Ruth Ferrer

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.

Sequential Amino Acid Exchange across b 0,+ -like System in Chicken Brush Border Jejunum

Abstract. In the small intestine, cationic amino acids are transported by y+-like and b0,+-like systems present in the luminal side of the epithelium. Here, we report the characterization of a b0,+-like system in the apical membrane of the chicken jejunum, and its properties as an amino acid exchanger. Analysis of the brush border membrane by Western blot points out the presence of rBAT (protein related to b0,+ amino acid transport system) in these membranes. A functional mechanism for amino acid exchange across this system was established by kinetic analysis measuring fluxes at varying substrate concentrations both in internal (in) and external (out) vesicle compartments. This intestinal b0,+-like system functions for l-arginine as an obligatory exchanger since its transport capacity increases 100–200 fold in exchange conditions, thus suggesting an important role in the intestinal absorption of cationic amino acids. The kinetic analysis of Argin efflux velocities is compatible with the formation of a ternary complex and excludes a model involving a ping-pong mechanism. The binding affinity of Argout is higher than that of Argin, suggesting a possible order of binding (Argout first) for the formation of the ternary complex during the exchange cycle. A model of double translocation pathways with alternating access is discussed.

Lipid peroxidation induced by DHA enrichment modifies paracellular permeability in Caco-2 cells protective role of taurine

Dietary enrichment with docosahexaenoic acid (DHA) has numerous beneficial effects on health. However, the intake of high doses of polyunsaturated fatty acids can promote lipid peroxidation and the subsequent propagation of oxygen radicals. The purpose of this study was to evaluate the effect of DHA on lipid peroxidation and tight junction structure and permeability in Caco-2 cell cultures. Moreover, the effects of taurine, a functional ingredient with antioxidant properties, were also tested. Differentiated Caco-2 cell monolayers were maintained in DHA-supplemented conditions with or without added taurine. Incubation with 100 μM DHA increased lipid peroxidation and paracellular permeability, in parallel with a redistribution of the tight junction proteins occludin and ZO-1. Taurine partially prevented all of these effects. The participation of reactive oxygen and nitrogen species in increased paracellular permeability was also examined using various agents that modify the formation of superoxide radical, hydrogen peroxide, nitric oxide, and peroxynitrite. We conclude that hydrogen peroxide and peroxynitrite may be involved in the DHA-induced increase in paracellular permeability and that the protective role of taurine may be in part related to its capacity to counteract the effects of hydrogen peroxide.

Effect of a lysine-enriched diet onl-lysine transport by the brush-border membrane of the chicken jejunum

The influx ofl-lysine into apical vesicles from the chicken jejunum occurs through two systems, one with low Michaelis constant ( K m) and features of system b0,+ and the other with relatively high K m forl-lysine and with properties of system y+. In the present study the effect of a lysine-enriched diet (Lys, containing 68 g l-lysine/kg dietary protein, control animals 48 g/kg) onl-lysine uptake through both transport systems was investigated. Results show that 1) lysine enrichment had no effect on either body weight or the efficiency of food utilization. 2) In Lys-fed animals, the mediatedl-lysine influx was best fitted to the two-system model with y+and b0,+ activity. 3) In the presence of an Na+ gradient, totall-lysine uptake is significantly higher in Lys-fed animals than in control birds (about 40% increase). 4) Lys diet increases K mb0,+6-fold (KSCN gradient) and 12-fold (NaSCN gradient) and maximum velocity ( V max) by 6- and 20-fold, respectively. The effects of Lys enrichment on the y+-like system are only observed on the V max and in the presence of a Na+ gradient (30% increase). 5) Na+ is involved in the activation of the transport process in the Lys-fed chickens, but there is no correlation between external Na+concentration and l-lysine influx. In conclusion, both b0,+- and y+-like transport systems are upregulated by dietary lysine but with different kinetic profiles; the high-capacity y+-like carrier shows a V maxincrease without changes in K m, whereas the low-capacity b0,+-like system shows an increase in V max as well as in the K m.The influx of L-lysine into apical vesicles from the chicken jejunum occurs through two systems, one with low Michaelis constant (K(m)) and features of system b0,+ and the other with relatively high K(m) for L-lysine and with properties of system y+. In the present study the effect of a lysine-enriched diet (Lys, containing 68 g L-lysine/kg dietary protein, control animals 48 g/kg) on L-lysine uptake through both transport systems was investigated. Results show that 1) lysine enrichment had no effect on either body weight or the efficiency of food utilization. 2) In Lys-fed animals, the mediated L-lysine influx was best fitted to the two-system model with y+ and b0,+ activity. 3) In the presence of an Na+ gradient, total L-lysine uptake is significantly higher in Lys-fed animals than in control birds (about 40% increase). 4) Lys diet increases K(m)b0,+ 6-fold (KSCN gradient) and 12-fold (NaSCN gradient) and maximum velocity (Vmax) by 6- and 20-fold, respectively. The effects of Lys enrichment on the y(+)-like system are only observed on the Vmax and in the presence of a Na+ gradient (30% increase). 5) Na+ is involved in the activation of the transport process in the Lys-fed chickens, but there is no correlation between external Na+ concentration and L-lysine influx. In conclusion, both b(0,+)- and y(+)-like transport systems are upregulated by dietary lysine but with different kinetic profiles; the high-capacity y(+)-like carrier shows a Vmax increase without changes in K(m), whereas the low-capacity b(0,+)-like system shows an increase in Vmax as well as in the K(m).

L-lysine Transport in Chicken Jejunal Brush Border Membrane Vesicles

Abstract.The properties of l-lysine transport in chicken jejunum have been studied in brush border membrane vesicles isolated from 6-wk-old birds. l-lysine uptake was found to occur within an osmotically active space with significant binding to the membrane. The vesicles can accumulate l-lysine against a concentration gradient, by a membrane potential-sensitive mechanism. The kinetics of l-lysine transport were described by two saturable processes: first, a high affinity-transport system (KmA= 2.4 ± 0.7 μmol/L) which recognizes cationic and also neutral amino acids with similar affinity in the presence or absence of Na+ (l-methionine inhibition constant KiA, NaSCN = 21.0 ± 8.7 μmol/L and KSCN = 55.0 ± 8.4 μmol/L); second, a low-affinity transport mechanism (KmB= 164.0 ± 13.0 μmol/L) which also recognizes neutral amino acids. This latter system shows a higher affinity in the presence of Na+ (KiB for l-methionine, NaSCN = 1.7 ± 0.3 and KSCN = 3.4 ± 0.9 mmol/L). l-lysine influx was significantly reduced with N-ethylmaleimide (0.5 mmol/L) treatment. Accelerative exchange of extravesicular labeled l-lysine was demonstrated in vesicles preloaded with 1 mmol/L l-lysine, l-arginine or l-methionine. Results support the view that l-lysine is transported in the chicken jejunum by two transport systems, A and B, with properties similar to those described for systems b0,+ and y+, respectively.

Hexose transport across the apical and basolateral membrane of enterocytes from different regions of the chicken intestine

The properties of hexose transport across the apical and basolateral membranes of chicken enterocytes have been studied in the small and large intestine. Results show that (a) isolated epithelial cells from all segments except the coprodeum can accumulate 3-O-methylglucose (Glc3Me) against a concentration gradient, by a Na+-dependent and phloridzin-sensitive mechanism, (b) The cell cumulative capacity for Glc3Me (control/phloridzin-incubated cells) is lower in the small intestine than in the large intestine (rectum = proximal caecum = ileum > jejunum > duodenum). (c) Theophylline enhances the cell Glc3Me cumulative capacity 2.9-fold in the duodenum and 2.4-fold in the jejunum but has no effect in the other segments studied. (d) Analysis of sugar uptake indicates that net hexose influx rates decrease from proximal to distal regions: jejunum > duodenum > ileum = proximal caecum = rectum for the apical transport system (α-methyl glucoside as substrate and phloridzin as inhibitor) and duodenum > jejunum > ileum = proximal caecum = rectum for the basolateral system (2-deoxyglucose; theophylline). (e) The duodenum and the jejunum show high apical and basolateral hexose transport rates, which confer a significant capacity for sugar absorption on the proximal intestine. More distal regions, including the ileum, the proximal caecum and the rectum, have transport systems analogous to those of the proximal intestine that keep a considerable potential capability to recover hexoses from the lumen.

Multiple pathways for L-methionine transport in brush-border membrane vesicles from chicken jejunum

1 The intestinal transport of L‐methionine has been investigated in brush‐border membrane vesicles isolated from the jejunum of 6‐week‐old chickens. L‐Methionine influx is mediated by passive diffusion and by Na+‐dependent and Na+‐independent carrier‐mediated mechanisms. 2 In the absence of Na+, cis‐inhibition experiments with neutral and cationic amino acids indicate that two transport components are involved in L‐methionine influx: one sensitive to L‐lysine and the other sensitive to 2‐aminobicyclo[2.2.1]heptane‐2‐carboxylic acid (BCH). The L‐lysine‐sensitive flux is strongly inhibited by L‐phenylalanine and can be broken down into two pathways, one sensitive to N‐ethylmaleimide (NEM) and the other to L‐glutamine and L‐cystine. 3 The kinetics of L‐methionine influx in Na+‐free conditions is described by a model involving three transport systems, here called a,b and c: systems a and b are able to interact with cationic amino acids but differ in their kinetic characteristics (system a: Km= 2.2 ± 0.3 μM and Vmax= 0.13 ± 0.005 pmol (mg protein)−1 (2 s)−1; system b: Km= 3.0 ± 0.3 mM and Vmax= 465 ± 4.3 pmol (mg protein)−1 (2 s)−1); system c is specific for neutral amino acids, has a Km of 1.29 ± 0.08 mM and a Vmax of 229 ± 5.0 pmol (mg protein)−1 (2 s)−1 and is sensitive to BCH inhibition. 4 The Na+‐dependent component can be inhibited by BCH and L‐phenylalanine but cannot interact either with cationic amino acids or with α‐(methylamino)isobutyrate (MeAIB). 5 The kinetic analysis of L‐methionine influx under a Na+ gradient confirms the activity of the above described transport systems a and b. System a is not affected by the presence of Na+ while system b shows a 3‐fold decrease in the Michaelis constant and a 1.4‐fold increase in Vmax. In the presence of Na+, the BCH‐sensitive component can be subdivided into two pathways: one corresponds to system c and the other is Na+ dependent and has a Km of 0.64 ± 0.013 mM and a Vmax of 391 ± 2.3 pmol (mg protein)−1 (2 s)−1. 6 It is concluded that L‐methionine is transported in the chicken jejunum by four transport systems, one with functional characteristics similar to those of system bo, + (system a); a second (system b) similar to system y+, which we suggest naming y+m to account for its high Vmax for L‐methionine transport in the absence of Na+; a third (system c) which is Na+ independent and has similar properties to system L; and a fourth showing Na+ dependence and tentatively identified with system B.

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.

Mucosal surface area in chicken small intestine during development

Abstract.The mucosal surface area of the chicken duodenum, jejunum, and ileum was determined during development (from 1-day to 12-week-old animals). The morphometric analysis was performed at three magnification levels. The nominal (serosal) surface area was determined at the macroscopic level, from intestinal length and perimeter. Villus and microvillus amplification factors were estimated at light-microscopic and transmission electron-microscopic levels, respectively. The results show, during the period considered: (1) a similar increase in nominal surface area for the three segments (6.5 to 7.2-fold), (2) a rise followed by a slight decrease in the villus amplification factor in the third week of age in the duodenum, a two-fold increase of this variable in the jejunum and no significant developmental variations in the ileum, (3) an increase in the microvillus amplification factor of 1.5-fold in the duodenum and jejunum and of 1.2-fold in the ileum, although a pronounced decrease in the first week of age was observed in the three segments. In conclusion, total mucosal surface area increased, from 1 day to 12 week, 12- to 13-fold in the duodenum and ileum and 20-fold in the jejunum.