Involvement of Ca2+in the H2O2-induced increase in endothelial permeability

We studied the role of Ca2+ in mediating the hydrogen peroxide (H2O2)-induced increase in endothelial permeability to I-125-labeled albumin using bovine pulmonary microvessel endothelial cells (BMVEC). Changes in cytosolic-free Ca2+ ([Ca2+](i)) were monitored in BMVEC monolayers loaded with the Ca2+-sensitive membrane permeant fluorescent dye fura 2-AM. H2O2 (100 mu M) produced a rise in [Ca2+](i) within 10 a that was reduced by the addition of EGTA to the medium. Uptake of Ca-45(2+) from the extracellular medium increased in the presence of H2O2 (100 mu M) compared with control mono-layers, suggesting that the H2O2-induced rise in [Ca2+](i) is partly the result of extracellular Ca2+ influx. The effects of [Ca2+](i) on endothelial permeability were addressed by pre-treatment of BMVEC monolayers with BAPTA-AM (3-5 mu M), a membrane permeant Ca2+ chelator, before the H2O2 exposure. BAPTA-AM produced an similar to 50% decrease in the H2O2-induced increase in endothelial permeability compared with endothelial cell monolayers exposed to H2O2 alone. The increase in endothelial permeability was independent of Ca2+ influx, since LaCl3 (0-100 mu M), which displaces Ca2+ from binding sites on the cell surface, did not modify the permeability response. These results indicate that the rise in [Ca2+](i) produced by H2O2 is a critical determinant of the increase in endothelial permeability.