Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: PepT1 mediates transport of the proinflammatory bacterial tripeptide l -Ala-?- d -Glu- meso -DAP in intestinal epithelial cells

doi: 10.1152/ajpgi.00527.2009

Figure Lengend Snippet: l-Ala-γ-d-Glu-meso-DAP (Tri-DAP) inhibits PepT1-mediated transport of [14C]glycine-sarcosine ([14C]Gly-Sar) into Caco2-BBE cells. Caco2-BBE cells (passages 20–30) were grown on plastic plates for 8 days. A: uptake of 20 μM [14C]Gly-Sar in the absence or presence of 20 mM glycine-leucine (Gly-Leu) or the indicated concentrations of Tri-DAP was determined as described undermaterials and methods. Results were expressed as real uptake of [14C]Gly-Sar. The Ki value, for Tri-DAP to PepT1, was determined from Dixon plot (competitive inhibition) by use of the SigmaPlot 8.0 software. Values represent means ± SE of n = 6 wells/condition. Data are representative of 3 determinations (*P < 0.05; ***P < 0.001) vs. no competitor. B: intracellular pH of Caco2-BBE cells in response to Tri-DAP stimulation. Caco2-BBE cells (passages 20–30) grown on glass-bottom microwell dishes for 8 days were loaded with the fluorescent dye BCECF-AM, and intracellular pH before and after addition of 5 mM Tri-DAP or vehicle (arrow) was measured in real time as described in materials and methods. For each condition, 3 independent experiments were performed. Graphs represent the mean of intracellular pH of 40 selected cells from 1 assay.

Article Snippet: Cells were then incubated with HBSS-10 mM MES (pH 6.2) containing 20 μM [ 14 C]glycine-sarcosine ([ 14 C]Gly-Sar; specific activity of 50 mCi/mM; American Radiolabeled Chemicals) in the presence or absence of 20 mM glycine-leucine (Gly-Leu, Sigma) or different concentrations of Tri-DAP (Anaspec) for 15 min at room temperature.

Techniques: Inhibition, Software

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: PepT1 mediates transport of the proinflammatory bacterial tripeptide l -Ala-?- d -Glu- meso -DAP in intestinal epithelial cells

doi: 10.1152/ajpgi.00527.2009

Figure Lengend Snippet: Tri-DAP inhibits the uptake of [14C]Gly-Sar in HT29-Cl.19A cells stably transfected with PepT1. HT29-Cl.19A cells stably transfected with a vector encoding PepT1 (HT29-Cl.19A/PepT1) or an empty vector (HT29-Cl.19A/vector) (passages 20–30) were cultured on plastic plates for 8 days. A: Western blot analysis of PepT1 expression in HT29-Cl.19A/PepT1 and HT29-Cl.19A/vector cells. Blots are representative of 3 similar determinations (top), and bars show means ± SD of relative intensity of the bands obtained from 3 determinations (bottom). **P < 0.005. B: uptake of 20 μM [14C]Gly-Sar in the absence or presence of 5 or 10 mM of Tri-DAP was determined by transport assay, and results were expressed as [14C]Gly-Sar real uptake. Values represent means ± SE of n = 6 wells/condition. Data are representative of 3 determinations. $$$P < 0.001; NS, not statically significant vs. untreated HT29-Cl.19A/vector; ***P < 0.001 vs. untreated HT29-Cl.19A/PepT1. C: HT29-Cl.19A/PepT1 and HT29-Cl.19A/vector cells (passages 20–30) grown on glass-bottom microwell dishes for 8 days were loaded with the fluorescent dye BCECF-AM, and intracellular pH before and after addition of 5 mM Tri-DAP (arrow) was measured in real time as described in materials and methods. For each condition, 3 independent experiments were performed. Graphs represent the mean of intracellular pH of 40 selected cells from 1 assay.

Article Snippet: Cells were then incubated with HBSS-10 mM MES (pH 6.2) containing 20 μM [ 14 C]glycine-sarcosine ([ 14 C]Gly-Sar; specific activity of 50 mCi/mM; American Radiolabeled Chemicals) in the presence or absence of 20 mM glycine-leucine (Gly-Leu, Sigma) or different concentrations of Tri-DAP (Anaspec) for 15 min at room temperature.

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Cell Culture, Western Blot, Expressing, Transport Assay

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: PepT1 mediates transport of the proinflammatory bacterial tripeptide l -Ala-?- d -Glu- meso -DAP in intestinal epithelial cells

doi: 10.1152/ajpgi.00527.2009

Figure Lengend Snippet: Downregulation of PepT1 expression in Caco2-BBE cells decreases Tri-DAP-induced inflammation. Caco2-BBE cells were stably transfected first with the pcDNA4TO/myc-His/LacZ vector, which encodes the tetracycline repressor (TetR), and then with the PepT1-short hairpin RNA (shRNA)1 construct (TetR Caco2-BBE/PepT1-shRNA1) or the empty vector (TetR Caco2-BBE/vector). Cells (passages 25–30) grown on plastic plates for 6 days were treated or not with 2.5 μg/ml tetracycline for 2 days. A: PepT1 and monocarboxylate transporter 1 (MCT-1) expression was analyzed by Western blot. B: uptake of 20 μM [14C]Gly-Sar ± 5 mM Tri-DAP and uptake of 20 μM [14C]butyrate ± 1 mM α-cyano-4-hydroxycinnamate (CHC) in these cells were determined. Data are presented as specific uptake: PepT1-mediated [14C]Gly-Sar specific uptake = (uptake of [14C]Gly-Sar) − (uptake of [14C]Gly-Sar + Tri-DAP); MCT-1-mediated [14C]butyrate specific uptake = (uptake of [14C]butyrate) − (uptake of [14C]butyrate + CHC). Values represent means ± SE of n = 6 wells/condition from 1 determination. ***P < 0.001. C and D: TetR Caco2-BBE/PepT1-shRNA1 and TetR Caco2-BBE/vector cells were treated with 2.5 μg/ml tetracycline for 2 days and then stimulated with 5 mM Tri-DAP for the indicated times. Degradation of IκB-α (C) and activation of the MAPKs ERK½ and p38 (D) were assessed by Western blot. All blots in A, C, and D are representative of 3 similar determinations, and bars show means ± SD of relative intensity of the bands obtained from 3 determinations. ***P < 0.001 (A and B); (*P < 0.05; **P < 0.005; ***P < 0.001) vs. untreated TetR Caco2-BBE/vector (C and D); $$P < 0.005 (C).

Article Snippet: Cells were then incubated with HBSS-10 mM MES (pH 6.2) containing 20 μM [ 14 C]glycine-sarcosine ([ 14 C]Gly-Sar; specific activity of 50 mCi/mM; American Radiolabeled Chemicals) in the presence or absence of 20 mM glycine-leucine (Gly-Leu, Sigma) or different concentrations of Tri-DAP (Anaspec) for 15 min at room temperature.

Techniques: Expressing, Stable Transfection, Transfection, Plasmid Preparation, shRNA, Construct, Western Blot, Activation Assay