THROMBIN-MEDIATED FOCAL ADHESION PLAQUE REORGANIZATION IN ENDOTHELIUM- ROLE OF PROTEIN-PHOSPHORYLATION

Endothelial cell (EC) gap formation and barrier function are subject t o dual regulation by (I) axial contractile forces, regulated by myosin light chain kinase activity, and (2) tethering forces, represented by cell-cell and cell-substratum adhesions. We examined whether focal ad hesion plaque proteins (vinculin and talin) and focal adhesion kinase, p125(FAK) (FAK), represent target regulatory sites involved in thromb in-mediated EC barrier dysfunction. Histologically, thrombin produced dramatic rearrangement of EC actin, vinculin, and FAK in parallel with the evolution of gap formation and barrier dysfunction. Vinculin and talin were in vitro substrates for phosphorylation by EC PKC, a key ef fector enzyme involved in thrombin-induced EC barrier dysfunction. Alt hough vinculin and talin were phosphorylated in situ under basal condi tions in P-32-labeled EC, thrombin failed to alter the basal level of phosphorylation of these proteins. Phosphotyrosine immunoblotting show ed that neither vinculin nor talin was significantly phosphorylated in situ on tyrosine residues in unstimulated ECs, and this was not furth er increased after thrombin. In contrast, both thrombin and the thromb in receptor-activating peptide (TRAP) produced an increase in FAK phos photyrosine levels (corrected for immunoreactive FAK content) present in EC immunoprecipitates. Ionomycin, which produces EC barrier dysfunc tion in a myosin Light chain kinase-independent manner, was used to in crease intracellular Ca2+ and evaluate the Ca2+ sensitivity of this ob servation. In contrast to thrombin, ionomycin effected a dramatic decr ease in the phosphotyrosine-to-immunoreactive FAK ratios, suggesting d istinct effects of the two agents on FAK phosphorylation and function. These data indicate that modulation of cell tethering via phosphoryla tion of focal adhesion proteins is complex, agonist-specific, and may be a relevant mechanism of EC barrier dysfunction in permeability mode ls that do not depend on an increase in myosin 20-kD regulatory Light chain phosphorylation.