SEPARATE ANALYSIS OF NUCLEAR AND CYTOSOLIC CA2-CELLS( CONCENTRATIONS IN HUMAN UMBILICAL VEIN ENDOTHELIAL)

Ca2+ concentration inside human umbilical vein endothelial cells was s tudied 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 ca libration, [Ca2+](n) was constantly higher than [Ca2+](i) before and a fter the chelation of extracellular Ca2+ suggesting an active Ca2+ acc umulation system on nuclear membrane. [Ca2+](n) was also constantly hi gher than [Ca2+](i) after the stimulation of thrombin (0.05 U/ml), FCS (10%), and thapsigargin (Tsg, 1 mu M). The temporal change of [Ca2+]( n) and [Ca2+](i) was identical, and [Ca2+](i) gradient towards the nuc leus 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 al so the generation of inositol-triphosphate. FCS caused heterogeneous [ Ca2+](n) or [Ca2+](i) rise from cell to cell; single spike or oscillat ory change of [Ca2+](n) and [Ca2+](i) was observed in about 56% of cel ls, which were completely abolished by the chelation of extracellular Ca2+, suggesting that FCS stimulated [Ca2+](n) and [Ca2+](i) rise sole ly depending on Ca2+ influx from extracellular medium. The higher conc entration of [Ca2+](n) and heterogeneous [Ca2+](n) rise may have impor tant roles in nuclear-specific cellular responses. (C) 1996 Wiley-Liss , Inc.