PI3Kinase/Akt activation trigger ROS production in a model of pulmonary ischemia

Abstract

We reported earlier that ischemia or loss of shear in the lung endothelium causes endothelial membrane depolarization (via KATP channel closure) that leads to NADPH oxidase activation and reactive oxygen species (ROS) production. ROS production was abolished in the presence of PI3Kinase/Akt inhibitor, wortmanin. We thus hypothesized that PI3K/Akt activation is the trigger for ROS production with pulmonary ischemia. We used pulmonary microvascular endothelial cells, either wild type (WT), KATP null (KIR6.2−/−), NADPH oxidase null (gp91phox−/−) or WT infected with dominant negative (DN) PI3K, and isolated WT and Akt‐1 null lungs to study signal transduction with ischemia. In WT lungs, the PI3K inhibitor wortmannin had no effect on ischemia mediated depolarization. Cells with DN PI3K and lungs with Akt1−/− depolarized with ischemia but failed to show NADPH oxidase assembly and ROS generation. Depolarization in WT and Akt−/− lungs was blocked upon addition of cromakalim (KATP channel agonist). In WT cells, Akt phosphorylation that was observed with ischemia and was abolished in KIR6.2−/− but not in gp91phox−/− cells. These results indicate that KATP induced depolarization leads to PI3K/Akt activation that triggers NADPH oxidase assembly and eventual ROS generation. Thus, ROS which has hitherto been shown to activate PI3K/Akt in other systems lies downstream of PI3K/Akt in our model of pulmonary ischemia.