scholarly journals Campylobacter concisus Impairs Sodium Absorption in Colonic Epithelium via ENaC Dysfunction and Claudin-8 Disruption

2020 ◽  
Vol 21 (2) ◽  
pp. 373 ◽  
Author(s):  
Praveen Kumar Nattramilarasu ◽  
Roland Bücker ◽  
Fábia Daniela Lobo de Sá ◽  
Anja Fromm ◽  
Oliver Nagel ◽  
...  
Keyword(s):  
Cell Model ◽  
Short Circuit ◽  
Intestinal Transport ◽  
Intestinal Lumen ◽  
Sodium Absorption ◽  
Epithelial Cell Line ◽  
Barrier Dysfunction ◽  
Campylobacter Concisus ◽  
Ussing Chambers ◽  
Ht 29

The epithelial sodium channel (ENaC) can increase the colonic absorptive capacity for salt and water. Campylobacter concisus is a common pathogenic epsilonproteobacterium, causing enteritis and diarrhea. It can induce barrier dysfunction in the intestine, but its influence on intestinal transport function is still unknown. Therefore, our study aimed to characterize C. concisus effects on ENaC using the HT-29/B6-GR/MR (epithelial cell line HT-29/B6 transfected with glucocorticoid and mineralocorticoid receptors) cell model and mouse colon. In Ussing chambers, C. concisus infection inhibited ENaC-dependent Na+ transport as indicated by a reduction in amiloride-sensitive short circuit current (−55%, n = 15, p < 0.001). This occurred via down-regulation of β- and γ-ENaC mRNA expression and ENaC ubiquitination due to extracellular signal-regulated kinase (ERK)1/2 activation, predicted by Ingenuity Pathway Analysis (IPA). In parallel, C. concisus reduced the expression of the sealing tight junction (TJ) protein claudin-8 and induced claudin-8 redistribution off the TJ domain of the enterocytes, which facilitates the back leakage of Na+ ions into the intestinal lumen. In conclusion, C. concisus caused ENaC dysfunction via interleukin-32-regulated ERK1/2, as well as claudin-8-dependent barrier dysfunction—both of which contribute to Na+ malabsorption and diarrhea.

Tin protoporphyrin induces intestinal chloride secretion by inducing light oxidation processes

2007 ◽  
Vol 292 (5) ◽  
pp. C1906-C1914 ◽  
Author(s):  
Aliye Uc ◽  
Krzysztof J. Reszka ◽  
Garry R. Buettner ◽  
John B. Stokes
Keyword(s):  
Short Circuit ◽  
Specific Inhibitor ◽  
Chloride Secretion ◽  
Epithelial Cell Line ◽  
Intestinal Epithelial ◽  
Paramagnetic Resonance ◽  
Ussing Chambers ◽  
Oxidative Processes ◽  
Tin Protoporphyrin ◽  
Intestinal Chloride Secretion

Heme induces Cl− secretion in intestinal epithelial cells, most likely via carbon monoxide (CO) generation. The major source of endogenous CO comes from the degradation of heme via heme oxygenase (HO). We hypothesized that an inhibitor of HO activity, tin protoporphyrin (SnPP), may inhibit the stimulatory effect of heme on Cl− secretion. To test this hypothesis, we treated an intestinal epithelial cell line (Caco-2 cells) with SnPP. In contrast to our expectations, Caco-2 cells treated with SnPP had an increase in their short-circuit currents ( Isc) in Ussing chambers. This effect was observed only when the system was exposed to ambient light. SnPP-induced Isc was caused by Cl− secretion because it was inhibited in Cl−-free medium, with ouabain or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). The Cl− secretion was not via activation of the CFTR, because a specific inhibitor had no effect. Likewise, inhibitors of adenylate cyclase and guanylate cyclase had no effect on the enhanced Isc. SnPP-induced Isc was inhibited by the antioxidant vitamins, α-tocopherol and ascorbic acid. Electron paramagnetic resonance experiments confirmed that oxidative reactions were initiated with light in cells loaded with SnPP. These data suggest that SnPP-induced effects may not be entirely due to the inhibition of HO activity but rather to light-induced oxidative processes. These novel effects of SnPP-photosensitized oxidation may also lead to a new understanding of how intestinal Cl− secretion can be regulated by the redox environment of the cell.

Protease-activated receptor-1 stimulates Ca2+-dependent Cl−secretion in human intestinal epithelial cells

2001 ◽  
Vol 281 (2) ◽  
pp. G323-G332 ◽  
Author(s):  
M. C. Buresi ◽  
E. Schleihauf ◽  
N. Vergnolle ◽  
A. Buret ◽  
J. L. Wallace ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Short Circuit ◽  
Intestinal Lumen ◽  
Epithelial Cell Line ◽  
Intracellular Camp ◽  
Intestinal Epithelial ◽  
Rt Pcr ◽  
Human Intestinal Epithelial Cells ◽  
Protease Activated Receptor 1

The thrombin receptor, protease-activated receptor-1 (PAR-1), has wide tissue distribution and is involved in many physiological functions. Because thrombin is in the intestinal lumen and mucosa during inflammation, we sought to determine PAR-1 expression and function in human intestinal epithelial cells. RT-PCR showed PAR-1 mRNA expression in SCBN cells, a nontransformed duodenal epithelial cell line. Confluent SCBN monolayers mounted in Ussing chambers responded to PAR-1 activation with a Cl−-dependent increase in short-circuit current. The secretory effect was blocked by BaCl2and the Ca2+-ATPase inhibitor thapsigargin, but not by the L-type Ca2+channel blocker verapamil or DIDS, the nonselective inhibitor of Ca2+-dependent Cl−transport. Responses to thrombin and PAR-1-activating peptides exhibited auto- and crossdesensitization. Fura 2-loaded SCBN cells had increased fluorescence after PAR-1 activation, indicating increased intracellular Ca2+. RT-PCR showed that SCBN cells expressed mRNA for the cystic fibrosis transmembrane conductance regulator (CFTR) and hypotonicity-activated Cl−channel-2 but not for the Ca2+-dependent Cl−channel-1. PAR-1 activation failed to increase intracellular cAMP, suggesting that the CFTR channel is not involved in the Cl−secretory response. Our data demonstrate that PAR-1 is expressed on human intestinal epithelial cells and regulates a novel Ca2+-dependent Cl−secretory pathway. This may be of clinical significance in inflammatory intestinal diseases with elevated thrombin levels.

Basolateral Mg2+/Na+ exchange regulates apical nonselective cation channel in sheep rumen epithelium via cytosolic Mg2+

2005 ◽  
Vol 288 (4) ◽  
pp. G630-G645 ◽  
Author(s):  
Sabine Leonhard-Marek ◽  
Friederike Stumpff ◽  
Inge Brinkmann ◽  
Gerhard Breves ◽  
Holger Martens
Keyword(s):  
Apical Membrane ◽  
Divalent Cations ◽  
Short Circuit ◽  
Ussing Chamber ◽  
External Solution ◽  
Sodium Absorption ◽  
High Potassium ◽  
Subsequent Increase ◽  
Ussing Chambers ◽  
Ruminal Epithelium

High potassium diets lead to an inverse regulation of sodium and magnesium absorption in ruminants, suggesting some form of cross talk. Previous Ussing chamber experiments have demonstrated a divalent sensitive Na+ conductance in the apical membrane of ruminal epithelium. Using patch-clamped ruminal epithelial cells, we could observe a divalent sensitive, nonselective cation conductance (NSCC) with K+ permeability > Cs+ permeability > Na+ permeability. Conductance increased and rectification decreased when either Mg2+ or both Ca2+ and Mg2+ were removed from the internal or external solution or both. The conductance could be blocked by Ba2+, but not by tetraethylammonium (TEA). Subsequently, we studied this conductance measured as short-circuit current ( Isc) in Ussing chambers. Forskolin, IBMX, and theophylline are known to block both Isc and Na transport across ruminal epithelium in the presence of divalent cations. When the NSCC was stimulated by removing mucosal calcium, an initial decrease in Isc was followed by a subsequent increase. The cAMP-mediated increase in Isc was reduced by low serosal Na+ and serosal addition of imipramine or serosal amiloride and depended on the availability of mucosal magnesium. Luminal amiloride had no effect. Flux studies showed that low serosal Na+ reduced 28Mg fluxes from mucosal to serosal. The data suggest that cAMP stimulates basolateral Na+/Mg2+ exchange, reducing cytosolic Mg. This increases sodium uptake through a magnesium-sensitive NSCC in the apical membrane. Likewise, the reduction in magnesium uptake that follows ingestion of high potassium fodder may facilitate sodium absorption, as observed in studies of ruminal osmoregulation. Possibly, grass tetany (hypomagnesemia) is a side effect of this useful mechanism.

DUSP16 IS A NOVEL IBD GENE IMPLICATED IN THE REGULATION OF DIFFERENTIATION AND HOMEOSTASIS OF INTESTINAL EPITHELIAL CELLS

2021 ◽  
Vol 27 (Supplement_1) ◽  
pp. S29-S30
Author(s):  
Jessy Ntunzwenimana ◽  
Azadeh Alikashani ◽  
Claudine Beauchamp ◽  
Jean Paquette ◽  
Gabrielle Boucher ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Map Kinases ◽  
Intestinal Epithelial Cells ◽  
Cell Model ◽  
Basolateral Membrane ◽  
Intestinal Lumen ◽  
Interleukin 17 ◽  
Intestinal Epithelial ◽  
Intestinal Pathogens ◽  
Ht 29

Abstract Inflammatory bowel disease (IBD) are chronic inflammatory diseases including Crohn’s disease (CD) and ulcerative colitis (UC). More than 200 genomic regions have been identified and validated (association values〈 5x10-8) to be associated with CD, UC or IBD. These regions may contain multiple genes and the current challenge lies in identifying the causal gene in each of these. To address this problem, we performed a functional genomic screen of 145 genes from validated IBD loci, in a relevant intestinal epithelial cell model (HT-29). The results of this transcriptome-based screening revealed that the candidate IBD gene DUSP16 (a dual specificity phosphatase targeting MAP kinases (MAPKs) phosphorylation) as well as the known IBD gene KSR1 (a scaffold protein regulating the spatiotemporal activation of the ERK) regulate the expression of genes involved in intestinal differentiation and homeostasis. They induce, among others, the expression of the PIGR gene that encodes the polymeric immunoglobulin receptor. PIGR plays a role in transporting dimeric IgA molecules from the basolateral membrane of epithelial cells to the intestinal lumen, via transcytosis, where they play an essential role in protecting the epithelium against intestinal pathogens. Our hypothesis is that DUSP16 and KSR1 modulate the activity of MAPKs in intestinal epithelial cells to induce PIGR expression, thus participating in the maintenance of homeostasis of the intestinal barrier. To better understand how DUSP16 modulates the expression of PIGR, we used an approach of over- expression (cDNA) and knockdown (shRNA) of DUSP16 in HT-29 cells. Our results confirmed that DUSP16 induction increases the expression of PIGR, whereas a knockdown of DUSP16 reduces the basal level of PIGR. Next we confirmed by Western Blot that the induction of DUSP16 was accompanied by a decrease in MAPK phosphorylation. The involvement of MAPKs was also confirmed through the use of chemical inhibitors specific for each MAPK, with inhibition of ERK and p38 showing the strongest induction of PIGR expression. We are currently analyzing known functional mutants of DUSP16 and KSR1 to determine their impact on MAPK activity and on PIGR expression. This work supports a role for PIGR in disease pathogenesis, adding to two recent studies that documented that patients suffering from UC accumulated somatic mutations in a group of genes regulating the expression of PIGR by Interleukin 17. The mutated genes, including PIGR, were positively selected in inflamed tissues, indicating the importance of the biological function occupied by this gene in the maintenance of homeostasis. In conclusion, our study successfully identified functional links between two genes from independent IBD loci, and suggests that these DUSP16 and KSR1 play a role in the process of epithelial transcytosis and the development of IBD.

Monocyte/macrophages evoke epithelial dysfunction: indirect role of tumor necrosis factor-α

1998 ◽  
Vol 275 (4) ◽  
pp. C932-C939 ◽  
Author(s):  
Mehri Zareie ◽  
Derek M. McKay ◽  
Garrett G. Kovarik ◽  
Mary H. Perdue
Keyword(s):  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Tumor Necrosis Factor Α ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Epithelial Cell Line ◽  
Ussing Chambers ◽  
Tnf Α ◽  
Factor Α ◽  
Necrosis Factor

We examined the ability of monocytes (MΦ) activated by bacterial products to alter epithelial physiology. Confluent monolayers of the T84 colonic epithelial cell line were grown on filter supports and then cocultured in the presence of human MΦ with or without the activating agents bacterial lipopolysaccharide and the bacterial tripeptide formyl-methionyl-leucyl-phenylalanine. After 24 or 48 h, monolayers were mounted in Ussing chambers where parameters of epithelial function were measured. Exposure to activated MΦ resulted in a significant increase ( P < 0.05) in baseline short-circuit current (250% after 48 h) that was associated with enhanced secretion of Cl−. In addition, epithelial permeability was significantly increased as shown by reduced transepithelial resistance and increased flux of51Cr-EDTA. Activated MΦ produced substantial amounts (∼3 ng/ml at 48 h) of tumor necrosis factor-α (TNF-α). TNF-α was identified as a key mediator acting via an autocrine mechanism to induce epithelial pathophysiology. Our data show that MΦ, when activated by common bacterial components, are potent effector cells capable of initiating significant changes in the transport and barrier properties of a model epithelium.

Development of L-glutamine-stimulated electroneutral sodium absorption in piglet jejunum

1990 ◽  
Vol 259 (1) ◽  
pp. G99-G107 ◽  
Author(s):  
J. M. Rhoads ◽  
E. O. Keku ◽  
L. E. Bennett ◽  
J. Quinn ◽  
J. G. Lecce
Keyword(s):  
Small Intestine ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Electrolyte Transport ◽  
Sodium Absorption ◽  
Nacl Transport ◽  
Ussing Chambers ◽  
Nacl Absorption ◽  
Intestinal Electrolyte Transport ◽  
Colonic Transport

Glutamine is the primary metabolic fuel of the small intestine. To determine the effects of glutamine on intestinal electrolyte transport, piglet (3 days to 3 wk old) jejunum was bathed in Ussing chambers in a buffer containing 10 mM serosal glucose, and the effects of different concentrations of mucosal L-glutamine and D-glucose on short-circuit current and transmucosal Na+ and Cl- transport were measured. Resting jejunum secreted Na+ and Cl- in an electrogenic manner. In contrast to mucosal D-glucose (30 mM), which promoted electrogenic Na+ absorption (1.8 mueq.cm-2.h-1), mucosal L-glutamine (30 mM) stimulated both Na+ (2.7 mueq.cm-2.h-1) and Cl- (2.2 mueq.cm-2.h-1) absorption. This NaCl-absorptive jejunal response depended on the presence of both Na+ and Cl-, did not appear until animals were greater than 7 days of age, and was not observed with glucose, phenylalanine, or mannitol. Serosal, as well as mucosal, glutamine (30 mM) promoted electroneutral NaCl absorption. A small electrogenic Na(+)-absorptive response to L-glutamine was also observed. The effect of L-glutamine on jejunal NaCl transport resembles that of other metabolic fuels on colonic transport; its mechanism remains to be determined. We conclude that glutamine promotes electroneutral salt absorption in the small intestine.

Chronic peripheral administration of corticotropin-releasing factor causes colonic barrier dysfunction similar to psychological stress

2008 ◽  
Vol 295 (3) ◽  
pp. G452-G459 ◽  
Author(s):  
Aaron A. Teitelbaum ◽  
Mélanie G. Gareau ◽  
Jennifer Jury ◽  
Ping Chang Yang ◽  
Mary H. Perdue
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Horseradish Peroxidase ◽  
Psychological Stress ◽  
Morphological Changes ◽  
Short Circuit ◽  
Light And Electron Microscopy ◽  
Bacterial Attachment ◽  
Barrier Dysfunction ◽  
Ussing Chambers

Chronic psychological stress causes intestinal barrier dysfunction and impairs host defense mechanisms mediated by corticotrophin-releasing factor (CRF) and mast cells; however, the exact pathways involved are unclear. Here we investigated the effect of chronic CRF administration on colonic permeability and ion transport functions in rats and the role of mast cells in maintaining the abnormalities. CRF was delivered over 12 days via osmotic minipumps implanted subcutaneously in wild-type (+/+) and mast cell-deficient (Ws/Ws) rats. Colonic segments were excised for ex vivo functional studies in Ussing chambers [short-circuit current ( Isc), conductance ( G), and macromolecular permeability (horseradish peroxidase flux)], and analysis of morphological changes (mast cell numbers and bacterial host-interactions) was determined by light and electron microscopy. Chronic CRF treatment resulted in colonic mucosal dysfunction with increased Isc, G, and horseradish peroxidase flux in +/+ but not in Ws/Ws rats. Furthermore, CRF administration caused mast cell hyperplasia and abnormal bacterial attachment and/or penetration into the mucosa only in +/+ rats. Finally, selective CRF agonist/antagonist studies revealed that stimulation of CRF-R1 and CRF-R2 receptors induced the elevated secretory state and permeability dysfunction, respectively. Chronic CRF causes colonic barrier dysfunction in rats, which is mediated, at least in part, via mast cells. This information may be useful in designing novel treatment strategies for stress-related gastrointestinal disorders.

Cl- secretion in epithelial monolayers of mucus-forming human colon cells (HT-29/B6)

1991 ◽  
Vol 261 (4) ◽  
pp. C574-C582 ◽  
Author(s):  
K. M. Kreusel ◽  
M. Fromm ◽  
J. D. Schulzke ◽  
U. Hegel
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Human Colon ◽  
Channel Blockers ◽  
Cellular Mechanisms ◽  
Colon Cells ◽  
Ussing Chambers ◽  
Cl Secretion ◽  
Mucus Cells ◽  
Ht 29

HT-29, an undifferentiated human colon cell line, is known to differentiate when cultured without glucose. This study aimed to characterize ion transport in the clone HT-29/B6, which was selected from HT-29 cells differentiated by glucose-free culture. HT-29/B6 cells seeded onto filter membranes grew as polarized monolayers, mainly consisting of mucus-forming cells and exhibiting high transepithelial resistance. Short-circuit current (Isc) of unstimulated HT-29/B6 monolayers in Ussing chambers was 0.1 +/- 0.01 mumol.h-1.cm-2, and conductance was 2.0 +/- 0.2 mS/cm2. Serosal forskolin (FSK; 10(-5) M) induced a sustained Isc of 1.9 +/- 0.1 mumol.h-1.cm-2, associated with a rise of intracellular adenosine 3',5'-cyclic monophosphate (cAMP). Isc was identified as Cl- secretion by tracer studies and by the inhibitory effects of serosal bumetanide and Ba2+. The Cl- channel blockers NPPB and DPC diminished FSK-induced Isc at respective doses of 3 x 10(-4) and 10(-3) M, being effective from either side of the monolayer. Cl- secretion could be triggered by vasoactive intestinal peptide (10(-8) M), prostaglandin E1 (10(-6) M), and dibutyryl cAMP (10(-3) M) as well. In conclusion, HT-29/B6 cells grow as polarized monolayers, forming mucus and secreting Cl- in response to secretagogues. This clone may not only serve as a model for investigation of cellular mechanisms of intestinal Cl- secretion but may also be helpful to elucidate the contribution of mucus cells to this process.

scholarly journals Luminal leptin inhibits l-glutamine transport in rat small intestine: involvement of ASCT2 and B0AT1

2010 ◽  
Vol 299 (1) ◽  
pp. G179-G185 ◽  
Author(s):  
Robert Ducroc ◽  
Yassine Sakar ◽  
Carmen Fanjul ◽  
Ana Barber ◽  
André Bado ◽  
...  
Keyword(s):  
Gene Expression ◽  
Brush Border ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Intestinal Lumen ◽  
Apical Plasma Membrane ◽  
Amino Acid Transporters ◽  
Dependent Manner ◽  
Ussing Chambers ◽  
Maximum Inhibition

l-glutamine is the primary metabolic fuel for enterocytes. Glutamine from the diet is transported into the absorptive cells by two sodium-dependent neutral amino acid transporters present at the apical membrane: ASCT2/SLC1A5 and B0AT1/SLC6A19. We have demonstrated that leptin is secreted into the stomach lumen after a meal and modulates the transport of sugars after binding to its receptors located at the brush border of the enterocytes. The present study was designed to address the effect of luminal leptin on Na+-dependent glutamine (Gln) transport in rat intestine and identify the transporters involved. We found that 0.2 nM leptin inhibited uptake of Gln and phenylalanine (Phe) (substrate of B0AT1) using everted intestinal rings. In Ussing chambers, 10 mM Gln absorption followed as Na+-induced short-circuit current was inhibited by leptin in a dose-dependent manner (maximum inhibition at 10 nM; IC50 = ∼0.1 nM). Phe absorption was also decreased by leptin. Western blot analysis after 3-min incubation of the intestinal loops with 10 mM Gln, showed marked increase of ASCT2 and B0AT1 protein in the brush-border membrane that was reduced by rapid preincubation of the intestinal lumen with 1 nM leptin. Similarly, the increase in ASCT2 and B0AT1 gene expression induced by 60-min incubation of the intestine with 10 mM Gln was strongly reduced after a short preincubation period with leptin. Altogether these data demonstrate that, in rat, leptin controls the active Gln entry through reduction of both B0AT1 and ASCT2 proteins traffic to the apical plasma membrane and modulation of their gene expression.

Membrane site of action of CO2 on colonic sodium absorption

1989 ◽  
Vol 256 (3) ◽  
pp. C584-C590 ◽  
Author(s):  
A. N. Charney ◽  
R. W. Egnor
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Sodium Absorption ◽  
Ussing Chambers ◽  
Chloride Absorption ◽  
Before And After ◽  
36Cl Fluxes ◽  
Tension Increase ◽  
Ambient Co2

Increases in ambient CO2 tension increase colonic sodium absorption by increasing mucosal to serosal sodium flux. We examined the membrane site of CO2 action by utilizing the polyene antibiotic nystatin to create aqueous pores in the apical membrane. Under these conditions, the basolateral rather than the apical membrane is rate limiting for sodium absorption. Pairs of stripped rat distal colonic segments were mounted in modified Ussing chambers in a Ringer-HCO3 solution gassed with either 3% CO2-97% O2 or 11% CO2-89% O2. Mucosal-to-serosal 22Na and 36Cl fluxes were measured under short-circuited conditions, and ouabain-sensitive absorption was calculated before and after the addition of mucosal nystatin 300 U/ml. Ouabain-sensitive sodium absorption was fivefold greater at 11% CO2 than at 3% CO2 before nystatin addition. Nystatin increased short-circuit current (Isc), transcolonic conductance (Gt) and ouabain-sensitive sodium absorption at 3% CO2 but only increased Isc and Gt at 11% CO2. The levels of sodium absorption at 3% and 11% CO2 after nystatin were equal and identical to the level measured at 11% CO2 in the absence of nystatin. Ouabain-sensitive chloride absorption was similar at 3% and 11% CO2 in the absence of nystatin and was not affected by nystatin addition. These findings suggest that ambient CO2 tension affects colonic sodium absorption by a selective action at the apical membrane.

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