Membrane depolarization is the trigger for PI3K/Akt activation and leads to the generation of ROS

Chatterjee S, Browning EA, Hong N, DeBolt K, Sorokina EM, Liu W, Birnbaum MJ, Fisher AB. Membrane depolarization is the trigger for PI3K/Akt activation and leads to the generation of ROS. Am J Physiol Heart Circ Physiol 302: H105-H114, 2012. First published October 14, 2011; doi:10.1152/ajpheart.00298.2011.-Loss of fluid shear stress (ischemia) to the lung endothelium causes endothelial plasma membrane depolarization via ATP-sensitive K+ (KATP) channel closure, initiating a signaling cascade that leads to NADPH oxidase (NOX2) activation and ROS production. Since wortmannin treatment significantly reduces ROS production with ischemia, we investigated the role of phosphoinositide 3-kinase (PI3K) in shear-associated signaling. Pulmonary microvascular endothelial cells in perfused lungs subjected to abrupt stop of flow showed membrane depolarization and ROS generation. Stop of flow in flow-adapted mouse pulmonary microvascular endothelial cells in vitro resulted in the activation of PI3K and Akt as well as ROS generation. ROS generation in the lungs in situ was almost abolished by the PI3K inhibitor wortmannin and the PKC inhibitor H7. The combination of the two (wortmannin and H7) did not have a greater effect. Activation of NOX2 was greatly diminished by wortmannin, knockout of Akt1, or dominant negative PI3K, whereas membrane depolarization was unaffected. Ischemia-induced Akt activation (phosphorylation) was not observed with KATP channel-null cells, which showed minimal changes in membrane potential with ischemia. Activation of Akt was similar to wild-type cells in NOX2-null cells, which do not generate ROS with ischemia. Cromakalim, a KATP channel agonist, prevented both membrane depolarization and Akt phosphorylation with ischemia. Thus, Akt1 phosphorylation follows cell membrane depolarization and precedes the activation of NOX2. These results indicate that PI3K/Akt and PKC serve as mediators between endothelial cell membrane depolarization and NOX2 assembly.