3 research outputs found
Trafficking of SLC26A3 in Intestinal Epithelial Cells: Effect of Enteropathogenic E. coli Infection
Trafficking of SLC26A3 (DRA) in Intestinal Epithelial Cells: Effect of Enteropathogenic E.coli (EPEC) Infection
Tarunmeet Gujral, Anoop Kumar, Shubha Priyamvada, Ravinder K Gill, Gail A Hecht, Waddah A Alrefai, & Pradeep K Dudeja
Enteropathogenic E.coli. (EPEC) via a type 3-secretion system decreases the activity of apical Cl-/HCO3- exchanger, DRA contributing to the pathophysiology of early diarrhea. Little is known about the mechanisms that regulate the cellular trafficking of DRA. We hypothesized that EPEC modulates endocytic and/or exocytic pathways involved in DRA trafficking. The aim of the current study was to study DRA trafficking events utilizing Caco-2 cells as an in vitro model. Dynasore, a dynamin inhibitor increased function and surface levels of DRA via decreased endocytosis. Disruption of microtubules followed by recovery in the presence or absence of nocodazole suggested that intact microtubules are essential for basal exocytic but not endocytic trafficking of DRA. Further, mice treated with colchicine showed a decrease in apical surface levels and an increase the sub-apical pool of DRA. EPEC deletion mutants EspG1/G2 (unable to disrupt microtubules) prevented both increased DRA endocytosis and decreased DRA exocytosis induced by wild-type EPEC indicating that intact microtubules are essential for surface membrane expression of DRA. EPEC induced decrease in apical Cl-/OH-(HCO3-) exchange activity and DRA association with lipid-rafts was unaltered in the presence of dynasore, suggesting a clathrin independent internalization of surface DRA during EPEC infection. Collectively, these data indicate that EPEC induces decrease in DRA surface levels by i) decreasing its association with rafts, ii) partly increasing endocytosis by clathrin independent mechanisms; and iii) by decreasing exocytosis of DRA via disruption of host microtubular network. In conclusion, our results highlight novel mechanisms of DRA cellular trafficking under physiological conditions and in response to EPEC infection. (Supported by NIDDK and Department of Veterans Affairs
TGF-β1 increases SERT levels on the luminal surface in 3D-culture of Caco-2 cells.
<p>Caco-2 cells in 3D culture system untreated or treated with TGF-β1 (10 ng/ml, 1h) were stained for SERT (green), Phalloidin (red) and DAPI (blue) and visualized by confocal microscopy. XY planar images and orthogonal XZ images Orthognal <i>xz</i> images were obtained with a Zeiss LSM 510 confocal microscope.</p
SERT function in response to TGF-β1 treatment of native mouse ileum.
<p>The distal small intestine was stripped off of the muscle layer, mounted in the Ussing chamber to expose tissue on both sides to Kreb’s solution supplemented with L-ascorbic acid (100 μM) and the monoamine oxidase inhibitor pargyline (100 μM). Tissues were treated with TGF-β1 for 1h from basolateral side and then incubated with <sup>3</sup>[H]-5-HT for 30 min. Fluoxetine (10 μM) was added 30 min prior to TGF-β1 treatment from the apical side. Values represent mean ± SEM of 3 different experiments. *<i>P</i><0.05 compared with untreated control.</p