Ikeda M, Ariyoshi H, Kambayashi J, Fujitani K, Shinoki N, Sakon M, Kawasaki T, Monden M
Department of Surgery II, Osaka University Medical School, Japan.
J Cell Biochem. 1996 Oct;63(1):23-36. doi: 10.1002/(sici)1097-4644(199610)63:1<23::aid-jcb2>3.0.co;2-1.
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.
使用荧光素-3通过共聚焦激光扫描显微镜分别研究了人脐静脉内皮细胞胞质溶胶和细胞核内的Ca2+浓度。胞质溶胶和细胞核的体内校准曲线显示荧光强度与胞质溶胶中Ca2+浓度（[Ca2+]i）和细胞核中Ca2+浓度（[Ca2+]n）之间具有良好的线性关系。校准后，在细胞外Ca2+螯合前后，[Ca2+]n始终高于[Ca2+]i，这表明核膜上存在活跃的Ca2+积累系统。在凝血酶（0.05 U/ml）、胎牛血清（FCS，10%）和毒胡萝卜素（Tsg，1 microM）刺激后，[Ca2+]n也始终高于[Ca2+]i。[Ca2+]n和[Ca2+]i的时间变化是相同的，并且在这些刺激后观察到朝向细胞核的[Ca2+]i梯度以及外周或中央[Ca2+]n的升高。从这些结果来看，[Ca2+]n不仅在静止时受核膜上活跃的Ca2+积累系统调节，还受肌醇三磷酸的生成调节。FCS导致细胞间[Ca2+]n或[Ca2+]i升高不均匀；在约56%的细胞中观察到[Ca2+]n和[Ca2+]i的单峰或振荡变化，细胞外Ca2+螯合可完全消除这些变化，这表明FCS刺激[Ca2+]n和[Ca2+]i升高完全依赖于细胞外介质中的Ca2+内流。较高的[Ca2+]n浓度和不均匀的[Ca2+]n升高可能在细胞核特异性细胞反应中起重要作用。
