Function analysis of Na^+ dependent ion transporters in bone metabolism

在许多哺乳动物细胞中，Na~(2+)/Ca~(2+)&gt；交换器(NCX)催化3 Na~+与1 Ca~(2+)的跨膜电生交换。我们先前已经报道，Na~+/Ca~(2+)和Gt；交换活性在CSF-1诱导破骨细胞分化中起重要作用。在本研究中，我们寻求机械应力的作用，以证实NCX1在成骨细胞骨形成和破骨细胞性骨吸收形成中的作用。机械应力诱导成骨细胞NCX1mRNA和F-肌动蛋白聚合。5~8周龄雄性ddy小鼠的胫骨和股骨取骨髓细胞，应用SA通道阻断剂Gadlinium ion抑制牵张激活的细胞反应。我们用Sephadex G-10柱(G10)去除巨噬细胞和间质细胞。未贴壁的G10传代细胞在含有终浓度为15%FBS、100 ng/mlRANKL、20 ng/mlCSF-1的α-MEM中培养。KB-R7943对破骨细胞分化和骨吸收有抑制作用，且呈剂量依赖关系。此外，NCX1反义寡核苷酸(ODN)减少了酒石酸抗性酸性磷酸酶阳性多核细胞的数量，并在蛋白水平上减少了NCX1的数量。8周龄雌性ddy小鼠接受假手术或卵巢摘除(OVX)，术后饲喂实验室饲料4周。每隔一天皮下注射KB-R7943或二甲基亚砜337.5 mg/kg，连续4周。KB-R7943治疗4周后，去卵巢组大鼠股骨骨密度明显低于假手术组。而KB-R7943治疗组大鼠骨密度与假手术组相近。这些结果表明，KB-R7943在体内也能阻止骨吸收。结论：NCX1的激活对破骨细胞的分化和激活是必不可少的。

Na^+/Ca^<2+> exchanger (NCX) catalyzes the electrogenic exchange of 3 Na^+ for 1 Ca2^+ across the plasma membrane in many mammalian cells. We have previously reported that the Na^+/Ca^<2+> exchange activity had an important role for CSF-1-induced osteoclast differentiation. In the present study, we sought the effect of mechanical stress to confirm the role of NCX1, in osteoblastic bone formation and osteoclastic bone resorption formation.Osteoblast were obtained from calvalia of new bone rat. Mechanical stress induced NCX1 mRNA and F-actin polyme ralization in osteoblast. The application of Gadlinium ion, a blocker of SAchannel, inhibited stretch-activated cell responces.Bone marrow cells were obtained from tibiae and femuor of 5- to 8-week-old male ddy mice. We used Sephadex G-10 column (G10) in order to remove macrophages and stroma cells. Non adherent G10-passed cells were cultured in a-MEM containing the final concentration of 15 % FBS, 100 ng/ml RANKL, 20 ng/ml CSF-1. KB-R7943 inhibited the osteoclast differentiation and bone resorption in vitro dose-dependently. In addition, NCX1 antisense oligo deoxy nucleotide (ODN) reduced the number of tartrate-resistant acid phosphatase-positive multinucleated cells and decreased the amount of NCX1 in protein level. Eight week-old female ddY mice received sham-operation or ovar iectomy (OVX) and were fed a laboratory chow for 4 weeks after the operation. Animals were given subcutaneously 337.5 mg/kg KB-R 7943 or DMSO every other day for 4 weeks. After 4 weeks treatment with KB-R7943, the BMD in the femur of OVX group was significantly decreased compared with sham group. However the BMD in KB-R7943 treated OVX group was quantitatively similar to the sham group. These results suggest that bone resorption was also blocked by treatment with KB-R7943 in vivo.In conclusion, the activation of NCX1 is essential for osteoclast differentiation and activation.