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

在许多哺乳动物细胞中，Na^+/Ca^2+交换器（NCX）催化3个Na^+通过质膜交换1个Ca ^2+。我们以前曾报道过Na^+/Ca^<2+>交换活性在CSF-1诱导的破骨细胞分化中具有重要作用。本研究从新生骨大鼠的胼胝体中分离成骨细胞，观察机械应力对成骨细胞成骨和成骨细胞骨吸收形成的影响，以证实NCX 1在成骨细胞骨形成和骨吸收形成中的作用。机械应力诱导成骨细胞NCX 1 mRNA和F-actin的多聚化。从5- 8周龄雄性ddy小鼠的胫骨和股骨中获得骨髓细胞，应用SA通道阻断剂钆离子抑制牵张激活的细胞反应。我们使用Sephadex G-10柱（G10）以去除巨噬细胞和基质细胞。在含有终浓度15% FBS、100 ng/ml RANKL、20 ng/ml CSF-1的a-MEM中培养非贴壁G10传代细胞。KB-R7943在体外呈剂量依赖性抑制破骨细胞分化和骨吸收。此外，NCX 1反义寡核苷酸（ODN）减少了抗酒石酸酸性磷酸酶阳性多核细胞的数量，并在蛋白水平上减少了NCX 1的含量。8周龄雌性ddY小鼠接受假手术或卵巢切除术（OVX），并在手术后喂养实验室食物4周。每隔一天皮下给予动物337.5 mg/kg KB-R 7943或DMSO，持续4周。KB-R7943治疗4周后，OVX组股骨BMD较Sham组显著降低。然而，KB-R7943处理的OVX组的BMD在数量上与假手术组相似。这些结果表明，骨吸收也被KB-R7943在体内处理的阻断。总之，NCX 1的激活是必要的破骨细胞的分化和激活。

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.