Disruption of epithelial tight junctions is prevented by cyclic nucleotide-dependent protein kinase inhibitors

Tight junctions (TJs), the most apical of the intercellular junctions, prev ent the passage of ions and molecules through the paracellular pathway. Int racellular signalling molecules are likely to be involved in the regulation of TJ integrity. In order to specifically investigate the role of protein kinase A (PKA) in the maintenance of epithelial TJ integrity, calcium-switc h experiments were performed, in which calcium was removed from EpH4 and MD CK culture medium, in the absence or presence of the PKA inhibitors H-89 or HA-1004. Removal of calcium from the culture media of the epithelial cells resulted in disruption of the TJs, characterised by a loss of membrane ass ociation of the TJ-associated proteins occludin, ZO-1 and ZO-2, by a loss o f TJ strands, by a marked decrease in the transepithelial electrical resist ance and by a dramatic increase in the transepithelial permeability to trac ers. The association of occludin, ZO-1 and ZO-2 with the actin cytoskeleton is not affected. In contrast, when the removal of calcium was performed in the presence of either the PKA inhibitor H-89 or HA-1004, all barrier char acteristics were preserved. Our data indicate that following the removal of calcium from the culture medium of epithelial cells in vitro, PKA is activ ated and subsequently is involved in the disruption of TJs.