNA synthesis induced by 10% FBS without cytotoxic or apoptotic activity. Interestingly, PGE2, LTB4 and 5-, 12- and 15-HETE at concentrations reached in 10% FBS Caco-2 cultures (1-10nM) were able to induce Caco-2 cell growth and DNA synthesis. This was due to the interaction of PGE2 with EP1 and EP4 receptors and LTB4 and HETEs with BLT1 and BLT2 receptors. Moreover, we provide evidence that PGE2 stimulates several cell signaling pathways such as ERK, P38α, CREB and GSKβ/β-catenin involved in the regulation of Caco-2 growth. Finally, we provide evidence that the mitogenic effects of LTB4 and HETEs can be dependent, at least in part, on PGE2 synthesis.

Liquid chromatography-tandem mass spectrometry analysis of eicosanoids and related compounds in cell models☆

Enzyme- and free radical-catalyzed oxidation of polyunsaturated fatty acids (PUFAs) produces the eicosanoids, docosanoids and octadecanoids. This large family of potent bioactive lipids is involved in many biochemical and signaling pathways which are implicated in physiological and pathophysiological processes and can be viable therapeutic targets. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers selectivity, sensitivity, robustness and high resolution and is able to analyze a large number of eicosanoids in biological samples in a short time. The present article reviews and discusses reported LC-MS/MS methods and the results obtained from their application in cell models. Reliable analytical outcomes are critically important for understanding physiological and pathophysiological cellular processes, such as inflammation, diseases with inflammatory components (e.g., cardiovascular disease, diabetes, metabolic syndrome), as well as cancer. Reported findings obtained by using the LC-MS/MS methodology in cell systems may have important predictive as well as nutritional and pharmacological implications. We conclude that the LC-MS/MS methodology is a versatile and reliable analytical tool for the simultaneous analysis of multiple PUFA-derived metabolites including the eicosanoids in cell culture samples at concentrations on the pM/nM threshold, i.e. at baseline and after stimulation.

The methionine precursor DL-2-hydroxy-(4-methylthio)butanoic acid protects intestinal epithelial barrier function.

DL-2-hydroxy-(4-methylthio)butanoic acid (HMTBA) is a source of dietary methionine (Met) that is widely used in poultry nutrition. We have previously shown that HMTBA is preferentially diverted to the transsulfuration pathway, which gives antioxidant metabolites such as taurine and glutathione. Therefore, here we hypothesize that this Met source can protect epithelial barrier function in an in vitro model of intestinal inflammation of Caco-2 cells. The results show that HMTBA prevents the increase in paracellular permeability induced by H2O2 or tumour necrosis factor-α. This effect can be attributed to the increased production of taurine and reduced glutathione. Similar results were obtained for DL-Met, although the protective role of the amino acid was less pronounced than that of the hydroxy analogue. In conclusion, the diversion to the transsulfuration pathway means that this Met precursor is of greater value than previously thought, due to its capacity to improve intestinal homeostasis and the quality of poultry products destined for human consumption.

In vitro study of the cytotoxicity and antiproliferative effects of surfactants produced by Sphingobacterium detergens

The application of biosurfactants in the biomedical field is growing due to their antimicrobial activity, low cytotoxicity and ability to induce apoptosis in cancer cells. In the light of this therapeutic potential, as well as possible applications in cosmetics or as drug vehicles in pharmaceutical products, a new biosurfactant produced by Sphingobacterium detergens was investigated for its haemolytic activity and cytotoxic and antiproliferative effects in different cell lines. Fraction A showed 100% haemolysis in rabbit erythrocytes, but in Fraction B the rate was only 83%. When comparing cytotoxicity values (IC50) of the two fractions in model fibroblast and keratinocyte cell cultures, Fraction B was less cytotoxic, showing lower values than the reference compound SDS, indicating low skin irritability. Finally, in non-differentiated intestinal Caco-2 cultures, Fractions A and B reduced cell proliferation and induced apoptosis by 44% and 75%, respectively. According to these results, biosurfactants produced by S. detergens have potential application in cosmetic and pharmaceutical formulations.

Intestinal cell conversion of DL-2-hydroxy-(4-methylthio)butanoic acid in vitro: dietary up-regulation by this methionine precursor.

DL-2-Hydroxy-(4-methylthio)butanoic acid (HMTBA) is a synthetic source of dietary methionine (Met) widely used in poultry nutrition. HMTBA is transported in the intestinal epithelium by the monocarboxylate transporter 1, after which its biological utilisation relies on its conversion to L-Met. This process involves stereospecific HMTBA oxidation to 2-keto-(4-methylthio)butanoic acid (KMB) and transamination to L-Met. In the present study, we examined HMTBA conversion to L-Met, further incorporation into cellular proteins and the regulation of both processes by HMTBA supplementation in differentiated intestinal Caco-2 cells. The results showed D- and L-HMTBA oxidation in the enterocytes, this process being up-regulated by HMTBA. The data also revealed that KMB transamination is not linked to a specific amino group donor. However, the branched-chain amino acid L-leucine is the preferred amino group donor. Furthermore, transamination was not affected by HMTBA availability. The incorporation of radioactivity from HMTBA into cellular proteins was not significantly different from that of L-Met and was not affected by HMTBA supplementation. In conclusion, the results reveal the capacity of Caco-2 cells to convert HMTBA to L-Met and the up-regulation of conversion by nutritional HMTBA supplementation, thus highlighting the contribution of the intestinal epithelium in the utilisation of HMTBA as a dietary source of Met.