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At least 127 records · Page 7Linked to original sources
PubMed1997

Effects of mesalamine on the hsp72 stress response in rat IEC-18 intestinal epithelial cells.

BACKGROUND & AIMS: Mesalamine has many effects and is commonly used for the treatment of inflammatory bowel diseases. Because sodium salicylate, a related compound, modulates the heat shock protein (hsp72) response in nonepithelial cells, the possibility that mesalamine confers cell protection by increasing intestinal epithelial hsp72 expression was examined. METHODS: Rat intestinal IEC-18 cells were treated with 0.3-3 mmol/L mesalamine and thermally stressed (39 degrees C-42 degrees C) for 23 minutes. The effects of mesalamine on basal expression and the threshold and time course of hsp72 thermal induction were determined. RESULTS: Although mesalamine had no effects on the basal hsp72 expression or its thermal activation threshold in IEC-18 cells, it accelerated and augmented thermal induction of hsp72 within the first 2 hours of exposure. This was associated with a transient increase in heat shock factor-heat shock element binding and enhanced cellular protection against oxidant-induced injury. In contrast, both mesalamine and sodium salicylate have been shown to lower the thermal induction threshold but not to enhance the hsp72 response in HeLa cells. CONCLUSIONS: Mesalamine augments thermal induction of the intestinal epithelial hsp72 expression in a manner that differs from that in nonintestinal epithelial cells. This effect is accompanied by increased cellular protection against oxidant injury.

Animals↗
PubMed2005

Measurement of the detective quantum efficiency (DQE) of digital X-ray detectors according to the novel standard IEC 62220-1.

A mobile measurement facility which complies with IEC 62220-1 has been set up to determine the detective quantum efficiency (DQE) of digital X-ray detector systems. Exemplary measurements were performed for two similar CR detector systems, a CsI-based indirect detector and an Se-based direct detector. The standardised radiation quality RQA 5 was applied for measurement and for three of these systems RQA 9 was also applied. A pronounced dependence of DQE on radiation quality was observed for the direct detector, where the DQEs for RQA 5 and RQA 9 differ by a factor of approximately 2. The uncertainty (95% confidence interval) associated with the measured DQE values is within 0.01 and 0.04 depending on, for example, the spatial frequency. Thus, it has been demonstrated that the DQE can be measured accurately and reliably with the accuracy required by the international standard IEC 62220-1. It is now possible to objectively measure and compare DQE values of digital X-ray detector systems.

Calibration↗
PubMed2001

Gene expression in intestinal epithelial cells, IEC-6, is altered by burn injury-induced circulating factors.

Severe burn injury is commonly associated with significant changes in intestinal epithelial function. These changes include mucosal atrophy and increased permeability. To date, the mechanism by which burn injury alters intestinal epithelium function are not clear. We used an in vitro model of intestinal epithelium, IEC-6 cells, and observed that the integrity of confluent culture is disrupted and cell growth and migration rates are reduced in the presence of serum collected from scald burn injury rats (SRS) (6). To identify gene products involved in mechanisms underlying these effects, we used the cDNA expression microarray analysis and found that genes whose expression was affected by SRS in IEC-6 cells were primarily associated with cell shape, growth and death, stress-response, protein turnover and transport of water and electrolytes. These data demonstrate that a burn-induced circulating factor(s) modulates expression of genes, which may affect intestinal epithelial cell survival and function. Thus, these findings provide clues to the nature of molecular mechanisms potentially involved in multiple-organ malfunction, in particular the atrophy and enhanced permeability of gut mucosa, after burn injury.

Actins↗
PubMed2005

Effects of human milk and spermine on hydrogen peroxide-induced oxidative damage in IEC-6 cells.

OBJECTIVE: Oxidative stress is intimately involved in the pathologic processes of serious diseases in the perinatal period. Human milk (HM) contains various bioactive substances, some of which are known as antioxidants, including polyamines such as spermine (SPM). We examined the antioxidative properties of HM and SPM in an intestinal epithelial cell line. METHOD: Confluent Intestinal Epithelial Cells-6 (IEC-6) cells were preincubated with 100-fold dilutions of defatted HM, bovine milk, or three artificial milks for 24 hours, followed by hydrogen peroxide (H2O2) challenge (0.5 mM, 30 min) for oxidative stress. Cells were preincubated with either HM or increasing concentrations (within the range of HM) of SPM for 24 hours followed by an H2O2 challenge (0.25 mM, 30 min). RESULTS: HM-treated cells showed the highest survival rate (50%) compared with no pretreatment (27%), bovine milk-treated (6%), or artificial formula-treated (13-16%) cells. Significantly higher survival rates were observed in the cells treated with HM (44.0%) and in those treated with 0.5, 1, or 5 microM of SPM (12.6, 13.1, or 22.2%, respectively) in comparison with the nontreated cells (7.0%). CONCLUSION: Our results demonstrated that HM and SPM alleviated H2O2-induced oxidative damage in IEC-6 cells, whereas bovine milk and artificial formula did not show any antioxidative capacity. These results suggest that HM acts as an antioxidant in the gastrointestinal tract of infants and that SPM plays an important role in the antioxidative properties of HM.

Animals↗
PubMed2004

Alpha-melanocyte stimulating hormone protects against H2O2-induced inhibition of wound restitution in IEC-6 cells via a Syk kinase- and NF-kappabeta-dependent mechanism.

Epithelial injury and repair are central consequences of ischemia and reperfusion of the gut. Intestinal mucosal wounds are repaired in part by epithelial restitution. However, the signaling mechanisms regulating restitution remain poorly understood, and few therapies to enhance restitution have been described. Previously we demonstrated that alpha-melanocyte-stimulating hormone (alpha-MSH) protected against postischemic gut injury in the rat. In this report, we tested the effects and mechanisms of alpha-MSH on wound restitution of rat small intestine (IEC-6) cells subjected to H2O2 stress with or without scrape wounding. H2O2 treatment resulted in tyrosine phosphorylation of Syk kinase and its downstream target IkappaBalpha, with subsequent NF-kappaB activation. Alpha-MSH and the Syk kinase inhibitor piceatannol blocked these processes. In scrape-wounded cells, H2O2 inhibited wound restitution, and this was partially restored by cotreatment with alpha-MSH or piceatannol. In contrast, overexpression of NF-kappaB p65 or Syk kinase, but not a dominant-negative mutant of Syk kinase, aggravated H2O2 inhibition of wound restitution, and inhibitors of c-Src tyrosine kinase or phosphatidylinositol-3 kinase were without effect. The results indicate an important role for Syk tyrosine kinase and the NF-kappaB pathway in the response to oxidant stress and the impairment of epithelial restitution in IEC-6 cells. The data also disclose that the beneficial effects of alpha-MSH on gut ischemia/reperfusion injury may relate to its acceleration of epithelial restitution.

Animals↗
PubMed2006

Modified vaccinia virus Ankara multiplies in rat IEC-6 cells and limited production of mature virions occurs in other mammalian cell lines.

Recombinant viruses based on modified vaccinia virus Ankara (MVA) are vaccine candidates against infectious diseases and cancers. Presently, multiplication of MVA has been demonstrated in chicken embryo fibroblast and baby hamster kidney (BHK-21) cells only. The multiplication and morphogenesis of a recombinant (MVA-HANP) and non-recombinant MVA strain in BHK-21 and 12 other mammalian cell lines have now been compared. Rat IEC-6 cells were fully permissive to MVA infection. The virus yield in IEC-6 cells was similar to that obtained in BHK-21 cells at low as well as high multiplicities of infection. Vero cells were semi-permissive to MVA infection. Mature virions were produced in supposedly non-permissive cell lines. The multiplication and morphogenesis of non-recombinant MVA and MVA-HANP were similar. These results are relevant to the production and biosafety of MVA-vectored vaccines.

Animals↗
PubMed2005

Temporal changes of multiple redox couples from proliferation to growth arrest in IEC-6 intestinal epithelial cells.

Changes in intracellular redox couples and redox reactive molecules have been implicated in the regulation of a variety of cellular processes, including cell proliferation and growth arrest by contact inhibition. However, the magnitude, direction, and temporal relationship of redox changes to cellular responses are incompletely defined. The present work sought to characterize redox and metabolic changes associated with proliferative stages to contact inhibition of growth in rat IEC-6 intestinal epithelial cells. From the first day of culture until 1 day before confluence, an increase in GSH concentrations and a significant reduction in the redox potential of the GSSG/2GSH couple were observed. These changes were accompanied by a decrease in relative reactive oxygen species (ROS) and nitric oxide (NO) concentrations and oxidation of the redox potential of the NADP(+)/reduced NADP and NAD(+)/NADH couples. Postconfluent cells exhibited a significant decrease in GSH concentrations and a significant oxidation of the GSSG/2GSH couple. When cell proliferation decreased, relative ROS concentrations increased (P < 0.01), whereas NO concentrations remained unchanged, and the NAD(+)/NADH couple became more reduced. Together, these data indicate that the redox potential of distinct couples varies differentially in both magnitude and direction during successive stages of IEC-6 growth. This finding points out the difficulty of defining intracellular redox status at particular stages of cell growth by examining only one redox species. In addition, the data provide a numerical framework for future research of regulatory mechanisms governed by distinct intracellular redox couples.

Adenosine Diphosphate↗
PubMed1994

Polyamine deficiency causes reorganization of F-actin and tropomyosin in IEC-6 cells.

In earlier work we have shown that polyamine-deficient IEC-6 cells lose most of their ability to migrate. In this report we describe the effect of polyamine deficiency on the cytoskeleton of migrating IEC-6 cells. Cells were grown on cover slips for 4 days. One-third of the monolayer was removed, and the remainder was incubated for 6 h. The monolayers were fixed and stained with rhodamine phalloidin for actin filaments and by immunocytochemistry for tropomyosin. In control cells, actin filaments were found as stress fibers traversing the cell, in a thin actin cortex often visible on only one edge of the cell, and in fine fibers extending into the lamellipodia. Tropomyosin was found in the same distribution. A Western blot showed that tropomyosin was present as 35- and 37-kDa isoforms. In polyamine-deficient cells, actin stress fibers were less dense, whereas the actin cortex was greatly increased in density and lamellipodia were less extensive. Tropomyosin distribution was similar and included a 30-kDa isoform not seen previously. In spite of the obvious changes in the distribution of these cytoskeletal proteins, the concentrations of filamentous actin, beta-actin mRNA, and the higher molecular weight tropomyosin isoforms did not change. In all cases the addition of putrescine to polyamine-deficient cells prevented the changes described. We conclude that polyamines are essential for migration in this system because of their effects on the organization of cytoskeletal actin, tropomyosin, and perhaps other proteins as well.

Actins↗
PubMed1996

Induction of heat shock protein 70 protects intestinal epithelial IEC-18 cells from oxidant and thermal injury.

The potential importance of inducible heat shock proteins (HSPs) in conferring protection to intestinal epithelial cells was investigated using IEC-18 cells. To establish optimal HSP induction, [35S]methionine and [35S]cysteine incorporation was assessed. Two 35S-labeled proteins of 70 and 90 kDa were preferentially synthesized. Western and Northern blot analyses confirmed induction of HSP70. This induction provided significant protection to the cells treated with the oxidant monochloramine or lethal heat (49 degrees C). To better establish the protective role of HSP70, cells were stably transfected with human HSP70 under control of the lac operator. When these cells were subjected to injury, they were protected by isopropylthiogalactoside-stimulated HSP70 induction under nonstress conditions. Additionally, the rate of protein synthesis (assessed by [3H]leucine incorporation) was protected. These results demonstrate that HSPs are preferentially and rapidly induced in IEC-18 cells and that induction of inducible HSP70 is important in promoting protection against cellular injury.

Animals↗
PubMed1999

Polyamine depletion arrests cell cycle and induces inhibitors p21(Waf1/Cip1), p27(Kip1), and p53 in IEC-6 cells.

The polyamines spermidine and spermine and their precursor putrescine are intimately involved in and are required for cell growth and proliferation. This study examines the mechanism by which polyamines modulate cell growth, cell cycle progression, and signal transduction cascades. IEC-6 cells were grown in the presence or absence of DL-alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, which is the first rate-limiting enzyme for polyamine synthesis. Depletion of polyamines inhibited growth and arrested cells in the G1 phase of the cell cycle. Cell cycle arrest was accompanied by an increase in the level of p53 protein and other cell cycle inhibitors, including p21(Waf1/Cip1) and p27(Kip1). Induction of cell cycle inhibitors and p53 did not induce apoptosis in IEC-6 cells, unlike many other cell lines. Although polyamine depletion decreased the expression of extracellular signal-regulated kinase (ERK)-2 protein, a sustained increase in ERK-2 isoform activity was observed. The ERK-1 protein level did not change, but ERK-1 activity was increased in polyamine-depleted cells. In addition, polyamine depletion induced the stress-activated protein kinase/c-Jun NH2-terminal kinase (JNK) type of mitogen-activated protein kinase (MAPK). Activation of JNK-1 was the earliest event; within 5 h after DFMO treatment, JNK activity was increased by 150%. The above results indicate that polyamine depletion causes cell cycle arrest and upregulates cell cycle inhibitors and suggest that MAPK and JNK may be involved in the regulation of the activity of these molecules.

Animals↗
PubMed2002

Heat shock induces intestinal-type alkaline phosphatase in rat IEC-18 cells.

We demonstrate a previously unknown regulation for intestinal-type alkaline phosphatase (IAP) as a heat shock protein (HSP). Heat shock to rat intestinal epithelial cells (IEC)-18 at 43 degrees C induced the expression of IAP-I and HSP72 mRNAs time dependently (<60 min) but did not induce expression of IAP-II, tissue nonspecific-type alkaline phosphatase (TNAP), or HSP90 as determined by the RT-PCR method. To confirm the identity of the IAP-I gene, we sequenced the amplification product of IAP-I and found the gene to have 99% homology with the sequence of the IAP-I gene in rat intestine. Under the subculture conditions used, no IAP protein was detected in IEC-18 cells, but it became detectable as a 62-kDa band on a Western blot after heat shock. IAP-I was also induced by sodium arsenite, which generates reactive oxygen species and is an inducer of members of the HSP family. Glutathione suppressed activating protein-1 and cAMP response element-binding protein activation caused by heat shock but did not suppress the expression of IAP-I. These results suggest that cellular stress induces the elevation of IAP-I mRNA and protein synthesis. IAP-I may play an important role as a dephosphorylating enzyme under stress conditions.

Alkaline Phosphatase↗
PubMed1992

Protein kinase C regulation of IEC-6 cell ornithine decarboxylase.

Experiments were performed to immunologically identify protein kinase C (PKC) in cultured IEC-6 cells. Polyclonal antibodies specific to PKC revealed an immunoreactive band of approximately 84 kDa in both cytosolic and solubilized particulate fractions. Treatment with phorbol 12-myristate 13-acetate (PMA; 10 nM x 60 min) increased the intensity of the 84-kDa band by 25% in the solubilized particulate fraction while decreasing it by 36% in the cytosolic fraction. Prolonged 24-h treatment with 300 nM PMA completely abolished the 84-kDa band in both fractions. Isoform-specific antisera demonstrated that alpha- and epsilon-isoforms of PKC were expressed in IEC-6 cells. Treatment of quiescent cultures with PMA induced a maximal 400% increase in ornithine decarboxylase (ODC) activity. Similarly, addition of exogenous phospholipase C (PLC) to quiescent cells stimulated ODC activity. Downregulation of PKC with 300 nM PMA x 24 h inhibited basal, serum, and PLC-stimulated ODC activity by 70%. Northern analysis revealed that PKC downregulation was correlated with a marked reduction in ODC mRNA levels, suggesting regulation of ODC enzyme at this level. Despite their ability to modulate ODC activity in quiescent cultures, neither PMA nor PLC induced [3H]thymidine incorporation at 24 h. Furthermore, downregulation of PKC did not attenuate thymidine incorporation. However, chronic PMA treatment caused the cells to contact-inhibit at a 30% lower cell density, 3.16 x 10(6) vs. 2.1 x 10(6) cells/35-mm plate, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Western↗
PubMed1993

Comparison of receptors for Escherichia coli heat-stable enterotoxin: novel receptor present in IEC-6 cells.

Enterotoxigenic Escherichia coli elaborate a heat-stable enterotoxin that causes diarrhea in humans and animals. The primary event in the diarrheal cascade is the binding of this enterotoxin to specific receptors on enterocytes and activation of guanylyl cyclase. Two intestinal cell lines, Caco-2 and IEC-6, were tested for the presence of these receptors. Although both cell lines exhibited specific binding, only the Caco-2 cell line responded to heat-stable enterotoxin with increased guanylyl cyclase activity. Cloning and expression studies confirmed that the receptor present in Caco-2 cells is a homologue of guanylyl cyclase C, a known transmembrane heat-stable enterotoxin receptor. Expression of the receptor in differentiating Caco-2 cells increases with cell maturation, indicating that these cells are a suitable model for future studies. However, Northern and polymerase chain reaction analyses demonstrated that guanylyl cyclase C is not expressed in IEC-6 cells, strongly suggesting the presence of a novel heat-stable enterotoxin receptor that is not coupled to guanylyl cyclase activity.

Amino Acid Sequence↗
PubMed1997

Polyamines are important for attachment of IEC-6 cells to extracellular matrix.

The inhibition of ornithine decarboxylase, a rate-limiting enzyme of polyamine biosynthesis, with alpha-difluoromethylornithine in IEC-6 cells (small intestinal crypt cell line) reduces cell migration by 70%, inhibits protein cross-linking, and affects the cytoskeletal assembly. The current study examines the effects of intracellular polyamine depletion on attachment of IEC-6 cells to different matrices. Polyamine deficiency inhibited cell attachment to plastic, laminin, fibronectin, collagen IV, and Matrigel by different extents. Intracellular putrescine restored attachment to all matrices. The presence of a specific inhibitor of protein cross-linking also inhibited attachment to laminin in a dose-dependent manner. The inhibition of cell attachment to plastic and Matrigel was correlated with the inhibition of cell migration. Immunofluorescence studies showed that polyamines are essential for the correct expression of the integrin subunit alpha 2 but not for the expression of the alpha 1-subunit. This study demonstrates that polyamines are important for cell attachment and expression of the integrin alpha 2 beta 1, a putative receptor for collagen and laminin. The impairment of protein cross-linking and the inhibition of the expression of cell surface receptors that bind extracellular matrix (ECM) proteins may be part of the mechanism by which polyamine deficiency retards cell migration in the small intestine.

Animals↗
PubMed2000

Putrescine does not support the migration and growth of IEC-6 cells.

The migration of IEC-6 cells is inhibited when the cells are depleted of polyamines by inhibiting ornithine decarboxylase with alpha-difluoromethylornithine (DFMO). Exogenous putrescine, spermidine, and spermine completely restore cell migration inhibited by DFMO. Because polyamines are interconverted during their synthesis and catabolism, the specific role of individual polyamines in intestinal cell migration, as well as growth, remains unclear. In this study, we used an inhibitor of S-adenosylmethionine decarboxylase, diethylglyoxal bis(guanylhydrazone)(DEGBG), to block the synthesis of spermidine and spermine from putrescine. We found that exogenous putrescine does not restore migration and growth of IEC-6 cells treated with DFMO plus DEGBG, whereas exogenous spermine does. In addition, the normal distribution of actin filaments required for migration, which is disrupted in polyamine-deficient cells, could be achieved by adding spermine but not putrescine along with DFMO and DEGBG. These results indicate that putrescine, by itself, is not essential for migration and growth, but that it is effective because it is converted into spermidine and/or spermine.

Actins↗
PubMed2001

Interleukin-1beta enhances retinoic acid-mediated expression of bone-type alkaline phosphatase in rat IEC-6 cells.

We previously showed that vitamin A upregulated the expression of bone-type alkaline phosphatase (ALP) in fetal rat small intestine and rat intestinal IEC-6 cells. In this study, we examined interactions between retinoic acid (RA) and several growth factors/cytokines on the isozyme expression in IEC-6 cells. Epidermal growth factor and interleukins (ILs)-2, -4, -5, and -6 completely blocked the RA-mediated increase in ALP activity. In contrast, IL-1beta markedly increased the activity, protein, and mRNA of the bone-type ALP only when RA was present. IL-1beta and/or RA did not change the type 1 IL-1 receptor transcript level, whereas IL-1beta enhanced the RA-induced expressions of retinoic acid receptor-beta (RAR-beta) and retinoid X receptor-beta (RXR-beta) mRNAs and RA-mediated RXR response element binding. The synergism of IL-1beta and RA on ALP activity was completely blocked by protein kinase C (PKC) inhibitors. Our results suggest that IL-1beta may modify the ALP isozyme expression in small intestinal epithelial cells by stimulating PKC-dependent, RAR-beta- and/or RXR-beta-mediated signaling pathways.

Alkaline Phosphatase↗
PubMed2004

Involvement of G protein-coupled receptor kinase 4 and 6 in rapid desensitization of dopamine D1 receptor in rat IEC-6 intestinal epithelial cells.

Dopamine-induced inhibition of Na(+)-K(+)-ATPase has been suggested to play a role in the regulation of Na(+) absorption at the intestinal level, and these effects were mediated by dopamine D(1)-like receptors. The aim of this work was to evaluate the effect of the activation of the D(1)-like receptors on the activity of the Na(+)/H(+) exchanger (NHE) in the rat intestinal epithelial cell line IEC-6. The presence of D(1) receptors was confirmed by immunoblotting. The dopamine D(1)-like receptor agonist SKF-38393 produced a concentration-dependent inhibition of NHE activity and stimulation of adenylyl cyclase (AC), this being antagonized by the D(1) selective antagonist SKF-83566. Effects of SKF-38393 on NHE and AC activities were maximal at 5 min of exposure to the agonist and rapidly diminished with no effect at 25 min. Exposure of cells for 25 min to dibutyryl-cAMP (0.5 mM) or to the AC activator forskolin (3 microM) effectively inhibited NHE activity. Pretreatment of cells with heparin (1 microM), a nonselective G protein-coupled receptor kinase (GRK) inhibitor, prevented the loss of effects on NHE activity after 25 min exposure to SKF-38393. The presence of GRK4, GRK6A, and GRK6B was confirmed by immunoblotting. Overnight treatment with the anti-GRK4-6 antibody complexed with Lipofectin was also effective in preventing loss of the effects of SKF-38393 on NHE and AC activities. It is concluded that dopamine D(1) receptors in IEC-6 rapidly desensitize to D(1)-like agonist stimulation and GRK4 and 6 appear to be involved in agonist-mediated responsiveness and desensitization.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1990

Apparent post-transcriptional modification of ornithine decarboxylase accounts for its induction in IEC-6 cells in culture.

Putrescine, the product of the ornithine decarboxylase (ODC)-catalyzed reaction, stimulates macromolecular synthesis in a duodenal crypt cell line, IEC-6 cells, grown in culture. In addition, supplementation of medium with putrescine alone reverses the inhibition of proliferation produced by inhibition of ODC with difluoromethylornithine (DFMO). A series of experiments was initiated in the IEC-6 cell line to study the regulation of induction of ODC, as this enzyme is rate-limiting in putrescine synthesis. Five percent fetal bovine serum (FBS) and 10 nM IGF-II stimulated a 21-fold and 6-fold induction of ODC activity, respectively. Kinetic analysis indicated that the effect was on the Vmax of the reaction and not on the Km, suggesting an increase in total ODC protein. This was verified by measuring [3H]DFMO binding; serum-stimulated induction of activity was accompanied by a corresponding 20-fold increase in the specific binding of DFMO to ODC. In contrast, Northern analysis demonstrated only a two-fold change in ODC mRNA level during induction. Measurement of enzyme stability showed that the half-life of the ODC protein was increased three-fold above basal level in the induced state. Inhibition of induction was produced by pretreatment with either the calmodulin antagonist, W-7, or the product of the ODC-catalyzed reaction, putrescine. Further analysis illustrated that the inhibition produced by these agents was partly the result of destabilization of the enzyme and not a decrease in message level. These results demonstrate that the induction of ODC by trophic agents is the result of post-transcriptional events rather than at the level of RNA synthesis.

Animals↗