Electrical parameters and water permeability properties of monolayers formed by T84 cells cultured on permeable supports

Abstract

T84 is an established cell line expressing an enterocyte phenotype whose permeability properties have been widely explored. Osmotic permeability (P OSM ), hydraulic permeability (P HYDR ) and transportassociated net water fluxes (J W -transp), as well as short-circuit current (I SC ), transepithelial resistance (R T ), and potential difference (∆V T ) were measured in T84 monolayers with the following results: P OSM 1.3 ± 0.1 cm.s -1 x 10 -3 ; P HYDR 0.27 ± 0.02 cm.s -1 ; R T 2426 ± 109 Ω.cm 2 , and ∆V T 1.31 ± 0.38 mV. The effect of 50 µM 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DCEBIO), a "net Cl -secretory agent", on T84 cells was also studied. We confirm the reported important increase in I SC induced by DCEBIO which was associated here with a modest secretory ∆J W -transp. The present results were compared with those reported using the same experimental approach applied to established cell lines originating from intestinal and renal epithelial cells (Caco-2, LLC-PK1 and RCCD-1). No clear association between P HYDR and R T could be demonstrated and high P HYDR values were observed in an electrically tight epithelium, supporting the view that a "water leaky" barrier is not necessarily an "electrically leaky" one. Furthermore, the modest secretory ∆J W -transp was not consistent with previous results obtained with RCCD-1 cells stimulated with vasopressin (absorptive fluxes) or with T84 cells secreting water under the action of Escherichia coli heat stable enterotoxin. We conclude that, while the presence of aquaporins is necessary to dissipate an external osmotic gradient, coupling between water and ion transport cannot be explained by a simple and common underlying mechanism.

Myosin 5b loss of function leads to defects in polarized signaling: implication for microvillus inclusion disease pathogenesis and treatment

“…The absolute values of net water flux in control cells (2.8 Ϯ 0.4 l·h Ϫ1 ·cm Ϫ2 mucosal to serosal) in the absence of external osmotic or hydrostatic pressures were almost identical to those reported in the original publication (30), and ϳ58-fold lower than those reported for basal isosmotic water absorption in the small intestine in vivo (4). This factor is consistent with the folding in the intestinal epithelium (56) when compared with a flat monolayer of epithelial cells in culture.…”

“…When untreated control (scrambled shRNA) cells were subjected to an apical 100 mosmol/kgH 2 O gradient, the net water flux was 11 Ϯ 2.3 l·h Ϫ1 ·cm Ϫ2 serosal to mucosal. This value is slightly below that observed in a previous publication (30) using T84 cells (8 l·h Ϫ1 ·cm Ϫ2 for the same gradient). Importantly, neither Myo5b kd nor PDK1 expression had any effect on osmotically driven water flows.…”

Inhibition of water absorption in human proximal tubular epithelial cells in response to Shiga toxin-2

“…Data are shown as means ± standard error of the mean (SEM) (n=6-10). *P<0.05 corresponds to the minimal doses of Stx2 necessary to produce a significant effect compared with the control epithelial cell lines grown on permeable supports [25,30] and rat renal proximal tubules [31]. A low P osm associated with a high P hydr indicates that water transport across HRTEC monolayers occurs by transcellular and paracellular pathways [32].…”

Mechanical Characterization of Microengineered Epithelial Cysts by Using Atomic Force Microscopy

“…The water could be transported either transcellularly, across the cell membranes (through molecules such as aquaporin) and the enclosed cytoplasm, or paracellularly through tight junctions (23,48,49). Previous studies evaluating the hydraulic permeability of transwell-cultured epithelial monolayers showed that the paracellular route is the most dominant route for hydrostatic-pressure-driven transepithelial water transport (50,51). This notion was supported by the results of our force-relaxation experiments performed on MDCK II cysts, in which the transcellular water-transport pathway was partially inhibited by adding HgCl 2 , a commonly used aquaporin blocker (Fig.…”