Role Of Pi3kinase/Akt And Rac In Activation Of The Endothelial Nadph Oxidase With Ischemia

We have shown previously that ischemia or abrupt cessation of flow in the lung endothelia in situ and in pulmonary microvascular endothelial cells (PMVEC) in vitro causes ROS production. This occured via NADPH oxidase as knockout of gp91phox prevented the response. We also reported that the trigger for NADPH oxidase activation is depolarization of the endothelial cell plasma membrane (via KATP channels) as preventing the depolarization with a channel agonist prevented the ROS generation. In phagocytes, the small G protein, rac (Rac1) is known to participate in NADPH assembly and activation. To investigate if Rac plays a similar role in endothelial NADPH oxidase assembly, we examined the link between endothelial cell membrane depolarization, Rac activation and NADPH oxidase assembly. Using PMVECs transfected with RacGFP, Rac translocation was monitored with membrane depolarization (achieved either by high external K+ or by ischemia). The significant increase in Rac translocation that was observed with depolarization was attenuated by C difficile Toxin B (Rac inhibitor) as also by wortmannin and LY294002 (PI3K inhibitors). PMVECs infected with dominant negative PI3K (mutant PI3K p85 subunit) showed no translocation of Rac to plasma membrane upon depolarization of the cells. These results indicate that PI3K/Akt may be upstream of Rac and that Rac 1 translocation is required for NADPH oxidase activation.