H2O2 and genistein differentially modulate protein tyrosine phosphorylation, endothelial morphology, and monolayer barrier function.
The effects of hydrogen peroxide (H2O2) and the protein tyrosine kinase (PTK) inhibitor, genistein, to modulate protein tyrosine phosphorylation (PTP) and endothelial barrier function were examined in bovine pulmonary artery endothelial cell (EC) monolayers. H2O2 stimulated a concentration (100-800 microM) and time-dependent increase in the phosphotyrosine (PY) content of multiple (56-72, 93-97, 113-142, and 161-183 kDa) EC proteins. A size-selected solute permeability assay of EC monolayer barrier function showed that (200 microM) H2O2 elevated EC monolayer permeability to large solutes. This effect was associated with paracellular hole formation and a loss of beta-catenin immunostaining at these sites. In contrast, genistein (100 microM, 1 h) reduced basal PY protein content and reorganized F-actin to beta-catenin containing cell-cell junctions, enhancing endothelial monolayer barrier function. In addition, genistein prevented the H2O2-induced increases in tyrosine phosphorylation, monolayer permeability, and paracellular hole formation. These data suggest that H2O2 and genistein differentially regulate PTP, endothelial morphology, and monolayer barrier function.
Department of Veteran Affairs Medical Center, University of Arizona, Tucson, Arizona, 85723, USA.
J M
JM
Carbajal
R C
RC
Schaeffer
D000199:Actins / Q000032:analysis; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000032:analysis; D004730:Endothelium, Vascular / Q000378:metabolism*; D019833:Genistein / Q000494:pharmacology*; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007700:Kinetics; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D011651:Pulmonary Artery; D015534:Trans-Activators; D051176:beta Catenin
D000199:Actins; D003598:Cytoskeletal Proteins; D010750:Phosphoproteins; D015534:Trans-Activators; D051176:beta Catenin; D019000:Phosphotyrosine; D006861:Hydrogen Peroxide; D019833:Genistein