Fig. 2. Membrane depolarization precedes ROS generation.

In subpleural endothelial cells in intact rat or mouse lungs. A. The time course of membrane potential change with ischemia. A decreased cell membrane potential with ischemia is indicated by increased fluorescence intensity of di-8-ANEPPS; the effect is blocked by the K_ATP channel agonist, lemakalim. The inset shows a rapid time frame recording of the initial 5 secs after stop of flow. Control (con) is the “ischemic start” point. B. Membrane depolarization with high K⁺ results in ROS generation in the absence of ischemia as detected by increased DCF fluorescence. Control was continuous flow with buffer containing physiological (5 mM) K⁺. C. Quantitation of ROS generation during ischemia by DCF fluorescence. The increased ROS production with ischemia is blocked by the presence of catalase to scavenge H₂O₂, cromakalim (a K_ATP channel agonist), or DPI (an inhibitor of NADPH oxidase) and is decreased in lungs from K_IR 6.2 null mice. The absence of ROS in gp91^phox null lungs indicates that ROS are generated by NADPH oxidase. For all panels, fluorescence intensity of 3 lungs (each representing the average value for 4-7 endothelial cells) are plotted as means ± SE. Reprinted with permission from [16, 38, 51].