1. Volume 119, Issue 2, Pages 420-430 (August 2000)
Gastrointestinal glutathione peroxidase prevents transport of lipid hydroperoxides in CaCo-2 cells 
Kirstin Wingler, Cordula Müller, Katrin Schmehl, Simone Florian, Regina Brigelius-Flohé 
Gastroenterology 
Volume 119, Issue 2, Pages (August 2000)
DOI: /gast
Copyright © 2000 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Selenium-dependent GPx activities in CaCo-2 cells. Cells were seeded into 175-cm2 cell culture flasks and grown until 2 days after confluence (8 days). Medium contained the concentrations of sodium selenite indicated from the beginning. To measure activity of total GPx (H2O2), PHGPx (phosphatidylcholine hydroperoxide), and the activity with 13-HPODE in a cell population, a flask of cells at the respective selenium concentration was needed. GPx activities were measured as described in Materials and Methods. Each value is the mean ± SD from 2 individual cell cultures measured in triplicate. *P < 0.01; **P <
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions

3. Fig. 2
Selenium-dependent levels of GPx proteins. (A) CaCo-2 cells were grown in 175-cm2 flasks until 2 days after confluence (8 days) supplemented with the indicated sodium selenite concentrations from the beginning. (B) Confluent selenium-deficient CaCo-2 cells were resupplemented with 50 nmol/L selenite for the indicated times. Lysates (150 μg protein/lane) were analyzed in Western blots with polyclonal antisera against GI-GPx and cGPx made visible by chemiluminescence. Relative intensities were calculated by means of densitometry. The strongest signal of each individual blot was taken as 100%. For further experimental details, see Materials and Methods.
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions

4. Fig. 3
HPLC analysis of basolateral lipids released by CaCo-2 cells. Polarized CaCo-2 cells grown on transwells were incubated with 100 μmol/L (A) [1-14C]linoleic acid or (B) [1-14C]13-HPODE for 12 hours. Then, basolateral lipids were extracted and run on an SP-HPLC column. Peaks corresponding to the indicated lipid classes are identified by retention times of authentic standards and made visible by radioactivity. For experimental details see Materials and Methods.
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions

5. Fig. 4
Hydroperoxides are only detected at the basolateral side of CaCo-2 cells after the cellular monolayer has been destroyed. (A) CaCo-2 cells were grown on polycarbonate filters until day 14 after confluence at 0, 2, and 50 nmol/L selenite supplementation. Filters were transferred into the diffusion chamber, and 500 μmol/L 13-HPODE complexed to bovine serum albumin was added to the apical medium. Aliquots of the basolateral medium were taken at the indicated times, and 13-HPODE concentration was determined by means of GSH peroxidase. (B) Diffusion of lucifer yellow (LY) and TEER were determined as indicators for the integrity of the cell monolayer. Control values of TEER after 12 hours were 86% ± 7.9% of the initials. LY was not detected on the basolateral side when cells were not treated with 13-HPODE. Values are means ± SD of 2 experiments with 3 (TEER), 2 (13-HPODE), or 1 (LY) filter(s) per experiment.
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions

6. Fig. 5
GSH depletion abolishes the protection of monolayer damage by selenium. CaCo-2 cells were grown on polycarbonate filters as described in Materials and Methods and treated with 300 μmol/L BSO during the 6 last days of cultivation with the medium changed daily. Then filters were transferred to the diffusion chamber and incubated with 500 μmol/L 13-HPODE for the indicated times. (A) Basolateral 13-HPODE. (B) TEER and lucifer yellow (LY) permeation. For details, see Figure 2 and Materials and Methods. *P < 0.05, **P < vs. selenium-supplemented cells.
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions

7. Fig. 6
Levels of GI-GPx and cGPx protein in different intestinal parts of a selenium-deficient and a selenium-adequate rat. (A) GI-GPx and (B) cGPx of indicated areas of intestines of a selenium-deficient (−) and a selenium-adequate (+) rat were detected by means of Western blotting with polyclonal antisera made visible by chemiluminescence. Lysates of the indicated parts of the intestine were separated on SDS gels (100 μg protein/lane) and blotted on nitrocellulose membranes. Because of the extremely long blotting time necessary to detect cGPx,19 a background is completely absent in B. cGPx, commercial cGPx as a positive control. For further experimental details, see Materials and Methods.
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions

8. Fig. 7
Localization of GI-GPx in the intestine of a selenium-deficient and a selenium-adequate rat. (A and B) Duodenum; (C and D) jejunum. Anti–GI-GPx IR-positive structures show a brown staining (diaminobenzidine). (A and C) In selenium deficiency, a weak IR was detected in many epithelial cells both at the surface of the villi (arrow) and in the crypts (arrowhead). Only rarely, some stromal cells (double arrowhead) showed positive IR signals. In general, the crypt architecture seemed to be irregular in selenium deficiency. (B and D) A stronger positive IR was detected in the enterocytes on the epithelial surface under selenium supplementation. GI-GPx apparently is ubiquitously distributed along the whole small bowel crypt-villus axis independent from the stage of cellular differentiation and migration. Original magnification 200×.
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions