Effects of paralytic shellfish toxins on the middle intestine of Oncorhynchus mykiss: Glutathione metabolism, oxidative status, lysosomal function and ATP-binding cassette class C (ABCC) proteins activity.

We studied the absorption, cytotoxicity and oxidative stress markers of Paralytic Shellfish Toxins (PST) from three extracts from Alexandrium catenella and A. ostenfeldii, in middle Oncorhynchus mykiss intestine in vitro and ex vivo preparations. We measured glutathione (GSH) content, glutathione-S transferase (GST), glutathione reductase (GR) and catalase (CAT) enzymatic activity, and lipid peroxidation in isolated epithelium exposed to 0.13 and 1.3 µM PST. ROS production and lysosomal membrane stability (as neutral red retention time 50%, NRRT50) were analyzed in isolated enterocytes exposed to PST alone or plus 3 µM of the ABCC transport inhibitor MK571. In addition, the concentration-dependent effects of PST on NRRT50 were assayed in a concentration range from 0 to 1.3 µM PST. We studied the effects of three different PST extracts on the transport rate of the ABCC substrate DNP-SG by isolated epithelium. The extract with highest inhibition capacity was selected for studying polarized DNP-SG transport in everted and non-everted intestinal segments. We registered lower GSH content and GST activity, and higher GR activity, with no significant changes in CAT activity, lipid peroxidation or ROS level. PST exposure decreased NRRT50 in a concentration-depend manner (IC50 = 0.0045 µM), but PST effects were not augmented by addition of MK571. All the three PST extracts inhibited ABCC transport activity, but this inhibition was effective only when the toxins were applied to the apical side of the intestine and DNP-SG transport was measured at the basolateral side. Our results indicate that PST are absorbed by the enterocytes from the intestine lumen. Inside the enterocytes, these toxins decrease GSH content and inhibit the basolateral ABCC transporters affecting the normal functions of the cell. Furthermore, PST produce a strong cytotoxic effect to the enterocytes by damaging the lysosomal membrane, even at low, non-neurotoxic concentrations.

Microtubules regulate brush border formation.

Most epithelial cells contain apical membrane structures associated to bundles of actin filaments, which constitute the brush border. Whereas microtubule participation in the maintenance of the brush border identity has been characterized, their contribution to de novo microvilli organization remained elusive. Hereby, using a cell model of individual enterocyte polarization, we found that nocodazole induced microtubule depolymerization prevented the de novo brush border formation. Microtubule participation in brush border actin organization was confirmed in polarized kidney tubule MDCK cells. We also found that centrosome, but not Golgi derived microtubules, were essential for the initial stages of brush border development. During this process, microtubule plus ends acquired an early asymmetric orientation toward the apical membrane, which clearly differs from their predominant basal orientation in mature epithelia. In addition, overexpression of the microtubule plus ends associated protein CLIP170, which regulate actin nucleation in different cell contexts, facilitated brush border formation. In combination, the present results support the participation of centrosomal microtubule plus ends in the activation of the polarized actin organization associated to brush border formation, unveiling a novel mechanism of microtubule regulation of epithelial polarity.

Lithocholic acid: a new emergent protector of intestinal calcium absorption under oxidant conditions.

LCA and 1,25(OH)2D3 are vitamin D receptor ligands with different binding affinity. The secosteroid stimulates intestinal Ca2+ absorption. Whether LCA alters this process remains unknown. The aim of our work was to determine the effect of LCA on intestinal Ca2+ absorption in the absence or presence of NaDOC, bile acid that inhibits the cation transport. The data show that LCA by itself did not alter intestinal Ca2+ absorption, but prevented the inhibitory effect of NaDOC. The concomitant administration of LCA avoided the reduction of intestinal alkaline phosphatase activity caused by NaDOC. In addition, LCA blocked a decrease caused by NaDOC on gene and protein expression of molecules involved in the transcellular pathway of intestinal Ca2+ absorption. The oxidative stress and apoptosis triggered by NaDOC were abrogated by LCA co-treatment. In conclusion, LCA placed in the intestinal lumen protects intestinal Ca2+ absorption against the inhibitory effects caused by NaDOC. LCA avoids the reduction of the transcellular Ca2+ movement, apparently by blocking the oxidative stress and apoptosis triggered by NaDOC, normalizing the gene and protein expression of molecules involved in Ca2+ movement. Therefore, LCA might become a possible treatment to improve intestinal calcium absorption under oxidant conditions.

Lactic acid bacteria isolated from poultry protect the intestinal epithelial cells of chickens from in vitro wheat germ agglutinin-induced cytotoxicity.

Poultry fed on wheat-based diets regularly ingest wheat germ agglutinin (WGA) that has toxic effects in vitro on intestinal epithelial cells (IEC) obtained from 14-d-old broilers. Cytotoxicity and the potential role of 14 intestinal bacterial strains in the removal of bound lectins in epithelial cell cultures were investigated. Cytotoxicity was dependent on time and lectin concentration; the lethal dose (LD50) was 8.36 µg/ml for IEC exposed for 2 h to WGA. Complementary sugars to WGA were detected on the surface of one Enterococcus and 9 Lactobacillus strains isolated from poultry. These strains were evaluated as a lectin removal tool for cytotoxicity prevention. Incubation of lactic acid bacteria with WGA before IEC-lectin interaction caused a substantial reduction in the percentage of cell deaths. The protection was attributed to the amount of lectin bound to the bacterial surfaces and was strain-dependent. L. salivarius LET 201 and L. reuteri LET 210 were more efficient than the other lactic acid bacteria assayed. These results provide a basis for the development of probiotic supplements or cell-wall preparations of selected lactic acid bacteria intended to avoid harmful effects of a natural constituent of the grain in wheat-based diets.

Infrared Spectroscopy as a Tool to Study the Antioxidant Activity of Polyphenolic Compounds in Isolated Rat Enterocytes.

The protective effect of different polyphenols, catechin (Cat), quercetin (Qc) (flavonoids), gallic acid (GA), caffeic acid (CfA), chlorogenic acid (ChA) (phenolic acids), and capsaicin (Cap), against H2O2-induced oxidative stress was evaluated in rat enterocytes using Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) Spectroscopy and Fourier Transform Infrared Microspectroscopy (FTIRM), and results were compared to standard lipid peroxidation techniques: conjugated dienes (CD) and Thiobarbituric Acid Reactive Substances (TBARS). Analysis of ATR-FTIR and FTIRM spectral data allowed the simultaneous evaluation of the effects of H2O2 and polyphenols on lipid and protein oxidation. All polyphenols showed a protective effect against H2O2-induced oxidative stress in enterocytes, when administered before or after H2O2. Cat and capsaicin showed the highest protective effect, while phenolic acids had weaker effects and Qc presented a mild prooxidative effect (IR spectral profile of biomolecules between control and H2O2-treated cells) according to FTIR analyses. These results demonstrated the viability to use infrared spectroscopy to evaluate the oxidant and antioxidant effect of molecules in cell systems assays.

Ursodeoxycholic and deoxycholic acids: Differential effects on intestinal Ca(2+) uptake, apoptosis and autophagy of rat intestine.

The aim of this work was to study the effect of sodium deoxycholate (NaDOC) and ursodeoxycholic acid (UDCA) on Ca(2+) uptake by enterocytes and the underlying mechanisms. Rats were divided into four groups: a) controls, b) treated with NaDOC, c) treated with UDCA d) treated with NaDOC and UDCA. Ca(2+) uptake was studied in enterocytes with different degrees of maturation. Apoptosis, autophagy and NO content and iNOS protein expression were evaluated. NaDOC decreased and UDCA increased Ca(2+) uptake only in mature enterocytes. The enhancement of protein expression of Fas, FasL, caspase-8 and caspase-3 activity by NaDOC indicates triggering of the apoptotic extrinsic pathway, which was blocked by UDCA. NO content and iNOS protein expression were enhanced by NaDOC, and avoided by UDCA. The increment of acidic vesicular organelles and LC3 II produced by NaDOC was also prevented by UDCA. In conclusion, the inhibitory effects of NaDOC on intestinal Ca(2+) absorption occur by decreasing the Ca(2+) uptake by mature enterocytes. NaDOC triggers apoptosis and autophagy, in part as a result of nitrosative stress. In contrast, UDCA increases the Ca(2+) uptake by mature enterocytes, and in combination with NaDOC acts as an antiapoptotic and antiautophagic agent normalizing the transcellular Ca(2+) pathway.

Acetaminophen inhibits intestinal p-glycoprotein transport activity.

Repeated acetaminophen (AP) administration modulates intestinal P-glycoprotein (P-gp) expression. Whether AP can modulate P-gp activity in a short-term fashion is unknown. We investigated the acute effect of AP on rat intestinal P-gp activity in vivo and in vitro. In everted intestinal sacs, AP inhibited serosal-mucosal transport of rhodamine 123 (R123), a prototypical P-gp substrate. R123 efflux plotted against R123 concentration adjusted well to a sigmoidal curve. Vmax decreased 50% in the presence of AP, with no modification in EC50, or slope, ruling out the possibility of inhibition to be competitive. Inhibition by AP was absent at 0°C, consistent with interference of the active transport of R123 by AP. Additionally, AP showed no effect on normal localization of P-gp at the apical membrane of the enterocyte and neither affected paracellular permeability. Consistent with absence of a competitive inhibition, two further strategies strongly suggested that AP is not a P-gp substrate. First, serosal-mucosal transport of AP was not affected by the classical P-gp inhibitors verapamil or Psc 833. Second, AP accumulation was not different between P-gp knock-down and wild-type HepG2 cells. In vivo intestinal absorption of digoxin, another substrate of P-gp, was assessed in the presence or absence of AP (100 µM). Portal digoxin concentration was increased by 214%, in average, by AP, as compared with digoxin alone. In conclusion, AP inhibited P-gp activity, increasing intestinal absorption of digoxin, a prototypical substrate. These results suggest that therapeutic efficacy of P-gp substrates can be altered if coadministered with AP.

Role of PTHrP in human intestinal Caco-2 cell response to oxidative stress.

We have previously demonstrated that parathyroid hormone (PTH) induces apoptosis in human colon adenocarcinoma Caco-2 cells but the effects of its tumoral analog PTH-related peptide (PTHrP) in this cell line are still unknown. In the present work we investigated whether PTHrP, as PTH, is able to induce Caco-2 cell apoptosis or if it exerts protective effects under apoptotic conditions. Using Caco-2 cells cultured under serum deprivation in the presence or absence of PTHrP we demonstrated that, differently to PTH, its analog employed at the same concentration (10(-8)M) is not a pro-apoptotic hormone. Cells were exposed to an oxidative insult in the form of hydrogen peroxide to induce apoptosis, which leads to a 50% loss of cell viability determined by MTS assay, morphological changes observed under fluorescence microscopy and Western blot analysis. Herein we demonstrate, for the first time, that pre-treatment with PTHrP prior to H2O2 incubation, prevents cell death induced by the apoptotic inductor; and using specific inhibitors we evidenced that protein kinase B (AKT), extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase 1/2 (JNK1/2) and p38 mitogen-activated protein kinase (MAPK) mediate this anti-apoptotic effect. Also, we found that PTHrP decreases the pro-apoptotic protein BAX levels and increases the protein expression of the anti-apoptotic HSP27. Immunoblot analysis revealed that H2O2 increases the phosphorylation levels of AKT and MAPKs, exhibiting a cellular defense response; and consequently increases phospho-BAD levels. The H2O2-induced activation of protein kinases is reverted when cells are pre-treated with PTHrP. Altogether these results evidence a protective effect of PTHrP under apoptotic conditions in intestinal cells, which may be mediated by AKT and MAPKs.

Polyphenols protect the epithelial barrier function of Caco-2 cells exposed to indomethacin through the modulation of occludin and zonula occludens-1 expression.

The aim of this study was to determine the protective effect of quercetin, epigallocatechingallate, resveratrol, and rutin against the disruption of epithelial integrity induced by indomethacin in Caco-2 cell monolayers. Indomethacin decreased the transepithelial electrical resistance and increased the permeability of the monolayers to fluorescein-dextran. These alterations were abolished by all the tested polyphenols but rutin, with quercetin being the most efficient. The protective effect of quercetin was associated with its capacity to inhibit the redistribution of ZO-1 protein induced in the tight junction by indomethacin or rotenone, a mitochondrial complex-I inhibitor, and to prevent the decrease of ZO-1 and occludin expression induced by indomethacin. The fact that the antioxidant polyphenols assayed in this study differ in their protective capacity against the epithelial damage induced by indomethacin suggests that this damage is due to the ability of this agent to induce not only oxidative stress but also mitochondrial dysfunction.

Intestinal epithelial restitution after TcdB challenge and recovery from Clostridium difficile infection in mice with alanyl-glutamine treatment.

BACKGROUND: Clostridium difficile is an anaerobic bacterium that causes antibiotic-associated diarrhea. It produces toxin A and toxin B (TcdB), which cause injury to the gut epithelium. Glutamine is a fundamental fuel for enterocytes, maintaining intestinal mucosal health. Alanyl-glutamine (AQ) is a highly soluble dipeptide derivative of glutamine. We studied whether administration of AQ ameliorates the effects of TcdB in the intestinal cells and improves the outcome of C. difficile infection in mice. METHODS: WST-1 proliferation and cell-wounding-migration assays were assessed in IEC-6 cells exposed to TcdB, with or without AQ. Apoptosis and necrosis were assessed using Annexin V and flow cytometry. C57BL/6 mice were infected with VPI 10463 and treated with either vancomycin, AQ, or vancomycin with AQ. Intestinal tissues were collected for histopathologic analysis, apoptosis staining, and determination of myeloperoxidase activity. RESULTS: AQ increased proliferation in intestinal cells exposed to TcdB, improved migration at 24 and 48 hours, and reduced apoptosis in intestinal cells challenged with TcdB. Infected mice treated with vancomycin and AQ had better survival and histopathologic findings than mice treated with vancomycin alone. CONCLUSIONS: AQ may reduce intestinal mucosal injury in C. difficile-infected mice by partially reversing the effects of TcdB on enterocyte proliferation, migration, and apoptosis, thereby improving survival from C. difficile infection.

Age-related changes in the response of intestinal cells to 1α,25(OH)2-vitamin D3.

The hormonally active form of vitamin D(3), 1α,25(OH)(2)-vitamin D(3), acts in intestine, its major target tissue, where its actions are of regulatory and developmental importance: regulation of intracellular calcium through modulation of second messengers and activation of mitogenic cascades leading to cell proliferation. Several causes have been postulated to modify the hormone response in intestinal cells with ageing, among them, alterations of vitamin D receptor (VDR) levels and binding sites, reduced expression of G-proteins and hormone signal transduction changes. The current review summarizes the actual knowledge regarding the molecular and biochemical basis of age-impaired 1α,25(OH)(2)-vitamin D(3) receptor-mediated signaling in intestinal cells. A fundamental understanding why the hormone functions are impaired with age will enhance our knowledge of its importance in intestinal cell physiology.

Effects of quercetin and menadione on intestinal calcium absorption and the underlying mechanisms.

Quercetin (QT) could be considered as a potential therapeutic agent for different diseases due to its antioxidant, anti-inflammatory, antiviral and anticancer properties. This study was designed to investigate the ability of QT to protect the chick intestine against menadione (MEN) induced injury in vivo and in vitro. Four-week old chicks (Gallus gallus) were treated i.p. with 2.5µmol of MEN/kg b.w. or with i.l. 50µM QT or both. QT protected the intestinal Ca(2+) absorption against the inhibition caused by MEN, but QT alone did not modify. Glutathione (GSH) depletion provoked by MEN in chick enterocytes was abolished by QT treatment, whereas QT alone did not modify the intestinal GSH content. The enhancement of GSH peroxidase activity produced by MEN was blocked by QT treatment. In contrast, superoxide dismutase activity remained high after simultaneous treatment of enterocytes with MEN and QT. The flavonol also avoided changes in the mitochondrial membrane permeability (swelling) produced by MEN. The FasL/Fas/caspase-3 pathway was activated by MEN, effect that was abrogated by QT. In conclusion, QT may be useful in preventing inhibition of chick intestinal Ca(2+) absorption caused by MEN or other substances that deplete GSH, by blocking the oxidative stress and the FasL/Fas/caspase-3 pathway activation.

Dietary iodide controls its own absorption through post-transcriptional regulation of the intestinal Na+/I- symporter.

Dietary I(-) absorption in the gastrointestinal tract is the first step in I(-) metabolism. Given that I(-) is an essential constituent of the thyroid hormones, its concentrating mechanism is of significant physiological importance. We recently described the expression of the Na(+)/I(-) symporter (NIS) on the apical surface of the intestinal epithelium as a central component of the I(-) absorption system and reported reduced intestinal NIS expression in response to an I(-)-rich diet in vivo. Here, we evaluated the mechanism involved in the regulation of NIS expression by I(-) itself in enterocytes. Excess I(-) reduced NIS-mediated I(-) uptake in IEC-6 cells in a dose- and time-dependent fashion, which was correlated with a reduction of NIS expression at the plasma membrane. Perchlorate, a competitive inhibitor of NIS, prevented these effects, indicating that an increase in intracellular I(-) regulates NIS. Iodide induced rapid intracellular recruitment of plasma membrane NIS molecules and NIS protein degradation. Lower NIS mRNA levels were detected in response to I(-) treatment, although no transcriptional effect was observed. Interestingly, I(-) decreased NIS mRNA stability, affecting NIS translation. Heterologous green fluorescent protein-based reporter constructs revealed a significant repressive effect of the I(-)-targeting NIS mRNA 3 untranslated region. In conclusion, excess I(-) downregulates NIS expression in enterocytes by virtue of a complex mechanism. Our data suggest that I(-) regulates intestinal NIS mRNA expression at the post-transcriptional level as part of an autoregulatory effect of I(-) on its own metabolism.

[Effect of dietary fiber in the quantitative expression of butyrate receptor GPR43 in rats colon]. / Efecto del consumo de la fibra dietética en la expresión cuantitativa del receptor de butirato GPR43 en colon de ratas.

INTRODUCTION: Short chain fatty acids (SCFA) acetate, propionate and butyrate are the major anions produced by the bacterial fermentation of dietary fiber (DF) in colon. Recently, butyrate has been recently studied because is important to maintain colonic functions and because it has been related with a protective effect in colorectal cancer, which is mainly, explained by its potential to regulate gene expression by inhibiting enzyme histonedeacetylase (HDAC). Several investigationsshown that SCFAreceptor GPR43 is involved insignal transduction mechanisms once they bind to ligands such as butyrate to generate different physiological effects in colonocytes. OBJECTIVE: Determine if dietary fiber consumption from nopal (Opuntia ficus I.) containing a ratio of soluble-insoluble fiber 40/60, has a direct influence on the quantitative expression of butyrate-specific receptor GPR43. METHODS: Wistar rats were fed with four different diets formulated at different concentrations of dietary fiber of 0, 5, 15 and 25% of dietary fiber from opuntia, respectively. RESULTS AND DISCUSSION: The results shown an increase in the expression of GPR43 (93.1%) when rats was fed with a 5% fiber diet, using ß-actin as a reference gene. The results of this investigation will contribute to determinate the relation of diet with intestinal health for the purpose of expanding the knowledge of butyric acid on colonic functions.

Induction of intestinal multidrug resistance-associated protein 2 by glucagon-like Peptide 2 in the rat.

The effects of glucagon-like peptide 2 (GLP-2) on expression and activity of jejunal multidrug resistance-associated protein 2 (Mrp2; Abcc2) and glutathione transferase (GST) were evaluated. After GLP-2 treatment (12 µg/100 g b.wt. s.c., every 12 h, for 5 consecutive days), Mrp2 and the α class of GST proteins and their corresponding mRNAs were increased, suggesting a transcriptional regulation. Mrp2 was localized at the apical membrane of the enterocyte in control and GLP-2 groups, as detected by confocal immunofluorescence microscopy. As a functional assay, everted intestinal sacs were incubated in the presence of 1-chloro-2,4-dinitrobenzene in the mucosal compartment, and the glutathione-conjugated derivative, dinitrophenyl-S-glutathione (DNP-SG; model Mrp2 substrate), was detected in the same compartment by high-performance liquid chromatography. A significant increase in apical secretion of DNP-SG was detected in the GLP-2 group, consistent with simultaneous up-regulation of Mrp2 and GST. GLP-2 also promoted an increase in cAMP levels as detected in homogenates of intestinal mucosa. Treatment of rats with 2',3'-dideoxyadenosine (DDA), a specific inhibitor of adenylyl cyclase, abolished the increase in cAMP levels and Mrp2 protein promoted by GLP-2, suggesting cAMP as a mediator of Mrp2 modulation. Increased expression of Mrp2 and cAMP levels in response to GLP-2 occurred not only at the tip but also at the middle region of the villus, where constitutive expression of Mrp2 is normally low. In conclusion, our study suggests a role for GLP-2 in the prevention of cell toxicity of the intestinal mucosa by increasing Mrp2 chemical barrier function.

Morphological alterations induced by boric acid and fipronil in the midgut of worker honeybee (Apis mellifera L.) larvae : Morphological alterations in the midgut of A. mellifera.

Morphological alterations, by means of histological and ultrastructural analysis, have been used to determine the effects of boric acid and fipronil on midgut tissues of honeybee worker, Apis mellifera L. larvae. In order to observe possible morphological alterations in the midgut, two groups of bioassays were performed. In the first one, the larvae were chronically treated with different concentrations of boric acid added to the food (1.0, 2.5 and 7.5 mg/g). In the second group, the larvae were fed with diets containing different concentrations of fipronil (0.1 and 1 microg/g) and compared with control groups without these chemical compounds. In the first bioassay, the larvae were collected on day 3 and in the second bioassay on day 4, when the mortality rate obtained in the toxicological bioassay was not very high. The larval midguts were removed and processed for morphological analyses using a light and transmission electron microscopy. We observed cytoplasmic vacuolizations, with the absence of autophagic vacuoles, and chromatinic compacting in most of the cells in the groups treated with pesticides. The morphological alterations were far greater in the larvae treated with boric acid than in the larvae treated with fipronil. Our data suggest that the midgut cell death observed was in response to boric acid and fipronil action. This study significantly improves the understanding of the toxicological effect of these insecticides from the ecotoxicological perspective.

Extracellular ATP activates MAP kinase cascades through a P2Y purinergic receptor in the human intestinal Caco-2 cell line.

BACKGROUND: ATP exerts diverse effects on various cell types via specific purinergic P2Y receptors. Intracellular signaling cascades are the main routes of communication between P2Y receptors and regulatory targets in the cell. METHODS AND RESULTS: We examined the role of ATP in the modulation of ERK1/2, JNK1/2, and p38 MAP kinases (MAPKs) in human colon cancer Caco-2 cells. Immunoblot analysis showed that ATP induces the phosphorylation of MAPKs in a time- and dose-dependent manner, peaking at 5 min at 10 microM ATP. Moreover, ATPgammaS, UTP, and UDP but not ADP or ADPbetaS increased phosphorylation of MAPKs, indicating the involvement of, at least, P2Y2/P2Y4 and P2Y6 receptor subtypes. RT-PCR studies and PCR product sequencing supported the expression of P2Y2 and P2Y4 receptors in this cell line. Spectrofluorimetric measurements showed that cell stimulation with ATP induced transient elevations in intracellular calcium concentration. In addition, ATP-induced phosphorylation of MAPKs in Caco-2 cells was dependent on Src family tyrosine kinases, calcium influx, and intracellular Ca2+ release and was partially dependent on the cAMP/PKA and PKC pathways and the EGFR. GENERAL SIGNIFICANCE: These findings provide new molecular basis for further understanding the mechanisms involved in ATP functions, as a signal transducer and activator of MAP kinase cascades, in colon adenocarcinoma Caco-2 cells.

Inhibitory effect of aluminium on calcium absorption in small intestine of rats with different thyroid hormone status.

To analyse the influence of thyroid status on the effect of aluminium (Al) upon intestinal calcium (Ca) absorption, adult male Wistar rats with experimentally altered thyroid hormones circulating levels, were orally treated (o.g.) with 0 (control), or 50 mg elemental Al (as chloride)/kg body weight (b.w.) per day, for a 14 d period. Hyper- and hypo-thyroid conditions were respectively achieved by means of administration of either sodium levothyroxine (50 microg/kg b.w. per day, o.g.) or methimazole, a thyroxine synthesis inhibitor (1mg/kg b.w. per day, o.g.). In duodenum-jejunum segments, in vitro mucosa-to-serosa (45)Ca flux (JCa(ms)) and kinetics of (45)Ca uptake in isolated enterocytes, were determined. In serum, concentrations of thyroxine (T4) and triiodothyronine (T3) were measured by chemiluminescent enzyme immunoassay. Unlike non-Al-treated rats, JCa(ms) of Al-exposed rats decreased as serum levels of T4 and T3 increased, showing a significant inverse correlation in both cases (T4: r(2)=0.414, P=0.024; T3: r(2)=0.443, P=0.018). Enterocytes isolated from rats treated with Al plus thyroxine showed a reduction of both maximum Ca uptake (4.86+/-0.44 vs. 6.85+/-1.04 nmol Ca/mg protein, P<0.05) and K(m) (0.84+/-0.18 vs. 1.05+/-0.36 mM, P<0.05) when compared to control. The observed variability in the Al effect on Ca transport with thyroid status of rats could be reflecting a negative interaction of Al with thyroid hormone action mechanisms on intestinal Ca absorption, which would take place mainly at Ca entry into enterocyte from lumen.

PTH regulation of c-Jun terminal kinase and p38 MAPK cascades in intestinal cells from young and aged rats.

In the present study, we examined the role of Parathyroid hormone (PTH) on the c-Jun N-terminal kinase (JNK) 1/2 and p38 mitogen-activated protein kinase (MAPK) members of the MAPK family as it relates to ageing by measuring hormone-induced changes in their activity in enterocytes isolated from young (3 month old) and aged (24 month old) rats. Our results show that PTH induces a transient activation of JNK 1/2, peaking at 1 min (+threefold). The hormone also stimulates JNK 1/2 tyrosine phosphorylation, in a dose-dependent fashion, this effect being maximal at 10 nM. PTH-induced JNK 1/2 phosphorylation was suppressed by its selective inhibitor SP600125. Moreover, hormone-dependent activation of JNK 1/2 was dependent on calcium, since pretreatment of cells with BAPTA-AM or EGTA blocked PTH effects. With ageing, the response to PTH was significantly reduced. JNK basal protein expression was not different in the enterocytes from young and aged rats, however, basal protein phosphorylation increased with ageing. PTH did not stimulate, within 1-10 min, the basal activity and phosphorylation of p38 MAPK in rat intestinal cells. The hormone increased enterocyte DNA synthesis; the response was dose-dependent and decreased (-40%) with ageing. In agreement with the mitogenic role of the MAPK cascades, this effect was blocked by specific inhibitors of extracellular signal-regulated protein kinase (ERK) 1/2 and JNK 1/2. The results obtained in this work expand our knowledge on the mechanism of action of PTH in duodenal cells.

Age-related alteration of 1alpha,25(OH)2-vitamin D3-dependent activation of p38 MAPK in rat intestinal cells.

In intestinal cells, 1alpha,25(OH)(2)-vitamin D(3) (1alpha,25(OH)(2)D(3)) regulates gene expression via the specific intracellular vitamin D receptor and induces fast non-transcriptional responses involving stimulation of transmembrane signal transduction pathways. In the present study, we analyzed, for the first time, alterations in p38 MAPK response to 1alpha,25(OH)(2)D(3) in rat enterocytes with ageing. In enterocytes from young rats, the hormone increased, in a time- and dose-dependent fashion, the phosphorylation of p38 MAPK, peaking at 3 min (+2-fold). Basal levels of p38 MAPK phosphorylation were lower in enterocytes from old rats and the hormone response was greatly diminished (+0.5-fold at 3 min). p38 MAPK phosphorylation impairment in old animals was not related to significant changes of the kinase protein expression and do not explain the decreased response to 1alpha,25(OH)(2)D(3). Extracellular and intracellular Ca(2+) chelation or c-Src pharmacological inhibition suppressed hormone activation of p38 MAPK in both, young and aged rats, demonstrating that Ca(2+) and the non-receptor tyrosine kinase c-Src are required for full activation of p38 MAPK in cells stimulated with 1alpha,25(OH)(2)D(3). Two other vitamin D(3) metabolites, 25(OH)D(3) and 24,25(OH)(2)D(3, )also enhanced p38 phosphorylation, and to a similar extent than 1alpha,25(OH)(2)D(3), an ability that is lost with ageing. Enterocyte exposure to the hormone also resulted in the rapid induction of c-fos protein (peaking at 5 min, +3-fold) and to a greater extent than that of mRNA induction. With ageing, 1alpha,25(OH)(2)D(3)-dependent increase of c-fos protein level was diminished, but c-fos mRNA expression was not different from young animals. Impairment of 1alpha,25(OH)(2)D(3) activation of p38 MAPK upon ageing and abnormal hormone regulation of the c-fos oncoprotein synthesis may affect intestinal cell function.