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

2008 ◽  
Vol 294 (2) ◽  
pp. G441-G451 ◽  
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.

Induction of Na+/K+/2Cl− cotransporter expression mediates chronic potentiation of intestinal epithelial Cl− secretion by EGF

2008 ◽  
Vol 294 (6) ◽  
pp. C1362-C1370 ◽  
Author(s):  
Fiona O'Mahony ◽  
Ferial Toumi ◽  
Magdalena S. Mroz ◽  
Gail Ferguson ◽  
Stephen J. Keely
Keyword(s):  
Time Course ◽  
Egf Receptor ◽  
Epithelial Transport ◽  
Short Circuit ◽  
Second Messengers ◽  
Short Circuit Current ◽  
Pcr Analysis ◽  
Intestinal Epithelial ◽  
Ussing Chambers ◽  
Intracellular Ca

Alterations in EGF receptor (EGFR) signaling occur in intestinal disorders associated with dysregulated epithelial transport. In the present study, we investigated a role for the EGFR in the chronic regulation of intestinal epithelial secretory function. Epithelial Cl− secretion was measured as changes in short-circuit current ( Isc) across voltage-clamped monolayers of T84 cells in Ussing chambers. Acute treatment of T84 cells with EGF (100 ng/ml, 15 min) chronically enhanced Isc responses to a broad range of secretagogues. This effect was apparent within 3 h, maximal by 6 h, and sustained for 24 h after treatment with EGF. The Na+/K+/2Cl− cotransporter (NKCC1) inhibitor bumetanide (100 μM) abolished the effect of EGF, indicating increased responses are due to potentiated Cl− secretion. Neither basal nor agonist-stimulated levels of intracellular Ca2+ or PKA activity were altered by EGF, implying that the effects of the growth factor are not due to chronic alterations in levels of second messengers. EGF increased the expression of NKCC1 with a time course similar to that of its effects on Cl− secretion. This effect of EGF was maximal after 6 h, at which time NKCC1 expression in EGF-treated cells was 199.9 ± 21.9% of that in control cells ( n = 21, P < 0.005). EGF-induced NKCC1 expression was abolished by actinomycin D, and RT-PCR analysis demonstrated EGF increased expression of NKCC1 mRNA. These data increase our understanding of mechanisms regulating intestinal fluid and electrolyte transport and reveal a novel role for the EGFR in the chronic regulation of epithelial secretory capacity through upregulation of NKCC1 expression.

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

2010 ◽  
Vol 298 (5) ◽  
pp. G714-G721 ◽  
Author(s):  
Raschid M. Hoda ◽  
Michael Scharl ◽  
Stephen J. Keely ◽  
Declan F. McCole ◽  
Kim E. Barrett
Keyword(s):  
Epithelial Cells ◽  
Rat Model ◽  
Intestinal Inflammation ◽  
Intestinal Epithelial Cells ◽  
Short Circuit ◽  
Chloride Secretion ◽  
Secretory Diarrhea ◽  
Intestinal Epithelial ◽  
Sprague Dawley ◽  
Ussing Chambers

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.

Protease-activated receptor-2 stimulates intestinal epithelial chloride transport through activation of PLC and selective PKC isoforms

2009 ◽  
Vol 296 (6) ◽  
pp. G1258-G1266 ◽  
Author(s):  
Jacques Q. van der Merwe ◽  
France Moreau ◽  
Wallace K. MacNaughton
Keyword(s):  
Serine Proteases ◽  
Chloride Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Immunoblot Analysis ◽  
Chloride Secretion ◽  
Intestinal Epithelial ◽  
Ussing Chambers ◽  
Pkc Isoforms ◽  
Gf 109203X

Serine proteases play important physiological roles through their activity at G protein-coupled protease-activated receptors (PARs). We examined the roles that specific phospholipase (PL) C and protein kinase (PK) C (PKC) isoforms play in the regulation of PAR2-stimulated chloride secretion in intestinal epithelial cells. Confluent SCBN epithelial monolayers were grown on Snapwell supports and mounted in modified Ussing chambers. Short-circuit current ( Isc) responses to basolateral application of the selective PAR2 activating peptide, SLIGRL-NH2, were monitored as a measure of net electrogenic ion transport caused by PAR2 activation. SLIGRL-NH2 induced a transient Isc response that was significantly reduced by inhibitors of PLC (U73122), phosphoinositol-PLC (ET-18), phosphatidylcholine-PLC (D609), and phosphatidylinositol 3-kinase (PI3K; LY294002). Immunoblot analysis revealed the phosphorylation of both PLCβ and PLCγ following PAR2 activation. Pretreatment of the cells with inhibitors of PKC (GF 109203X), PKCα/βI (Gö6976), and PKCδ (rottlerin), but not PKCζ (selective pseudosubstrate inhibitor), also attenuated this response. Cellular fractionation and immunoblot analysis, as well as confocal immunocytochemistry, revealed increases of PKCβI, PKCδ, and PKCε, but not PKCα or PKCζ, in membrane fractions following PAR2 activation. Pretreatment of the cells with U73122, ET-18, or D609 inhibited PKC activation. Inhibition of PI3K activity only prevented PKCδ translocation. Immunoblots revealed that PAR2 activation induced phosphorylation of both cRaf and ERK1/2 via PKCδ. Inhibition of PKCβI and PI3K had only a partial effect on this response. We conclude that basolateral PAR2-induced chloride secretion involves activation of PKCβI and PKCδ via a PLC-dependent mechanism resulting in the stimulation of cRaf and ERK1/2 signaling.

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.

Signaling pathway via TNF-α/NF-κB in intestinal epithelial cells may be directly involved in colitis-associated carcinogenesis

2009 ◽  
Vol 296 (4) ◽  
pp. G850-G859 ◽  
Author(s):  
Michio Onizawa ◽  
Takashi Nagaishi ◽  
Takanori Kanai ◽  
Ken-ichi Nagano ◽  
Shigeru Oshima ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Maintenance Therapy ◽  
Intestinal Epithelial Cells ◽  
Immunoblot Analysis ◽  
Intestinal Epithelial ◽  
Tnf Receptor ◽  
Tnf Α ◽  
Sequential Administration ◽  
Bowel Diseases ◽  
Inflammatory Bowel

Treatment with anti-TNF-α MAb has been accepted as a successful maintenance therapy for patients with inflammatory bowel diseases (IBD). Moreover, it has been recently reported that blockade of TNF receptor (TNFR) 1 signaling in infiltrating hematopoietic cells may prevent the development of colitis-associated cancer (CAC). However, it remains unclear whether the TNF-α signaling in epithelial cells is involved in the development of CAC. To investigate this, we studied the effects of anti-TNF-α MAb in an animal model of CAC by administration of azoxymethane (AOM) followed by sequential dextran sodium sulfate (DSS) ingestion. We observed that the NF-κB pathway is activated in colonic epithelia from DSS-administered mice in association with upregulation of TNFR2 rather than TNFR1. Immunoblot analysis also revealed that the TNFR2 upregulation accompanied by the NF-κB activation is further complicated in CAC tissues induced in AOM/DSS-administered mice compared with the nontumor area. Such NF-κB activity in the epithelial cells is significantly suppressed by the treatment of MP6-XT22, an anti-TNF-α MAb. Despite inability to reduce the severity of colitis, sequential administration of MP6-XT22 reduced the numbers and size of tumors in association with the NF-κB inactivation. Taken together, present studies suggest that the TNFR2 signaling in intestinal epithelial cells may be directly involved in the development of CAC with persistent colitis and imply that the maintenance therapy with anti-TNF-α MAb may prevent the development of CAC in patients with long-standing IBD.

Morphogenic protein epimorphin protects intestinal epithelial cells from oxidative stress by the activation of EGF receptor and MEK/ERK, PI3 kinase/Akt signals

2007 ◽  
Vol 292 (1) ◽  
pp. G39-G52 ◽  
Author(s):  
Masahiro Iizuka ◽  
Kenji Sasaki ◽  
Yohei Hirai ◽  
Kenichi Shindo ◽  
Shiho Konno ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Cell Death ◽  
Epithelial Cells ◽  
Egf Receptor ◽  
Intestinal Epithelial Cells ◽  
Pi3 Kinase ◽  
Intestinal Epithelial ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal

Epimorphin is a mesenchymal protein that regulates morphogenesis of epithelial cells. Our preliminary study suggested a novel function of epimorphin in enhancing survival of intestinal epithelial cells (IEC). Oxidative stress leads to cell injury and death and is suggested to be a key contributor to pathogenesis of inflammatory bowel disease. This study was conducted to determine whether epimorphin protects IEC from oxidative stress. Rat intestinal epithelial cell line IEC-6 was cultured with epimorphin (10 and 20 μg/ml), and the life span of IEC was assessed. The mean life span of IEC-6 cells was prolonged 1.9-fold ( P < 0.0006) by treatment with epimorphin. We then examined the epimorphin signaling pathways. Epimorphin phosphorylated epidermal growth factor (EGF) receptor, activated the MEK/extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase and phosphatidylinositol 3 (PI3) kinase/Akt pathways, phosphorylated Bad, and induced Bcl-XL and survivin. Hydrogen peroxide (1 mM) induced cell death in 92% of IEC-6 cells, but epimorphin dramatically diminished (88.7%) cell death induced by hydrogen peroxide ( P < 0.0001). This protective effect of epimorphin was significantly attenuated by inhibitors of MEK and PI3 kinase ( P < 0.0001) or EGF receptor-neutralizing antibody ( P = 0.0007). In wound assays, the number of migrated cells in the wound area decreased (72.5%) by treatment with 30 μM hydrogen peroxide, but epimorphin increased the number of migrated cells 3.18-fold ( P < 0.0001). These results support a novel function of epimorphin in protecting IEC from oxidative stress. This anti-oxidative function of epimorphin is dramatic and is likely mediated by the activation of EGF receptors and the MEK/extracellular signal-regulated kinase and PI3 kinase/Akt signaling pathways and through the induction of anti-apoptotic factors.

P2Y11, a purinergic receptor acting via cAMP, mediates secretion by pancreatic duct epithelial cells

2001 ◽  
Vol 280 (5) ◽  
pp. G795-G804 ◽  
Author(s):  
T. D. Nguyen ◽  
S. Meichle ◽  
U. S. Kim ◽  
T. Wong ◽  
M. W. Moody
Keyword(s):  
Epithelial Cells ◽  
Pancreatic Duct ◽  
Purinergic Receptor ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Exocrine Secretion ◽  
Type I ◽  
Acetoxymethyl Ester ◽  
Ussing Chambers ◽  
Receptor Blockers

Pancreatic duct epithelial cells (PDEC) mediate the exocrine secretion of fluid and electrolytes. We previously reported that ATP and UTP interact with P2Y2 receptors on nontransformed canine PDEC to increase intracellular free Ca2+ concentration ([Ca2+]i) and stimulate Ca2+-activated Cl− and K+channels. We now report that ATP interacts with additional purinergic receptors to increase cAMP and activate Cl−channels. ATP, 2-methylthio-ATP, and ATP-γ-S stimulated a 4- to 10-fold cAMP increase with EC50 of 10–100 μM. Neither UTP nor adenosine stimulated a cAMP increase, excluding a role for P2Y2 or P1 receptors. Although UTP stimulated an125I− efflux that was fully inhibited by 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM), ATP stimulated a partially resistant efflux, suggesting activation of additional Cl−conductances through P2Y2-independent and Ca2+-independent pathways. In Ussing chambers, increased cAMP stimulated a much larger short-circuit current ( I sc) increase from basolaterally permeabilized PDEC monolayers than increased [Ca2+]i. Luminal ATP and UTP and serosal UTP stimulated a small Ca2+-type I sc increase, whereas serosal ATP stimulated a large cAMP-type I scresponse. Serosal ATP effect was inhibited by P2 receptor blockers and unaffected by BAPTA-AM, supporting ATP activation of Cl−conductances through P2 receptors and a Ca2+-independent pathway. RT-PCR confirmed the presence of P2Y11 receptor mRNA, the only P2Y receptor acting via cAMP.

Profile of IGF-binding proteins secreted by intestinal epithelial cells changes with differentiation

1994 ◽  
Vol 267 (5) ◽  
pp. G843-G850 ◽  
Author(s):  
S. Oguchi ◽  
W. A. Walker ◽  
I. R. Sanderson
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Epithelial Cells ◽  
Binding Proteins ◽  
Egf Receptor ◽  
Transforming Growth Factor ◽  
Intestinal Epithelial Cells ◽  
Alpha Activity ◽  
Intestinal Epithelial ◽  
Igf I

Previous reports have shown that gastrointestinal epithelial cells produce insulin-like growth factor-binding proteins (IGF-BP), which modulate the actions of IGF. This study aims to examine the relationship between differentiation and IGF-BP secretion by human intestinal epithelial cells and the effect of growth factors on their production. Caco-2 cells were cultured in serum-free media. IGF-BP secretion into the incubation media was analyzed by Western ligand blotting and immunoblotting. Caco-2 cells produced IGF-BP-2, IGF-BP-3, and IGF-BP-4. Secretion of IGF-BP-2 and IGF-BP-3 increased with differentiation, but IGF-BP-4 secretion diminished. The effect of exogenous growth factors on IGF-BP secretion was maximal at earlier stages of differentiation. IGF-I stimulated mainly IGF-BP-3 production, but epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) stimulated predominantly IGF-BP-4 secretion. Adding an anti-EGF receptor antibody to block autocrine TGF-alpha activity inhibited IGF-BP-4 production but stimulated IGF-BP-2 and IGF-BP-3. In conclusion, the profile of IGF-BP secretion changes with differentiation. IGF-I and EGF (or TGF-alpha) stimulate different types of IGF-BP, with autocrine TGF-alpha activity being a factor affecting IGF-BP production during differentiation.

Na+/Ca2+ exchange regulates Ca2+-dependent duodenal mucosal ion transport and HCO3− secretion in mice

2005 ◽  
Vol 288 (3) ◽  
pp. G457-G465 ◽  
Author(s):  
Hui Dong ◽  
Zachary M. Sellers ◽  
Anders Smith ◽  
Jimmy Y. C. Chow ◽  
Kim E. Barrett
Keyword(s):  
Western Blot ◽  
Ion Transport ◽  
Muscarinic Receptors ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Duodenal Mucosa ◽  
Ussing Chambers ◽  
Intracellular Ca ◽  
Ph Stat ◽  
Stimulation Of

Stimulation of muscarinic receptors in duodenal mucosa raises intracellular Ca2+, which regulates ion transport, including HCO3− secretion. However, the underlying Ca2+ handling mechanisms are poorly understood. The aim of the present study was to determine whether Na+/Ca2+ exchanger (NCX) plays a role in the regulation of duodenal mucosal ion transport and HCO3− secretion by controlling Ca2+ homeostasis. Mouse duodenal mucosa was mounted in Ussing chambers. Net ion transport was assessed as short-circuit current ( Isc), and HCO3− secretion was determined by pH-stat. Expression of NCX in duodenal mucosae was analyzed by Western blot, and cytosolic Ca2+ in duodenocytes was measured by fura 2. Carbachol (100 μM) increased Isc in a biphasic manner: an initial transient peak within 2 min and a later sustained plateau starting at 10 min. Carbachol-induced HCO3− secretion peaked at 10 min. 2-Aminoethoxydiphenylborate (2-APB, 100 μM) or LiCl (30 mM) significantly reduced the initial peak in Isc by 51 or 47%, respectively, and abolished the plateau phase of Isc without affecting HCO3− secretion induced by carbachol. Ryanodine (100 μM), caffeine (10 mM), and nifedipine (10 μM) had no effect on either response to carbachol. In contrast, nickel (5 mM) and KB-R7943 (10–30 μM) significantly inhibited carbachol-induced increases in duodenal mucosal Isc and HCO3− secretion. Western blot analysis showed expression of NCX1 proteins in duodenal mucosae, and functional NCX in duodenocytes was demonstrated in Ca2+ imaging experiments where Na+ depletion elicited Ca2+ entry via the reversed mode of NCX. These results indicate that NCX contributes to the regulation of Ca2+-dependent duodenal mucosal ion transport and HCO3− secretion that results from stimulation of muscarinic receptors.

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