H2O2-mediated permeability II: importance of tyrosine phosphatase and kinase activity

We previously reported that exposure of endothelial cells to H2O2 results i n a loss of cell-cell apposition and increased endothelial solute permeabil ity. The purpose of this study was to determine how tyrosine phosphorylatio n and tyrosine phosphatases contribute to oxidant-mediated disorganization of endothelial cell junctions. We found that H2O2 caused a rapid decrease i n total cellular phosphatase activity that facilitates a compensatory incre ase in cellular phosphotyrosine residues. H2O2 exposure also results in inc reased endothelial monolayer permeability, which was attenuated by pp60, an inhibitor of src kinase. Inhibition of protein tyrosine phosphatase activi ty by phenylarsine oxide (PAO) demonstrated a similar permeability profile compared with H2O2, suggesting that tyrosine phosphatase activity is import ant in maintaining a normal endothelial solute barrier. Immunofluorescence shows that H2O2 exposure caused a loss of pan-reactive cadherin and beta -c atenin from cell junctions that was not blocked by the src kinase inhibitor PP1. H2O2 also caused beta -catenin to dissociate from the endothelial cyt oskeleton, which was not prevented by PP1. Finally, we determined that PP1 did not prevent cadherin internalization. These data suggest that oxidants like H2O2 produce biological effects through protein phosphotyrosine modifi cations by decreasing total cellular phosphatase activity combined with inc reased src kinase activity, resulting in increased endothelial solute perme ability.