Stereochemistry of Dihydroxy Bile Acids Dictates Their Role in Barrier Dysfunction in Human Colon T84 Cells

Changes in cytosolic free Ca2+ ([Ca2+]i) in response to the secretagogue carbachol have been characterized in the human colon cancer cell line T84, a model Cl- secretory cell. In this study, [Ca2+]i was determined with the fluorescence indicator fura-2 at the single-cell level with a fluorescent microscope-imaging system. Basal [Ca2+]i in T84 cells in Ringer-HCO3 solution was 76 +/- 4 nM and was decreased by exposure to Ca2+ free solution or 25 microM verapamil. The cholinergic agonist carbachol caused a concentration-dependent rise in [Ca2+]i with a Km of 4 microM and a peak increase in [Ca2+]i of approximately 50 nM. The onset of the [Ca2+]i increase was within 3 s, occurred uniformly among cells, and peaked at 10-15 s. The increase in [Ca2+]i was heterogenous in the length of time the [Ca2+]i remained elevated above basal, and cell responses could be divided into at least two groups on that basis. Blocking the contributions of intracellular Ca2+ with dantrolene inhibited the increase in [Ca2+]i as early as could be determined, whereas blocking the extracellular contribution with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), verapamil, or nifedipine inhibited a slightly later increase in [Ca2+]i. In conclusion, the initial detectable increase in [Ca2+]i caused by carbachol is due to the release of Ca2+ from internal stores, whereas the contribution of extracellular Ca2+ occurs later and at least partially involves a nifedipine- and verapamil-sensitive process.

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Effects of specific bile acids on c-fos messenger RNA levels in human colon carcinoma Caco-2 cells

European Journal of Pharmaceutical Sciences ◽

10.1016/s0928-0987(00)00111-1 ◽

2000 ◽

Vol 11 (4) ◽

pp. 291-298 ◽

Cited By ~ 14

Author(s):

Rosanna Di Toro ◽

Gabriele Campana ◽

Giovanna Murari ◽

Santi Spampinato

Keyword(s):

Bile Acids ◽

Colon Carcinoma ◽

Messenger Rna ◽

Human Colon ◽

Human Colon Carcinoma ◽

Rna Levels

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Enterohemorrhagic Escherichia coli Colonization of Human Colonic EpitheliumIn VitroandEx Vivo

Infection and Immunity ◽

10.1128/iai.02928-14 ◽

2014 ◽

Vol 83 (3) ◽

pp. 942-949 ◽

Cited By ~ 35

Author(s):

Steven B. Lewis ◽

Vivienne Cook ◽

Richard Tighe ◽

Stephanie Schüller

Keyword(s):

Escherichia Coli ◽

Human Colon ◽

Enterohemorrhagic Escherichia Coli ◽

Care Needs ◽

T84 Cells ◽

Host Cell Membrane ◽

Content Type ◽

Colonic Biopsy

EnterohemorrhagicEscherichia coli(EHEC) is an important foodborne pathogen causing gastroenteritis and more severe complications, such as hemorrhagic colitis and hemolytic uremic syndrome. Pathology is most pronounced in the colon, but to date there is no direct clinical evidence showing EHEC binding to the colonic epithelium in patients. In this study, we investigated EHEC adherence to the human colon by usingin vitroorgan culture (IVOC) of colonic biopsy samples and polarized T84 colon carcinoma cells. We show for the first time that EHEC colonizes human colonic biopsy samples by forming typical attaching and effacing (A/E) lesions which are dependent on EHEC type III secretion (T3S) and binding of the outer membrane protein intimin to the translocated intimin receptor (Tir). A/E lesion formation was dependent on oxygen levels and suppressed under oxygen-rich culture conditions routinely used for IVOC. In contrast, EHEC adherence to polarized T84 cells occurred independently of T3S and intimin and did not involve Tir translocation into the host cell membrane. Colonization of neither biopsy samples nor T84 cells was significantly affected by expression of Shiga toxins. Our study suggests that EHEC colonizes and forms stable A/E lesions on the human colon, which are likely to contribute to intestinal pathology during infection. Furthermore, care needs to be taken when using cell culture models, as they might not reflect thein vivosituation.

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Enteroaggregative Escherichia coli Disrupts Epithelial Cell Tight Junctions

Infection and Immunity ◽

10.1128/iai.00580-10 ◽

2010 ◽

Vol 78 (11) ◽

pp. 4958-4964 ◽

Cited By ~ 57

Author(s):

Maura C. Strauman ◽

Jill M. Harper ◽

Susan M. Harrington ◽

Erik Juncker Boll ◽

James P. Nataro

Keyword(s):

Escherichia Coli ◽

Tight Junction ◽

Tight Junctions ◽

Cell Model ◽

Barrier Dysfunction ◽

T84 Cells ◽

E Coli ◽

Eaec Strain ◽

Epithelial Barrier Dysfunction

ABSTRACT Enteroaggregative Escherichia coli (EAEC) is responsible for inflammatory diarrhea in diverse populations, but its mechanisms of pathogenesis have not been fully elucidated. We have used a previously characterized polarized intestinal T84 cell model to investigate the effects of infection with EAEC strain 042 on tight junction integrity. We find that infection with strain 042 induces a decrease in transepithelial electrical resistance (TER) compared to uninfected controls and to cells infected with commensal E. coli strain HS. When the infection was limited after 3 h by washing and application of gentamicin, we observed that the TER of EAEC-infected monolayers continued to decline, and they remained low even as long as 48 h after the infection. Cells infected with the afimbrial mutant strain 042aafA exhibited TER measurements similar to those seen in uninfected monolayers, implicating the aggregative adherence fimbriae II (AAF/II) as necessary for barrier dysfunction. Infection with wild-type strain 042 induced aberrant localization of the tight junction proteins claudin-1 and, to a lesser degree, occludin. EAEC-infected T84 cells exhibited irregular shapes, and some cells became elongated and/or enlarged; these effects were not observed after infection with commensal E. coli strain HS or 042aafA. The effects on tight junctions were also observed with AAF/I-producing strain JM221, and an afimbrial mutant was similarly deficient in inducing barrier dysfunction. Our results show that EAEC induces epithelial barrier dysfunction in vitro and implicates the AAF adhesins in this phenotype.

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Effect of Bile Acids on Butyrate-Sensitive and -Resistant Human Colon Adenocarcinoma Cells

Nutrition and Cancer ◽

10.1207/s15327914nc5302_10 ◽

2005 ◽

Vol 53 (2) ◽

pp. 208-219 ◽

Cited By ~ 8

Author(s):

Pablo Perez-Ramos ◽

Nieves Olmo ◽

Javier Turnay ◽

Emilio Lecona ◽

Gonzalo Gonzalez de Buitrago ◽

...

Keyword(s):

Bile Acids ◽

Colon Adenocarcinoma ◽

Human Colon ◽

Adenocarcinoma Cells ◽

Human Colon Adenocarcinoma ◽

Colon Adenocarcinoma Cells

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Sulphation of lithocholic acid in the colon-carcinoma cell line CaCo-2

Biochemical Journal ◽

10.1042/bj3430533 ◽

1999 ◽

Vol 343 (3) ◽

pp. 533-539 ◽

Cited By ~ 6

Author(s):

Bente HALVORSEN ◽

Bengt Frode KASE ◽

Kristian PRYDZ ◽

Sedegheh GARAGOZLIAN ◽

Marianne S. ANDRESEN ◽

...

Keyword(s):

Bile Acids ◽

Cell Line ◽

Colon Carcinoma ◽

Carcinoma Cell ◽

Lithocholic Acid ◽

Human Colon ◽

Carcinoma Cell Line ◽

Gel Chromatography ◽

Colon Carcinoma Cell Line ◽

Increased Risk

High levels of bile acids in the colon may correlate with an increased risk of colon cancer, but the underlying mechanisms are not known. Proteoglycan structures have been shown to change when human colon cells differentiate in vitro. The expression of [35S]sulphated molecules was used as a phenotypic marker to study the effects of bile acids on the human-colon-carcinoma cell line CaCo-2. [35S]sulphated compounds were isolated from the medium of cell fractions of cells metabolically labelled with [35S]sulphate in the absence and presence of cholic acid, deoxycholic acid, chenodeoxycholic acid and lithocholic acid (LA). Labelled molecules were analysed by gel chromatography, HPLC and SDS/PAGE in combination with chemical and enzymic methods. The expression of 35S-labelled proteoglycans was not affected by any of the bile acids tested. However, the level of sulphated metabolites increased 7-18-fold in different experiments during a 22 h labelling period in the presence of an LA concentration of 10 μg/ml (26.6 nmol/ml) compared with controls. Further analyses showed that this was due, at least in part, to the sulphation of LA itself. This sulphation of LA was a rapid process followed by secretion back to the medium. Brefeldin A did not reduce the sulphation of LA, indicating that this conversion takes place in the cytosol, rather than in the Golgi apparatus of the CaCo-2 cells. LA in colon may be sulphated efficiently by the colonocytes to reduce the toxic effects of this particular bile acid. Sulphation may possibly be an important protective mechanism in the colon.

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