Apical leptin induces chloride secretion by intestinal epithelial cells and in a rat model of acute chemotherapy-induced colitis

The purpose of this study was to investigate whether luminal leptin alters ion transport properties of the intestinal epithelium under acute inflammatory conditions. Monolayers of human intestinal T84 epithelial cells and a rat model of chemotherapy-induced enterocolitis were used. Cells were treated with leptin and mounted in Ussing chambers to measure basal and secretagogue-induced changes in transepithelial short-circuit current ( Isc). Furthermore, the role of MAPK and phosphatidylinositol 3-kinase (PI3K) signaling pathways in mediating responses to leptin was investigated. Acute colitis in Sprague-Dawley rats was induced by intraperitoneal injection of 40 mg/kg methotrexate. Leptin (100 ng/ml) induced a time-dependent increase in basal Isc in T84 intestinal epithelial cells ( P < 0.01). Moreover, pretreatment of T84 cells with leptin for up to 1 h significantly potentiated carbachol- and forskolin-induced increases in Isc. Pretreatment with an inhibitor of MAPK abolished the effect of leptin on basal, carbachol- and forskolin-induced chloride secretion ( P < 0.05). However, the PI3K inhibitor, wortmannin, only blunted the effect of leptin on forskolin-induced increases in Isc. Furthermore, leptin treatment evoked both ERK1/2 and Akt1 phosphorylation in T84 cells. In the rat model, luminal leptin induced significant increases in Isc across segments of proximal and, to a lesser extent, distal colon ( P < 0.05). We conclude that luminal leptin is likely an intestinal chloride secretagogue, particularly when present at elevated concentrations and/or in the setting of inflammation. Our findings may provide a mechanistic explanation, at least in part, for the clinical condition of secretory diarrhea both in hyperleptinemic obese patients and in patients with chemotherapy-induced intestinal inflammation.

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EGF receptor transactivation and MAP kinase mediate proteinase-activated receptor-2-induced chloride secretion in intestinal epithelial cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00303.2007 ◽

2008 ◽

Vol 294 (2) ◽

pp. G441-G451 ◽

Cited By ~ 26

Author(s):

Jacques Q. van der Merwe ◽

Morley D. Hollenberg ◽

Wallace K. MacNaughton

Keyword(s):

Epithelial Cells ◽

Tyrosine Kinases ◽

Egf Receptor ◽

Intestinal Epithelial Cells ◽

Short Circuit ◽

Short Circuit Current ◽

Immunoblot Analysis ◽

Intestinal Epithelial ◽

Ussing Chambers ◽

Intracellular Ca

We examined the stimulus-secretion pathways whereby proteinase-activated receptor 2 (PAR-2) stimulates Cl− secretion in intestinal epithelial cells. SCBN and T84 epithelial monolayers grown on Snapwell supports and mounted in modified Ussing chambers were activated by the PAR-2-activating peptides SLIGRL-NH2 and 2-furoyl-LIGRLO-NH2. Short-circuit current ( Isc) was used as a measure of net electrogenic ion transport. Basolateral, but not apical, application of SLIGRL-NH2 or 2-furoyl-LIGRLO-NH2 caused a concentration-dependent change in Isc that was significantly reduced in Cl−-free buffer and by the intracellular Ca2+ blockers thapsigargin and BAPTA-AM, but not by the Ca2+ channel blocker verapamil. Inhibitors of PKA (H-89) and CFTR (glibenclamide) also significantly reduced PAR-2-stimulated Cl− transport. PAR-2 activation was associated with increases in cAMP and intracellular Ca2+. Immunoblot analysis revealed increases in phosphorylation of epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase, Src, Pyk2, cRaf, and ERK1/2 in response to PAR-2 activation. Pretreatment with inhibitors of cyclooxygenases (indomethacin), tyrosine kinases (genistein), EGFR (PD-153035), MEK (PD-98059 or U-0126), and Src (PP1) inhibited SLIGRL-NH2-induced increases in Isc. Inhibition of Src, but not matrix metalloproteinases, reduced EGFR phosphorylation. Reduced EGFR phosphorylation paralleled the reduction in PAR-2-stimulated Isc. We conclude that activation of basolateral, but not apical, PAR-2 induces epithelial Cl− secretion via cAMP- and Ca2+-dependent mechanisms. The secretory effect involves EGFR transactivation by Src, leading to subsequent ERK1/2 activation and increased cyclooxygenase activity.

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Phosphatidylinositol 3-Kinase-dependent Pathways Oppose Fas-induced Apoptosis and Limit Chloride Secretion in Human Intestinal Epithelial Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m106226200 ◽

2001 ◽

Vol 276 (50) ◽

pp. 47563-47574 ◽

Cited By ~ 16

Author(s):

Maria T. Abreu ◽

Elizabeth T. Arnold ◽

Jimmy Y. C. Chow ◽

Kim E. Barrett

Keyword(s):

Epithelial Cells ◽

Barrier Function ◽

Intestinal Inflammation ◽

Short Circuit ◽

Chloride Secretion ◽

Pi3 Kinase ◽

Secretory Diarrhea ◽

Cross Linking ◽

Barrier Dysfunction ◽

Kinase Inhibition

The epithelial lining of the intestine serves as a barrier to lumenal bacteria and can be compromised by pathologic Fas-mediated epithelial apoptosis. Phosphatidylinositol (PI)3-kinase signaling has been described to limit apoptosis in other systems. We hypothesized that PI3-kinase-dependent pathways regulate Fas-mediated apoptosis and barrier function in intestiynal epithelial cells (IEC). IEC lines (HT-29 and T84) were exposed to agonist anti-Fas antibody in the presence or absence of chemical inhibitors of PI3-kinase (LY294002 and wortmannin). Apoptosis, barrier function, changes in short circuit current (ΔIsc), and expression of adhesion molecules were assessed. Inhibition of PI3-kinase strongly sensitized IEC to Fas-mediated apoptosis. Expression of constitutively active Akt, a principal downstream effector of the PI3-kinase pathway, protected against Fas-mediated apoptosis to an extent that was comparable with expression of a genetic caspase inhibitor, p35. PI3-kinase inhibition sensitized to apoptosis by increasing and accelerating Fas-mediated caspase activation. Inhibition of PI3-kinase combined with cross-linking Fas was associated with increased permeability to molecules that were <400 Da but not those that were >3,000 Da. Inhibition of PI3-kinase resulted in chloride secretion that was augmented by cross-linking Fas. Confocal analyses revealed polymerization of actin and maintenance of epithelial cell adhesion molecule-mediated interactions in monolayers exposed to anti-Fas antibody in the context of PI3-kinase inhibition. PI3-kinase-dependent pathways, especially Akt, protect IEC against Fas-mediated apoptosis. Inhibition of PI3-kinase in the context of Fas signaling results in increased chloride secretion and barrier dysfunction. These findings suggest that agonists of PI3-kinase such as growth factors may have a dual effect on intestinal inflammation by protecting epithelial cells against immune-mediated apoptosis and limiting chloride secretory diarrhea.

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Adenylyl Cyclase 6 Expression Is Essential for Cholera Toxin–Induced Diarrhea

The Journal of Infectious Diseases ◽

10.1093/infdis/jiz013 ◽

2019 ◽

Vol 220 (11) ◽

pp. 1719-1728 ◽

Cited By ~ 4

Author(s):

Robert A Fenton ◽

Sathish K Murali ◽

Izumi Kaji ◽

Yasutada Akiba ◽

Jonathan D Kaunitz ◽

...

Keyword(s):

Epithelial Cells ◽

Adenylyl Cyclase ◽

Cholera Toxin ◽

Knockout Mice ◽

Intestinal Epithelial Cells ◽

Short Circuit ◽

Secretory Diarrhea ◽

Intestinal Epithelial ◽

Α Subunit ◽

Small Intestinal

AbstractBackgroundCholera toxin (CT)–induced diarrhea is mediated by cyclic adenosine monophosphate (cAMP)–mediated active Cl– secretion via the cystic fibrosis transmembrane conductance regulator (CFTR). Although the constitutive activation of adenylyl cyclase (AC) in response to CT is due to adenosine diphosphate ribosylation of the small G protein α-subunit activating CFTR with consequent secretory diarrhea, the AC isoform(s) involved remain unknown.MethodsWe generated intestinal epithelial cell–specific adenylyl cyclase 6 (AC6) knockout mice to study its role in CT-induced diarrhea.ResultsAC6 messenger RNA levels were the highest of all 9 membrane-bound AC isoforms in mouse intestinal epithelial cells. Intestinal epithelial-specific AC6 knockout mice (AC6loxloxVillinCre) had undetectable AC6 levels in small intestinal and colonic epithelial cells. No significant differences in fluid and food intake, plasma electrolytes, intestinal/colon anatomy and morphology, or fecal water content were observed between genotypes. Nevertheless, CT-induced fluid accumulation in vivo was completely absent in AC6loxloxVillinCre mice, associated with a lack of forskolin- and CT-induced changes in the short-circuit current (ISC) of the intestinal mucosa, impaired cAMP generation in acutely isolated small intestinal epithelial cells, and significantly impaired apical CFTR levels in response to forskolin.ConclusionsAC6 is a novel target for the treatment of CT-induced diarrhea.

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Rotavirus infection induces increased chloride secretion, altered barrier function and epidermal growth factor receptor (EGF-R) polyubiquitination in intestinal epithelial cells (IEC)

Gastroenterology ◽

10.1016/s0016-5085(01)83508-3 ◽

2001 ◽

Vol 120 (5) ◽

pp. A704-A705

Author(s):

S RESTALENERT ◽

K BARRETT

Keyword(s):

Epidermal Growth Factor Receptor ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Epithelial Cells ◽

Barrier Function ◽

Intestinal Epithelial Cells ◽

Growth Factor Receptor ◽

Chloride Secretion ◽

Intestinal Epithelial ◽

Epidermal Growth

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PI3'-kinase- and fas-dependent pathways regulate apoplosis, barrier function and chloride secretion in intestinal epithelial cells (IEC): Implications for inflammatory diarrheal states

Gastroenterology ◽

10.1016/s0016-5085(01)80613-2 ◽

2001 ◽

Vol 120 (5) ◽

pp. A125-A125

Author(s):

M ABREU ◽

E ARNOLD ◽

J CHOW ◽

K BARRETT

Keyword(s):

Epithelial Cells ◽

Barrier Function ◽

Intestinal Epithelial Cells ◽

Chloride Secretion ◽

Pi3 Kinase ◽

Intestinal Epithelial

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Corrigendum to “Let-7b ameliorates Crohn’s disease-associated adherent-invasive E coli induced intestinal inflammation via modulating Toll-Like Receptor 4 expression in intestinal epithelial cells” [Biochem. Pharmacol. 15 (2018) 196–203]

Biochemical Pharmacology ◽

10.1016/j.bcp.2018.09.025 ◽

2018 ◽

Vol 158 ◽

pp. 35-36

Author(s):

Zhen Guo ◽

Xingchen Cai ◽

Xian Guo ◽

Yihan Xu ◽

Jianfeng Gong ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Epithelial Cells ◽

Intestinal Inflammation ◽

Intestinal Epithelial Cells ◽

Intestinal Epithelial ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

E Coli

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Unique Regulation of Enterocyte Brush Border Membrane Na-Glutamine and Na-Alanine Co-Transport by Peroxynitrite during Chronic Intestinal Inflammation

International Journal of Molecular Sciences ◽

10.3390/ijms20061504 ◽

2019 ◽

Vol 20 (6) ◽

pp. 1504 ◽

Cited By ~ 1

Author(s):

Subha Arthur ◽

Palanikumar Manoharan ◽

Shanmuga Sundaram ◽

M Rahman ◽

Balasubramanian Palaniappan ◽

...

Keyword(s):

Epithelial Cells ◽

Brush Border ◽

Brush Border Membrane ◽

Intestinal Inflammation ◽

Intestinal Epithelial Cells ◽

Rabbit Model ◽

Intestinal Epithelial ◽

Mechanism Of Inhibition ◽

Chronic Intestinal Inflammation

Na-amino acid co-transporters (NaAAcT) are uniquely affected in rabbit intestinal villus cell brush border membrane (BBM) during chronic intestinal inflammation. Specifically, Na-alanine co-transport (ASCT1) is inhibited secondary to a reduction in the affinity of the co-transporter for alanine, whereas Na-glutamine co-transport (B0AT1) is inhibited secondary to a reduction in BBM co-transporter numbers. During chronic intestinal inflammation, there is abundant production of the potent oxidant peroxynitrite (OONO). However, whether OONO mediates the unique alteration in NaAAcT in intestinal epithelial cells during chronic intestinal inflammation is unknown. In this study, ASCT1 and B0AT1 were inhibited by OONO in vitro. The mechanism of inhibition of ASCT1 by OONO was secondary to a reduction in the affinity of the co-transporter for alanine, and secondary to a reduction in the number of co-transporters for B0AT1, which were further confirmed by Western blot analyses. In conclusion, peroxynitrite inhibited both BBM ASCT1 and B0AT1 in intestinal epithelial cells but by different mechanisms. These alterations in the villus cells are similar to those seen in the rabbit model of chronic enteritis. Therefore, this study indicates that peroxynitrite may mediate the inhibition of ASCT1 and B0AT1 during inflammation, when OONO levels are known to be elevated in the mucosa.

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7 LACTOBACILLUS REUTERI SUPPRESSES PRO-INFLAMMATORY DRIVEN REACTIVE OXYGEN SPECIES IN VITRO IN HUMAN INTESTINAL EPITHELIAL CELLS AND IN VIVO IN A TNBS COLITIS MOUSE MODEL

Inflammatory Bowel Diseases ◽

10.1093/ibd/zaa010.106 ◽

2020 ◽

Vol 26 (Supplement_1) ◽

pp. S41-S41 ◽

Cited By ~ 1

Author(s):

Wenly Ruan ◽

Melinda Engevik ◽

Alexandra Chang-Graham ◽

Joseph Hyser ◽

James Versalovic

Keyword(s):

Reactive Oxygen Species ◽

Epithelial Cells ◽

Lactobacillus Reuteri ◽

Intestinal Inflammation ◽

Intestinal Epithelial Cells ◽

Oxygen Species ◽

Intestinal Epithelial ◽

Colitis Model

Abstract Background Reactive oxygen species (ROS) play a role in maintaining intestinal epithelial homeostasis and are normally kept at low levels via antioxidant compounds. Dysregulation of ROS can lead to intestinal inflammation and contribute to inflammatory bowel disease (IBD). Select gut microbes possess the enzymatic machinery to produce antioxidants whereas others can dysregulate levels of ROS. Our model microbe, Lactobacillus reuteri (ATCC PTA 6475), has been demonstrated to reduce intestinal inflammation in mice models. It contains the genes encoding two distinct GshA-like glutamylcysteine ligases. We hypothesize that L. reuteri can secrete γ-glutamylcysteine to suppress ROS, minimize NFκB activation and regulate secretion of e pithelial cytokines. Methods & Results Conditioned media from L. reuteri was analyzed via mass spectrometry to confirm the presence of γ-glutamylcysteine. All cysteine containing products including γ-glutamylcysteine were fluorescently tagged in the conditioned media and then incubated with HT29 cell monolayers as well as human jejunal enteroid (HJE) monolayers. γ-glutamylcysteine was demonstrated to enter intestinal epithelial cells based on microscopy. Next, a Thioltracker assay was used to show increased intracellular glutathione levels by L. reuteri secreted γ-glutamylcysteine. HT29 cells and HJEs were then treated with IL-1β or hydrogen peroxide, and L. reuteri metabolites as well as γ-glutamylcysteine significantly suppressed pro-inflammatory cytokine driven ROS and IL-8 production. L. reuteri secreted products also reduced activity of NFκB as determined by a luciferase reporter assay. γ-glutamylcysteine deficient mutants were generated by targeted mutagenesis of GshA genes, and these mutant L. reuteri strains had a diminished ability to suppress IL-8 production and ROS. To further test the role of L. reuteri secreted γ-glutamylcysteine in vivo, a 2,4,6-Trinitrobenzenesulfonic acid (TNBS)- induced mouse colitis model was used. Adolescent mice were orogavaged with PBS, L. reuteri, L. reuteri GshA2 mutant, or γ-glutamylcysteine for a week after which TNBS was rectally administered to induce colitis. We demonstrate that L. reuteri and γ-glutamylcysteine can suppress histologic inflammation compared to PBS control and L. reuteri GshA2 mutant groups. Conclusions Together these data indicate that L. reuteri secretes γ-glutamylcysteine which can enter the intestinal epithelial cells and modulate epithelial cytokine production. It acts via suppression of ROS and NFκB which then decreases IL-8 production. We are able to demonstrate this in vitro in both HT 29 cells and HJEs. We now also demonstrate this in vivo in a mouse colitis model. These experiments highlight a prominent role for ROS intermediates in microbiome-mammalian cell signaling processes involved in immune responses and intestinal inflammation.

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Tin protoporphyrin induces intestinal chloride secretion by inducing light oxidation processes

AJP Cell Physiology ◽

10.1152/ajpcell.00550.2006 ◽

2007 ◽

Vol 292 (5) ◽

pp. C1906-C1914 ◽

Cited By ~ 5

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.

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