DIBUTYRYL CYCLIC ADENOSINE-MONOPHOSPHATE AND FORSKOLIN ALTER THE PARACELLULAR PATHWAY IN CULTURED CORNEAL ENDOTHELIAL-CELLS

Purpose. This study describes the effects of the cyclic adenosine mono phosphate (cAMP) pathway on the tight junctional barrier of the cornea l endothelium, which plays a critical role in maintaining the corneal stroma in an underhydrated, transparent state. Methods. Subcultured bo vine corneal endothelial cells grown on filters were used to study the effects of dibutyryl-cAMP and forskolin on transendothelial electrica l resistance and [H-3]inulin flux. The tight junction-associated prote in ZO-1 (zonula occludens protein-1) and F-actin were visualized by in direct immunofluorescence, and the ultrastructural organization of jun ctional complexes was studied by freeze-fracture electron microscopy. Results. Cells formed a continuous monolayer of closely apposed hexago nal-type cells separated by a discontinuous belt of tight junctions wi th a transendothelial electrical resistance of 20.8 +/- 0.6 Omega . cm (2). Dibutyryl-cAMP (10(-4) M) and forskolin (10(-5) M) increased cell cAMP, significantly decreased the transendothelial resistance by 54% and 43%, respectively, and increased the flux of [3H]inulin from the a pical to the basal side of the cells by 56% and 40%, respectively. Bot h agents also induced condensation of F-actin at the cell borders with out any marked changes in the immunostaining of ZO-1 that delineated c ell peripheries. However, freeze-fracture studies showed that dibutyry l-cAMP and forskolin induced dispersion of the tight junction network. Conclusions. These data suggest that activation of the cAMP-dependent pathway, leading to structural changes of the tight junctional networ k, may modulate the passive fluxes mediated by the paracellular pathwa y of the corneal endothelial barrier.