Human umbilical vein endothelial cells (HUVECs) show Ca2+ mobilization as well as Ca2+ influx upon hypoxia

Bleb formation is an early event of cellular damage observed in a variety o f cell types upon hypoxia. Although we previously found that the [Ca2+](i) rise before bleb formation only at the same loci of HUVECs upon hypoxia (lo calized [Ca2+](i) rise), the mode of the [Ca2+](i) rise remains ill-defined . In order to clarify the mechanisms causing the localized [Ca2+](i) rise i n hypoxia challenged HUVECs, we studied the effects of several Ca2+ channel blockers or a Ca2+ chelator, EGTA, which reduces extracellular Ca2+ concen tration on the hypoxia-induced localized [Ca2+](i) rise and bleb formation by employing a confocal laser scanning microscopy (CLSM). After the initiat ion of hypoxia, [Ca2+](i) rose gradually in a localized fashion up to 15 mi n, which was associated with bleb formation at the same loci. The maximal [ Ca2+](i) rise was 435 +/- 84 nM at the loci of bleb formation. Ca2+ channel blockers including Ni2+ (non-specific, 1 mM), nifedipine (L type, 10 mu M) , nicardipine (L + T type, 10 mu M), and cilnidipine (L + N type, 10 mu M) did not inhibit either the localized [Ca2+](i) rise or bleb formation. Alth ough both the localized [Ca2+](i) rise and bleb formation were inhibited by lowering extracellular Ca2+ concentration below 100 nM, a diffuse [Ca2+](i ) rise through the cytoplasm remained without bleb formation, which was inh ibited by a phospholipase C (PLC) inhibitor, U73122. In conclusion, hypoxia causes both the Ca2+ mobilization and the Ca2+ influx in HUVECs and the Ca 2+ influx through unknown Ca2+ channels is responsible for the localized [C a2+](i) rise integral to bleb formation. (C) 2000 Wiley-Liss, Inc.