Outline:

Claudins, constituing the majority of tight junction proteins, in some cases form paracellular channels but mostly exert sealing properties. Whereas single claudins have been functionally characterized in cell monolayers so far, information on the functional interplay with secretory mechanisms in native tissue is limited. Therefore, in our study, effects of cholera toxin and theophylline were analyzed in rat colon including detection of sealing tight junction proteins.

Methods:

Tissue specimens were stripped off submucosal tissue layers and mounted in Ussing chambers. Short circuit current (I_sc) and transepithelial resistance (TER) were recorded, and expression and localization of claudins were analyzed by Western blotting and immunostaining.

Results:

Application of theophylline induced a strong increase of I_sc in colon tissue specimens over a time course of 120 min. In parallel, a decrease of TER was observed. In contrast, cholera toxin did not alter I_sc, but increased TER. Protein expression of claudin-3 and -4 was increased after incubation with cholera toxin while theophylline induced an increase of claudin-1. Confocal immunofluorescence microscopy revealed an increased appearance of claudin-3 and -4 within the tight junction complex, whereas abundance of claudin-1 was increased in subjunctional compartments only.

Conclusion:

Cholera toxin and theophylline have different effects on the regulation of sealing tight junction proteins in native colon preparations. Whereas theophylline induces a subjunctional increase of claudin-1, incubation with cholera toxin leads to an increased appearance of claudin-3 and -4 within the tight junction. In rat colon source tissue, both perturbators of transcellular Cl^- secretion also affect paracellular permeability.