Depolarization-associated iron release with abrupt reduction in pulmonary endothelial shear stress in situ.

This study evaluated the roles of endothelial cell membrane potential and reactive oxygen species (ROS) in the increase of tissue free iron during lung ischemia. Oxygenated ischemia was produced in the isolated rat lung by discontinuing perfusion while ventilation with O2 was maintained. We have shown previously that tissue oxygenation is maintained in this model of ischemia and that biochemical changes are the result of an abrupt reduction in endothelial shear stress. With 1 hr oxygenated ischemia, generation of ROS, evaluated by oxidation of dichlorodihydrofluorescein (H2DCF) to fluorescent dichlorofluorescein, increased 8.0-fold, lung thiobarbituric acid reactive substances (TBARS) increased 3.4-fold, and lung protein carbonyl content increased 2.4-fold. Lung tissue free iron, measured in the lung homogenate with a fluorescent desferrioxamine derivative, increased 4.0-fold during ischemia. Pretreatment of lungs with thapsigargin abolished the increase in free iron with ischemia indicating that this effect is dependent on Ca2+ release from intracellular stores. Perfusion of lungs with high (25 mM) K+ to depolarize the endothelium also led to a significant increase in tissue free iron. Pretreatment of lungs with 35 microM cromakalim, a K+-channel agonist, significantly inhibited both ischemia-induced tissue oxidant injury and the increase in free iron with ischemia or with high K+ perfusion. A similar increase in free iron was observed when lungs were ventilated with either O2 or N2 during the ischemic period or were pre-perfused with an inhibitor of ROS production (diphenyleneiodonium). These results indicate that ROS generation is not required for ischemia-mediated iron release. Thus, ROS generation and iron release with ischemia are independent although both are subsequent to endothelial cell membrane depolarization.