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

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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.