Ana Isabel Alcalde

Role of thyroid hormone in regulation of renal phosphate transport in young and aged rats.

In the present study, we have examined the cellular mechanisms mediating the regulation of renal proximal tubular sodium-coupled inorganic phosphate (Na/Pi) transport by thyroid hormone (T3) in young and aged rats. Young hypothyroid rats showed a marked decrease in Na/Pi cotransport activity, which was associated with parallel decreases in type II Na/Pi cotransporter (NaPi-2) protein and messenger RNA (mRNA) abundance. In contrast, administration of long-term physiological and supraphysiological doses of T3 resulted in significant increases in Na/Pi cotransport activity, protein, and mRNA levels. Nuclear run-on experiments indicated that thyroid hormone regulates NaPi-2 mRNA levels by a transcriptional mechanism. In aged rats, although there were no changes in T3 serum levels (when compared with young animals), there were significant decreases in serum Pi concentration, renal Na/Pi cotransport activity, and NaPi-2 protein and mRNA abundance. These effects were mediated, at least in part, by a reduction in...

Antibiotic-Induced Depletion of Murine Microbiota Induces Mild Inflammation and Changes in Toll-Like Receptor Patterns and Intestinal Motility

We examine the impact of changes in microbiota induced by antibiotics on intestinal motility, gut inflammatory response, and the function and expression of toll-like receptors (TLRs). Alterations in mice intestinal microbiota were induced by antibiotics and evaluated by q-PCR and DGGE analysis. Macroscopic and microscopic assessments of the intestine were performed in control and antibiotic-treated mice. TLR expression was determined in the intestine by q-RT-PCR. Fecal parameter measurements, intestinal transit, and muscle contractility studies were performed to evaluate alterations in intestinal motility. Antibiotics reduced the total bacterial quantity 1000-fold, and diversity was highly affected by treatment. Mice with microbiota depletion had less Peyer’s patches, enlarged ceca, and mild gut inflammation. Treatment with antibiotics increased the expression of TLR4, TLR5, and TLR9 in the ileum and TLR3, TLR4, TLR6, TLR7, and TLR8 in the colon, and it reduced the expression of TLR2, TLR3, and TLR6 in the ileum and TLR2 and TLR9 in the colon. Antibiotics decreased fecal output, delayed the whole gut and colonic transit, and reduced the spontaneous contractions and the response to acetylcholine (ACh) in the ileum and colon. Activation of TLR4 by lipopolysaccharide (LPS) reverted the reduction of the spontaneous contractions induced by antibiotics in the ileum. Activation of TLR4 by LPS and TLR5 by flagellin reduced the response to ACh in the ileum in control mice. Our results confirm the role of the microbiota in the regulation of TLRs expression and shed light on the microbiota connection to motor intestinal alterations.

Lipopolysaccharide induces alteration of serotonin transporter in human intestinal epithelial cells

Intestinal serotoninergic activity and serotonin transporter (SERT) function have been shown to be altered in intestinal inflammatory diseases. Serotonin (5-HT) plays a critical role in the regulation of gastrointestinal physiology. Activity of 5-HT depends on its extracellular availability, partly modulated by SERT that transports 5-HT into the cell. Lipopolysaccharide (LPS) is a component of Gram-negative bacteria outer membrane, which acts as a potent activator of the inflammatory system in the intestine. The aim of this work was to determine, in the enterocyte-like cell line Caco-2, whether LPS treatment affects serotoninergic activity by acting on SERT. The results demonstrate that LPS treatment diminishes SERT activity in a dose- and period-dependent way. The kinetic study shows that Vmax was significantly reduced after treatment with LPS. The LPS effect on 5-HT uptake was, in part, mediated by protein kinase C (PKC) activation. The molecular expression of SERT revealed that LPS treatment did not affect the mRNA level or the SERT protein content in cell homogenate. The level of SERT protein, however, was reduced on brush border membrane. The LPS effect might be due to an alteration of the intracellular traffic of SERT which may, in part, be mediated by PKC activity.

Regulation of the human serotonin transporter mediated by long-term action of serotonin in Caco-2 cells.

Aim:  The aim of this study was to determine the effect of long‐term serotonin (5‐hydroxytryptamine, 5‐HT) treatment on the human serotonin transporter (hSERT) function and its expression.

5-HT RECEPTOR SUBTYPES INVOLVED IN THE SEROTONIN-INDUCED INHIBITION OF L-LEUCINE ABSORPTION IN RABBIT JEJUNUM

The aim of the present study was to determine the 5-HT receptor subtypes involved in the serotonin-induced inhibition of L-leucine absorption across rabbit jejunum in vitro. A number of agonists and antagonists were used to characterize the receptors through which serotonin inhibits this absorption. The results show that 2.5x10(-6) M 5-HT inhibits the amino acid absorption by about 20%. The 5-HT receptor agonists, alpha-methyl-5-HT (5-HT2), 2-methyl-5-HT (5-HT3) and zacopride (5-HT4) at concentrations 2.5x10(-6) and 2.5x10(-5) M produced 10-30% inhibition on L-leucine intestinal absorption. 5-carboxyamidotryptamine (5-HT1) did not produce any inhibition. The 5-HT antagonists, GR 113808A (5-HT4) at 2.5x10(-6) M and ritanserin (5-HT2) and ondansetron (5-HT3) at 2.5x10(-5) M completely blocked the effect of 5-HT. However, methiothepin (5-HT1) did not produce any effect on serotonin action in the intestinal absorption of amino acid. It can be concluded that 5-HT2, 5-HT3 and 5-HT4 receptors could mediate inhibition of L-leucine absorption across rabbit jejunum.

IL-10 modulates serotonin transporter activity and molecular expression in intestinal epithelial cells

Serotonin is a neuromodulator mainly synthesized by intestinal enterochromaffin cells that regulate overall intestinal physiology. The serotonin transporter (SERT) determines the final serotonin availability and has been described as altered in inflammatory bowel diseases. IL-10 is an anti-inflammatory cytokine that is involved in intestinal inflammatory processes and also contributes to intestinal mucosa homeostasis. The regulation of SERT by pro-inflammatory factors is well known; however, the effect of IL-10 on the intestinal serotoninergic system mediated by SERT remains unknown. Therefore, the aim of the present study is to determine whether IL-10 affects SERT activity and expression in enterocyte-like Caco-2 cells. Treatment with IL-10 was assessed and SERT activity was determined by 5-HT uptake. SERT mRNA and protein expression was analyzed using quantitative RT-PCR and western blotting. The results showed that IL-10 induced a dual effect on SERT after 6h of treatment. On one hand, IL-10, at a low concentration, inhibited SERT activity, and this effect might be explained by a non-competitive inhibition of SERT. On the other hand, IL-10, at a high concentration, increased SERT activity and molecular expression in the membrane of the cells. This effect was mediated by the IL-10 receptor and triggered by the PI3K intracellular pathway. Our results demonstrate that IL-10 modulates SERT activity and expression, depending on its extracellular conditions. This study may contribute to understand serotoninergic responses in intestinal pathophysiology.

Regulation of serotonin transporter activity by adenosine in intestinal epithelial cells

Serotonin plays a critical role in the regulation of intestinal physiology. The serotonin transporter (SERT) expressed in the intestinal epithelium determines 5-HT availability and activity. The serotoninergic system and SERT activity have been described as being altered in chronic intestinal pathologies such as inflammatory diseases. Adenosine has also been shown to be involved in a variety of intestinal functions and to play a central role in the regulation of inflammatory responses of injured tissue. Since the modulation of SERT by adenosine in the intestine remains unknown, the aim of the present work was to study the effect of adenosine on SERT activity and expression and to determine the molecular mechanism involved. The study has been carried out using human enterocyte-like Caco-2 cells which endogenously express SERT. The results show that adenosine diminishes SERT activity in both the apical and basal membranes by acting in the intrinsic molecule with no alteration of either SERT mRNA or protein levels. The effect of adenosine appears to be mediated by A(2) receptors and activation of the cAMP/PKA signalling pathway. Moreover, the adenosine effect did not seem to involve the activation of AMP activated protein kinase. Adenosine effects are reached at high concentrations, which suggests that adenosine modulation of SERT may be significant under conditions of inflammation and tissue injury.

Toll-like Receptor 3 Activation Affects Serotonin Transporter Activity and Expression in Human Enterocyte-like Caco-2 Cells

Serotonin, a neurotransmitter/autocrineagent mainly synthesized by intestinal enterochromaffin cells, regulates the whole intestinal physiology. Toll-like receptor 3 (TLR3) also contributes to the intestinal physiology by modulating intestinal innate immunity responses. Both serotonin and TLR3 are involved in intestinal inflammatory processes; however, the role of TLR3 in the regulation of intestinal 5-HT availability remains unexplored. The present study analyzes the effect of TLR3 activation on serotonin transporter (SERT) activity in Caco-2 cells. Treatment with poly(I:C), dsRNA synthetic analogue and TLR3 ligand, was assayed and SERT activity determined by 5-HT uptake and transepithelial flux. SERT expression was analyzed by qRT-PCR and western blotting. Poly(I:C) short-term treatment inhibited SERT activity in the apical and basal membrane of epithelial cells and diminished SERT protein content in the membrane. SERT total protein and mRNA levels were not affected by poly(I:C), suggesting a post-translational alteration of SERT. The poly(I:C) effect on SERT activity did not appear to be mediated by PKC, cAMP, PKR or JNK signaling pathways; however, the p38 MAPK pathway seemed to be involved. Our results demonstrate that TLR3 inhibits SERT activity, which may increase 5-HT extracellular levels and contribute to the inflammatory response; however, 5-HT treatment did not affect TLR3 expression.

Melatonin inhibits serotonin transporter activity in intestinal epithelial cells

Abstract:  Gastrointestinal serotonin (5‐HT) and melatonin are two closely related neuromodulators which are synthesised in the enterochromaffin cells of the intestinal epithelium and which have been shown to be involved in the physiopathology of the gastrointestinal tract. The effects of 5‐HT depend on 5‐HT availability which is, in part, modulated by the serotonin transporter (SERT). This transporter provides an efficient 5‐HT uptake after release and is expressed in the membrane of the enterocytes. Although the origin and effects of 5‐HT and melatonin are similar, the interrelationship between them in the gastrointestinal tract is unknown. The main aim of this study was to determine whether melatonin affects SERT activity and expression, and, if so, to elucidate the mechanisms involved. Caco‐2 cell line was used to carry out the study as these cells have been shown to endogenously express SERT. The results showed that melatonin inhibits SERT activity by affecting both Vmax and kt kinetic constants although SERT synthesis or intracellular trafficking did not appear to be affected. The melatonin effect seemed to be independent of melatonin receptors MT1 and MT2 and protein kinase C and cAMP intracellular pathways. Our results suggest that the inhibition of SERT might be due to a catalytic effect of melatonin on the allosteric citalopram‐sensitive site in SERT. This study shows, for the first time, that melatonin modulates SERT activity, thus demonstrating the feedback system between melatonin and the serotoninergic system in the gastrointestinal tract.

Expression and Molecular Characterization of Rat Renal d-Mannose Transport in Xenopus Oocytes

Abstract. Renal reabsorption appears to play a major role in d-mannose homeostasis. Here we show that in rat kidney, the transport of d-mannose by brush border membrane vesicles from tubular epithelial cells involves an uphill and rheogenic Na-dependent system, which is fully inhibited by d-mannose itself, incompletely inhibited by d-glucose, d-fructose, phloridzin, and phloretin, and noninhibited by l-mannose or disaccharides. In addition, this system exhibits both low capacity (112.9 ± 15.6 pmol/mg/second) and high affinity (0.18 ± 0.04 mm), with a 2:1 stoichiometry for the Na:d-mannose interaction, and low affinity for sodium (16.6 ± 3.67 mm).We also show expression of d-mannose transport by Xenopus laevis oocytes injected with rat renal polyA+ RNA. Kinetic analysis of the expressed transport was performed after RNA enrichment by fractionation through a sucrose density gradient and was shown to be identical to that measured in membrane vesicles. The RNA species encoding the expressed transport has a small mean size, 1 kb approximately, and shows no homology with the SGLT family of Na-dependent d-glucose transporters, as shown by low stringent RT-PCR and northern analysis. The expressed transport is specific for d-mannose, since in spite of a significant inhibition by d-glucose and d-fructose, neither of these two substrates was transported above the level of the water-injected oocytes.