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 Ca 2+ ([Ca 2+] 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 [Ca 2+] i or nuclear free Ca 2+ ([Ca 2+] n), we analyzed [Ca 2+] i and [Ca 2+] i in HUVEC during hypoxic injury using confocal laser scanning microscopy. [Ca 2+], and [Ca 2+]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, [Ca 2+] i and [Ca 2+]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 123 fluorescence and an increase in PI-stained cells. Bleb formation was observed after [Ca 2+] i and [Ca 2+] n had reached their peak values and the number of blebs increased thereafter. Confocal sectioning images revealed a localized increase in [Ca 2+] i at the bleb forming site and this localized elevation in [Ca 2+] i was observed before bleb formation in the corresponding area. In conclusion, bleb formation induced by hypoxic stress appears to involve Ca 2+-dependent reactions that are linked to a regional elevation of [Ca 2+] i.