In situ imaging of intracellular calcium with ischemia in lung subpleural microvascular endothelial cells

We propose that generation of reactive oxygen species (ROS) during ischemia is associated with an increase in intracellular calcium ([Ca2+]i) in pulmonary capillary endothelial cells. We used an isolated rat lung model and epifluorescence microscopy to evaluate [Ca2+]i in subpleural microvascular endothelial cells in situ by ratio imaging of the fluorophores, Calcium Green and Fura Red (CG/FR). Lungs were ventilated continuously under control (continuously perfused) or global ischemia (no perfusion) and thus remained adequately oxygenated even with ischemia. Ischemia for 5 min led to increase in CG/FR, indicating increase in [Ca2+]i in endothelial cells in situ; CG/FR remained elevated during a subsequent 10 min of ischemia. Ca(2+)-free perfusion and gadolinium (100 microM) inhibited the increase in [Ca2+]i, while thapsigargin (250 nM) had no effect. These results indicate that increase in endothelial cell [Ca2+]i with ischemia was due to influx from the extracellular medium. Perfusion with N-acetyl-L-cysteine (20 mM) or diphenyleneiodonium chloride (10 microM) prevented the ischemia-mediated [Ca2+]i increase, suggesting a role for ROS in the Ca2+ changes with ischemia. Membrane depolarization by perfusion with high potassium (K+) or glyburide also resulted in increased [Ca2+]i whereas the K(+)-channel agonist cromakalim, inhibited ischemia-mediated Ca2+ influx. We conclude that increased ROS generation with 'oxygenated' lung ischemia is associated with influx of Ca2+ and an increase in endothelial cell cytosolic calcium concentration.