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 123 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 Ca(2+)-dependent reactions that are linked to a regional elevation of [Ca2+]i.
Ikeda
M
M
Department of Surgery II, Osaka University Medical School, Japan.
Ariyoshi
H
H
Sakon
M
M
Kambayashi
J
J
Yoshikawa
N
N
Shinoki
N
N
Kawasaki
T
T
Monden
M
M
D000703:Analog-Digital Conversion; D002118:Calcium / Q000378:metabolism*; D015687:Cell Hypoxia; D002470:Cell Survival; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008564:Membrane Potentials; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism; D020112:Rhodamine 123; D014471:Umbilical Veins
D020112:Rhodamine 123; D002118:Calcium