INVOLVEMENT OF CA2-INDUCED INCREASE IN ENDOTHELIAL PERMEABILITY( IN THE H2O2)

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 wa s reduced by the addition of EGTA to the medium. Uptake of Ca-45(2+) f rom the extracellular medium increased in the presence of H2O2 (100 mu M) compared with control mono-layers, suggesting that the H2O2-induce d rise in [Ca2+](i) is partly the result of extracellular Ca2+ influx. The effects of [Ca2+](i) on endothelial permeability were addressed b y pre-treatment of BMVEC monolayers with BAPTA-AM (3-5 mu M), a membra ne permeant Ca2+ chelator, before the H2O2 exposure. BAPTA-AM produced an similar to 50% decrease in the H2O2-induced increase in endothelia l permeability compared with endothelial cell monolayers exposed to H2 O2 alone. The increase in endothelial permeability was independent of Ca2+ influx, since LaCl3 (0-100 mu M), which displaces Ca2+ from bindi ng sites on the cell surface, did not modify the permeability response . These results indicate that the rise in [Ca2+](i) produced by H2O2 i s a critical determinant of the increase in endothelial permeability.