Intracellular signal transaction of osteoblastic cells cultured in a low calcium environments

众所周知，体液中的低钙（Ca）条件导致硬组织的各种畸形。我们以前的研究表明，在体内和体外的大鼠股骨的畸形和消除之间的相关性。结果清楚地表明，细胞暴露于低钙环境中会发生钙化机制的紊乱。成骨细胞在骨形成的钙调节中起核心作用。在本研究中，我们研究了低Ca环境对从Wistar大鼠胚胎颅骨分离的成骨细胞细胞内信号转导的影响。细胞在BGJ_B培养基中培养10天。培养基中的Ca浓度对于对照细胞为1.87 mM，对于低Ca细胞为0.34 mM。低钙细胞的变化如下：两种细胞组分中Ca^<2+>含量均降低。细胞内游离Ca（[Ca^<2+]i）和1，4，5-三磷酸肌醇（IP_3）含量显著降低。在伴刀豆球蛋白A刺激或加入外源性Ca后，[Ca^2+]i迅速增加。胞浆和胞膜组分中蛋白激酶C（PKC）活性均降低。当培养基改为对照培养基时，细胞膜组分中PKC活性的增加明显高于胞浆组分。两种细胞中PKC（Ⅰ、Ⅱ、Ⅲ型）的免疫细胞化学染色结果相似。Ⅰ型细胞胞浆内可见。尤其是部分细胞质染色更为明显。而且，它还存在于质膜的内部。Ⅱ型为强雌激素。Typel几乎没有染色或仅显示微弱的染色。这些发现表明，在非生理条件下，如低钙，细胞可能执行功能替代，以维持正常的钙代谢在硬组织。科学研究补助金资助（编号：02670822，63480408）。

It is widely known that low calcium(Ca)conditions in body fluids cause various malformations of hard tissue. Our previous studies have shown a corelation between the malfortation and the elnvironment for the rat-femur in vivo and in vitro. The results clearly showed that disturbances to the calcification mechanism occur with cells exposed to the low-Ca environment. Osteoblasts play a central role in the Ca regulation of bone formation. In the present study, we examined the effect of a lowCa environment on intracellular sggnal trunsduction ofosteoblastic cells isolated from the embryonic calvaria of Wistar rats. The cells were cultured up to 10 days in BGJ_b medlum. The Ca concentration in the medium was 1.87 mM for control cells and 0.34 mM for low-Ca cells. The changes in the low-Ca cells were as follows : The Ca^<2+> contents decreased in both total the cellular fractions. Intracellular free Ca([Ca^<2+>]i)and'inositol 1, 4, 5-triphosphate(IP_3)contents decreased markedly. The[Ca^2+]i increased rapidly after stimulation with concanavalin A or the addition of exogenous Ca. The activity of protein kinase C(PKC)decreased in both cytosol and membrane fractions. When the medium was changed to the control medium, the increase of PKC activity in the membrane fraction was much higher than that in cytosol one. The immunocytochemical stinning of PKC(subspecies : TypeI, II andIII)was observed by similar in the both cells. TypeI was cleary observed in the cytoplasm. Especially, the staining was more markedly in a part of cytoplasm. Also, it was presented at the inside of the plasmamembrane. TypeII was the strongestisozne. Typel showed almost no staining or only a faint staining. These findings suggest that the cells under non-physiological conditions such as low-Ca, may perform a functional replacement to maintain normal calcium metabolism in hard tissue. Supported by Grant-in-Aid for Scientific Research(Nos. 02670822, 63480408)of Japan.