Objective: 

Chemical compounds such as EGTA and sodium caprate are widely used as additional components in drug administration. As specific information on molecular mechanisms supporting absorption is scarce, we have analyzed effects on paracellular permeability in the intestinal epithelial cell model Caco-2.

Methods: 

Caco-2 cells were grown on permeable supports to confluence. Supports were then mounted in Ussing-type chambers, and transepithelial resistance and flux of permeability markers was measured. Two-path impedance spectroscopy was employed to differentiate between transcellular and paracellular resistance. Subsequently, cells were harvested from the supports and tight junction proteins as well as cytoskeletal markers were analyzed by means of Western blotting. Subcellular localization was explored by confocal laser scanning microscopy.

Results: 

Both compounds, EGTA as well as sodium caprate, lead to a strong and reversible decrease of transepithelial resistance. Two-path impedance spectroscopy revealed that this decrease was based on a specific effect on the paracellular pathway. Flux of FITC-labeled 4K dextran was markedly increased with both substances. Whereas Western blots of crude membrane fractions detecting tight junction markers as occludin and claudins did not show changes, a more distinctive effect was observed in immunofluorescence stainings: while the perijunctional actin ring condenses during Ca²⁺ depletion, it remains stable with sodium caprate. Condensation of the perijunctional actin ring affects tight junction proteins by withdrawal from the tight junction, whereas sodium caprate leads to a specific internalization of tight junction proteins, including occludin and claudin-4.

Conclusions: 

Analyses of the two compounds reveal two different mechanisms on the paracellular permeability: Whereas a condensation of the cytoskeleton during Ca²⁺ depletion generally perturbs tight junction integrity, a specific internalization of tight junction proteins occurs during incubation with sodium caprate. Both compounds are suitable to perturb the paracellular pathway and by this to reversibly enhance the flux of paracellular markers, which represent permeability for pharmacological compounds at least up to a molecular weight of 4000.