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.

Carrageenan induces interleukin-8 production through distinct Bcl10 pathway in normal human colonic epithelial cells

2007 ◽  
Vol 292 (3) ◽  
pp. G829-G838 ◽  
Author(s):  
Alip Borthakur ◽  
Sumit Bhattacharyya ◽  
Pradeep K. Dudeja ◽  
Joanne K. Tobacman
Keyword(s):  
Molecular Weight ◽  
Epithelial Cells ◽  
High Molecular Weight ◽  
Intestinal Epithelial Cells ◽  
Epithelial Cell Line ◽  
Intestinal Epithelial ◽  
Human Intestinal Epithelial Cells ◽  
Normal Rat ◽  
Normal Human ◽  
Inflammatory Bowel

Carrageenan is a high molecular weight sulfated polygalactan used to improve the texture of commercial food products. Its use increased markedly during the last half century, although carrageenan is known to induce inflammation in rheumatological models and in intestinal models of colitis. We performed studies to determine its direct effects on human intestinal cells, including normal human intestinal epithelial cells from colonic surgeries, the normal intestinal epithelial cell line NCM460, and normal rat ileal epithelial cells. Cells were treated with high molecular weight λ-carrageenan at a concentration of 1 μg/ml for 1–96 h. IL-8, IL-8 promoter activity, total and nuclear NF-κB, IκBα, phospho-IκBα, and Bcl10 were assessed by immunohistochemistry, Western blot, ELISA, and cDNA microarray. Increased Bcl10, nuclear and cytoplasmic NF-κB, IL-8 promoter activation, and IL-8 secretion were detected following carrageenan exposure. Knockdown of Bcl10 by siRNA markedly reduced the increase in IL-8 that followed carrageenan exposure in the NCM460 cells. These results show, for the first time, that exposure of human intestinal epithelial cells to carrageenan triggers a distinct inflammatory pathway via activation of Bcl10 with NF-κB activation and upregulation of IL-8 secretion. Since Bcl10 contains a caspase-recruitment domain, similar to that found in NOD2/CARD15 and associated with genetic predisposition to Crohn's disease, the study findings may represent a link between genetic and environmental etiologies of inflammatory bowel disease. Because of the high use of carrageenan as a food additive in the diet, the findings may have clinical significance.

scholarly journals PEPT1-Mediated Cefixime Uptake into Human Intestinal Epithelial Cells Is Increased by Ca2+ Channel Blockers

2002 ◽  
Vol 46 (5) ◽  
pp. 1375-1380 ◽  
Author(s):  
Uwe Wenzel ◽  
Sabine Kuntz ◽  
Simone Diestel ◽  
Hannelore Daniel
Keyword(s):  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Peptide Transporter ◽  
Epithelial Cell Line ◽  
Channel Blockers ◽  
Intestinal Epithelial ◽  
Regulatory Systems ◽  
Human Intestinal Epithelial Cells ◽  
Acid Load ◽  
Molecular Mode

ABSTRACT Ca2+ channel blockers like nifedipine have been shown to increase the oral bioavailability of β-lactam antibiotics, such as cefixime, in humans. The molecular mode of action of Ca2+ channel blockers on β-lactam absorption, however, has not yet been defined. Using the Caco-2 human intestinal epithelial cell line, we assessed whether alterations in intracellular free Ca2+ ion (Ca2+ in) concentrations by Ca2+ channel blockers or by Ca2+ ionophores affect [14C]cefixime absorption. Reduction of Ca2+ in levels by Ca2+ channel blockers (nifedipine, verapamil, diltiazem, or bepridil) at concentrations of 100 μM led to 35 to 50% increases in the cellular uptake of 1 mM [14C]cefixime. Increases in Ca2+ in levels by Ca2+ ionophores, on the other hand, led to 40% reductions in [14C]cefixime absorption. Nifedipine increased the V max of cefixime transport by 67%, whereas the Km of cefixime transport remained unaffected. By measuring the pH in Caco-2 cells loaded with the pH-sensitive fluorescent dye 2′,7′-bis(2-carboxyethyl)-5-(6)-carboxyfluorescein, we show that cefixime transport mediated by the intestinal H+-coupled peptide transporter PEPT1 leads to intracellular acidification. This acid load was reduced by nifedipine, although the Ca2+ channel blocker increased the level of H+ and cefixime cotransport. Increases in Ca2+ in levels by ionomycin enhanced the decline in intracellular pH induced by cefixime alone, although ionomycin reduced the level of H+ and cefixime cotransport. In conclusion, our studies demonstrate that alterations of Ca2+ in levels, e.g., by Ca2+ channel blockers, affect pH regulatory systems, such as apical Na+ and H+ exchange, and thereby alter the H+ gradient that serves as the driving force for uptake of β-lactams into intestinal epithelial cells.

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