Separate analysis of nuclear and cytosolic Ca2+ concentrations in human umbilical vein endothelial cells.
Ca2+ concentration inside human umbilical vein endothelial cells was studied separately in cytosol and nucleus by a confocal laser scanning microscopy using fluo-3. The in vivo calibration curve for cytosol and nucleus showed good linearity between fluorescence intensity and Ca2+ concentration in cytosol ([Ca2+]i) and nuclei ([Ca2+]n). After calibration, [Ca2+]n was constantly higher than [Ca2+]i before and after the chelation of extracellular Ca2+ suggesting an active Ca2+ accumulation system on nuclear membrane. [Ca2+]n was also constantly higher than [Ca2+]i after the stimulation of thrombin (0.05 U/ml), FCS (10%), and thapsigargin (Tsg, 1 microM). The temporal change of [Ca2+]n and [Ca2+]i was identical, and [Ca2+]i gradient towards the nucleus and peripheral or central [Ca2+]n rise was observed after these stimulations. From these results, [Ca2+]n is not only regulated by the active Ca2+ accumulation system on nuclear membrane at rest but also the generation of inositol-triphosphate. FCS caused heterogeneous [Ca2+]n or [Ca2+]i rise from cell to cell; single spike or oscillatory change of [Ca2+]n and [Ca2+]i was observed in about 56% of cells, which were completely abolished by the chelation of extracellular Ca2+, suggesting that FCS stimulated [Ca2+]n and [Ca2+]i rise solely depending on Ca2+ influx from extracellular medium. The higher concentration of [Ca2+]n and heterogeneous [Ca2+]n rise may have important roles in nuclear-specific cellular responses.
Department of Surgery II, Osaka University Medical School, Japan.
M
M
Ikeda
H
H
Ariyoshi
J
J
Kambayashi
K
K
Fujitani
N
N
Shinoki
M
M
Sakon
T
T
Kawasaki
M
M
Monden
D000814:Aniline Compounds; D002118:Calcium / Q000032:analysis*; D002467:Cell Nucleus / Q000737:chemistry; D002478:Cells, Cultured; D003600:Cytosol / Q000737:chemistry; D004533:Egtazic Acid / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000737:chemistry* / Q000187:drug effects; D005456:Fluorescent Dyes; D006801:Humans; D018613:Microscopy, Confocal; D019284:Thapsigargin / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D014471:Umbilical Veins; D014966:Xanthenes
D000814:Aniline Compounds; D005456:Fluorescent Dyes; D014966:Xanthenes; C059715:Fluo-3; D004533:Egtazic Acid; D019284:Thapsigargin; D013917:Thrombin; D002118:Calcium