Hydrogen peroxide stimulates tyrosine phosphorylation of focal adhesion kinase in vascular endothelial cells

Reactive oxygen species (ROS) are implicated in the pathophysiology of seve ral vascular disorders including atherosclerosis. Although the mechanism(s) of ROS-induced vascular damage remains unclear, there is increasing eviden ce for ROS-mediated modulation of signal transduction pathways. Exposure of bovine pulmonary artery endothelial cells to hydrogen peroxide (H2O2) enha nced tyrosine phosphorylation of 60- to 80- and 110- to 130-kDa cellular pr oteins, which were determined by immunoprecipitation with specific antibodi es focal adhesion kinase (p125(FAK)) and paxillin (p68). Brief exposure of cells to a relatively high concentration of H2O2 (1 mM) resulted in a time- and dose-dependent tyrosine phosphorylation of FAK, which reached maximum levels within 10 min (290% of basal levels). Cytoskeletal reorganization as evidenced by the appearance of actin stress fibers preceded H2O2-induced t yrosine phosphorylation of FAK, and the microfilament disrupter cytochalasi n D also attenuated the tyrosine phosphorylation of FAK. Treatment of BPAEC s with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM attenuat ed H2O2-induced increases in intracellular Ca2+ but did not show any consis tent effect on H2O2-induced tyrosine phosphorylation of FAK. Several tyrosi ne kinase inhibitors, including genistein, herbimycin, and tyrphostin, had no detectable effect on tyrosine phosphorylation of FAK but attenuated the H2O2-induction of mitogen-activated protein kinase activity. We conclude th at H2O2-induced increases in FAK tyrosine phosphorylation may be important in H2O2-mediated endothelial cell activation.