Studies on the alteration of calcium ions in osteoblastic cell line using a fluorescence dye

在小鼠成骨细胞克隆MC3T3-E1细胞中，用荧光分光光度计和荧光显微镜测量系统研究了细胞内钙离子的内流和变化。在人表皮样癌细胞系A431中观察到对表皮生长因子(EGF)有反应的Ca~(2+)~(2+)和Gt~(2+)离子只有一次内流，而成骨细胞系MC3T_3-E_1则未见此现象。然而，两种细胞系对胎牛血清(FCS)的反应均使细胞内游离钙离子浓度升高。此外，FCS处理后的成骨细胞MC3T3-E1出现了对EGF有反应的Ca~(2+)和Gt~(2+)离子内流，提示在EGF和FCS中的任一因子(S)的协同作用下，有少量的Ca~(2+)和Gt~(2+)离子进入成骨细胞。这些结果表明，在成骨细胞的生长初期，EGF-EGF受体系统传递的信号是通过协同作用发挥作用的。另一方面，我们还发现神经营养因子-3(NT-3：一种神经生长因子)及其受体(TrkC)在成骨细胞MC3T3-E1中表达，并且成骨细胞中存在对NT-3反应的钙离子内流。这表明，在MC3T3-E1细胞的生长初期，NT-3与TrkC相互作用所致的信号转导系统起作用。此外，荧光显微镜测量系统对细胞内Ca~(2+)浓度变化的测绘数据表明，MC3T3-E1细胞内Ca~(2+)的积累量高于A431细胞。这表明成骨细胞系统中存在一种成骨细胞特有的细胞内钙蓄积机制，其作用方式不同于其他细胞，如表皮细胞。

In mouse osteoblastic clone MC3T3-E1 cells, influx and intracellular alteration of Ca^<2+>ions were investigated with the fluorescence-spectrophotometer and fluorrescence-microscopic measurement system using a fluorescence reagent for intracellular Ca^<2+>ions. One passing influx of Ca^<2+>ions responsive to epidermal growth factor (EGF) was observed in human epidermoid carcinoma cell line : A431, but not in the osteoblastic cell line : MC3T3-E1 with both the fluorescence measurements. However, the increase of intracellular concentration of free Ca^<2+>ions responsive to fetal calf serum (FCS) was detected in both cell lines. With the manner of one passage, futhermore, influx of Ca^<2+>ions responsive to EGF appeared in the osteblastic MC3T3-E1 cells after the FCS-treatment, suggesting that a slight influx of Ca^<2+>ions into the MC3T3-E1 cells is induced with the synergistic function of EGF and any factor (s) in FCS.These results indicate that a very silght signal transduing due to the EGF-EGF receptor system is functioning by the synergistic effect during the initial growth phase of MC3T3-E1 cells. On the other hand, we also found that neurotrophin-3 (NT-3 : one of nerve growth factors) and its receptor (TRKC) is expressed in the osteoblastic MC3T3-E1 cells and the influx of Ca^<2+>ions responsive to NT-3 occurs in the osteoblastic cells. This implies that the signal transdicing sytem due to the interaction between NT-3 and TRKC is acting during the initial growth of MC3T3-E1.Moreover, photomapping data on the alteration of intracellular Ca^<2+>concentration detected with a fluorescence-microscopic measurement system indicated that the intracellular Ca^<2+>accumulation in MC3T3-E1 cells is higher than that in A431 cells. This suggests that an osteoblast-specific mechanism of intracellular Ca^<2+> accumulation plays in the osteoblastic cell system with the different manner from other cells such as epidermal cells.