title: Dibutyryl cyclic adenosine monophosphate and forskolin alter the paracellular pathway in cultured corneal endothelial cells.
abstract: OBJECTIVE
  This study describes the effects of the cyclic adenosine monophosphate (cAMP) pathway on the tight junctional barrier of the corneal endothelium, which plays a critical role in maintaining the corneal stroma in an underhydrated, transparent state.
  
  
  METHODS
  Subcultured bovine corneal endothelial cells grown on filters were used to study the effects of dibutyryl-cAMP and forskolin on transendothelial electrical resistance and [3H]inulin flux. The tight junction-associated protein ZO-1 (zonula occludens protein-1) and F-actin were visualized by indirect immunofluorescence, and the ultrastructural organization of junctional complexes was studied by freeze-fracture electron microscopy.
  
  
  RESULTS
  Cells formed a continuous monolayer of closely apposed hexagonal-type cells separated by a discontinuous belt of tight junctions with a transendothelial electrical resistance of 20.8 +/- 0.6 omega.cm2. 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 apical to the basal side of the cells by 56% and 40%, respectively. Both agents also induced condensation of F-actin at the cell borders without any marked changes in the immunostaining of ZO-1 that delineated cell peripheries. However, freeze-fracture studies showed that dibutyryl-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 network, may modulate the passive fluxes mediated by the paracellular pathway of the corneal endothelial barrier.
authors.affiliation: Laboratoire de Recherches Ophtalmologiques, Unité 86, Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France.
authors.forename: B
authors.initials: B
authors.lastname: Le Varlet
authors.forename: R
authors.initials: R
authors.lastname: Ducroc
authors.forename: F B
authors.initials: FB
authors.lastname: Dagonet
authors.forename: Y
authors.initials: Y
authors.lastname: Pouliquen
authors.forename: A
authors.initials: A
authors.lastname: Vandewalle
authors.forename: M
authors.initials: M
authors.lastname: Hirsch
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology*; D002417:Cattle; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D004553:Electric Conductivity; D004728:Endothelium, Corneal / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D019084:Fluorescent Antibody Technique, Indirect; D005614:Freeze Fracturing; D007444:Inulin / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D062826:Zonula Occludens-1 Protein
chemical_list: D000199:Actins; D008565:Membrane Proteins; D010750:Phosphoproteins; D062826:Zonula Occludens-1 Protein; D005576:Colforsin; D003994:Bucladesine; D007444:Inulin; D000242:Cyclic AMP