Krieger N S
Department of Medicine, Box 675, University of Rochester School of Medicine, 601 Elmwood Avenue, Rochester, New York 14642, USA.
Calcif Tissue Int. 1997 May;60(5):473-8. doi: 10.1007/s002239900265.
We previously described Na+-Ca2+ exchange in osteoblastic rat osteosarcoma cells (UMR-106) and demonstrated that Na+-dependent Ca2+ transport was inhibited by 24-hour treatment of cells with parathyroid hormone (PTH), prostaglandin E2 (PGE2), or 1,25(OH)2D3. To determine whether this inhibition of Na+-Ca2+ exchange is at the level of exchanger protein synthesis we have examined exchanger protein levels using immunoblot analysis. UMR-106 cells were treated for 24 hours with or without PTH, PGE2, or 1,25(OH)2D3. Plasma membrane fractions (7500 g) were obtained and proteins were separated by SDS-PAGE, transferred to nylon membranes, and immunoblotted with a polyclonal antibody to the canine cardiac Na+-Ca2+ exchanger. In rat cardiac membranes, we detected 125 and 75 kD bands, similar to findings for the canine exchanger. In the osteoblastic UMR cell membranes, a specific band was detected at 90 kD that decreased 65% after treatment of cells with PTH. Inhibition by PTH was dose dependent, was maximal with 10(-7) M PTH, and required 16-24 hour treatment time. Similar inhibition was observed after a 24 hour treatment with 10(-6) M PGE2 or 10(-8) M 1,25(OH)2D3. These results demonstrate the presence of a specific protein in UMR cells that cross-reacts with antibody directed against the cardiac Na+-Ca2+ exchanger. Thus, the previously reported inhibition of Na+-Ca2+ exchange activity by calcemic agents in osteoblasts appears to be due to regulation of exchanger protein levels in these osteoblastic cells.
我们之前描述了大鼠成骨样骨肉瘤细胞（UMR - 106）中的钠钙交换，并证明用甲状旁腺激素（PTH）、前列腺素E2（PGE2）或1,25(OH)2D3对细胞进行24小时处理可抑制钠依赖性钙转运。为了确定这种对钠钙交换的抑制是否发生在交换蛋白合成水平，我们使用免疫印迹分析检测了交换蛋白水平。UMR - 106细胞在有或无PTH、PGE2或1,25(OH)2D3的情况下处理24小时。获得质膜组分（7500g），蛋白质通过SDS - PAGE分离，转移至尼龙膜，并用针对犬心脏钠钙交换体的多克隆抗体进行免疫印迹。在大鼠心脏膜中，我们检测到125kD和75kD条带，与犬交换体的检测结果相似。在成骨样UMR细胞膜中，在90kD处检测到一条特异性条带，在用PTH处理细胞后该条带减少了65%。PTH的抑制作用呈剂量依赖性，在10(-7)M PTH时最大，且需要16 - 24小时的处理时间。在用10(-6)M PGE2或10(-8)M 1,25(OH)2D3处理24小时后观察到类似的抑制作用。这些结果表明UMR细胞中存在一种与针对心脏钠钙交换体的抗体发生交叉反应的特异性蛋白。因此，先前报道的成骨细胞中钙调节因子对钠钙交换活性的抑制似乎是由于这些成骨细胞中交换蛋白水平的调节。
