A role for local calcium gradients upon hypoxic injury in human umbilical vein endothelial cells (HUVEC)

Upon hypoxic injury, bleb formation is an early event of cell damage observed in a variety of cell types. Although a rise in cytosolic free Ca2+ ([Ca2+](i)) has been considered to be involved in this process, the exact relationship between these phenomena remains ill-defined. In order to examine the relationship between bleb formation, and [Ca2+](i) or nuclear free Ca2+ ([Ca2+](n)), we analyzed [Ca2+](i) and [Ca2+](n) in HUVEC during hypoxic injury using confocal laser scanning microscopy. [Ca2+](i) and [Ca2+](n) were measured using Fluo-3, and cell viability and mitochondrial membrane potential were assessed by the exclusion of propidium iodide (PI) and rhodamine 123, respectively. After the initiation of hypoxia, [Ca2+](i) and [Ca2+](n) rose gradually up to 15 min reaching peak values of 447 +/- 62 and 516 +/- 105 nM, respectively which was accompanied by a decrease in rhodamine 1.23 fluorescence and an increase in PI-stained cells. Bleb formation was observed after [Ca2+](i) and [Ca2+](n) had reached their peak values and the number of blebs increased thereafter. Confocal z-sectioning images revealed a localized increase in [Ca2+](i) at the bleb forming site and this localized elevation in [Ca2+](i) was observed before bleb formation in the corresponding area. In conclusion, bleb formation induced by hypoxic stress appears to involve Ca2+-dependent reactions that are linked to a regional elevation of [Ca2+](i).