Gupta A, Miyauchi A, Fujimori A, Hruska K A
Renal Division, Jewish Hospital of St. Louis, Washington University School of Medicine, Missouri, USA.
Kidney Int. 1996 Apr;49(4):968-74. doi: 10.1038/ki.1996.137.
Osteoclasts are polarized cells involved in bone resorption. They are exposed to high ambient concentrations of inorganic phosphate (Pi) during the active process of bone resorption. We hypothesize that osteoclasts may possess specific Pi-transport system(s) for transcellular movement of Pi released from bone into the resorption cavity. We have previously reported the existence of a Na-dependent Pi cotransporter in the avian osteoclast, which provides a model culture system for the fully differentiated phenotype capable of bone resorption. In whole cell Pi-uptake studies, the rate of Pi transport was sensitive to both ouabain and 2,4-DNP, an inhibitor of aerobic ATP production. When these osteoclasts were exposed to bone particles, there was an immediate stimulation of Pi transport, independent of de novo protein synthesis. The stimulatory effect of bone particles was inhibited by peptides with the Arg-Gly-Asp-Ser (RGDS) motif, an effect which implicates integrins and cell-matrix interaction in the regulation of Pi transport. We performed Western blots on both whole cell lysates and membrane fractions using a polyclonal antibody to the N-terminal of NaPi-2 (the rat variant) and found a single approximately 100 kDa protein; the non-immune serum was used as control. Immunofluorescence studies using the same N-terminal antibody to NaPi-2 detected the protein in discrete vesicles. There was an induction of the protein in membrane fractions isolated from osteoclasts cultured in the presence of bone particles. Our preliminary studies indicate that a Na-Pi cotransporter may exist in the avian osteoclast, immunologically related to the NaPi-2 family, and which may be regulated through integrin-mediated pathways in the presence of bone. We also hypothesize that there may be a redistribution of vesicular pools containing the Na-Pi cotransporter toward discrete plasma membrane sites on the polarized osteoclast for transcellular movement of Pi during active bone resorption.
破骨细胞是参与骨吸收的极化细胞。在骨吸收的活跃过程中，它们暴露于高环境浓度的无机磷酸盐（Pi）中。我们假设破骨细胞可能拥有特定的Pi转运系统，用于将从骨中释放的Pi跨细胞转运到吸收腔中。我们之前报道过在禽类破骨细胞中存在一种Na依赖性Pi共转运体，它为能够进行骨吸收的完全分化表型提供了一个模型培养系统。在全细胞Pi摄取研究中，Pi转运速率对哇巴因和2,4-二硝基苯酚（一种有氧ATP产生的抑制剂）均敏感。当这些破骨细胞暴露于骨颗粒时，Pi转运立即受到刺激，且与从头蛋白质合成无关。骨颗粒的刺激作用被具有精氨酸-甘氨酸-天冬氨酸-丝氨酸（RGDS）基序的肽所抑制，这一效应表明整合素和细胞-基质相互作用参与了Pi转运的调节。我们使用针对NaPi-2（大鼠变体）N端的多克隆抗体对全细胞裂解物和膜组分进行了蛋白质免疫印迹分析，发现了一条约100 kDa的单一蛋白条带；使用非免疫血清作为对照。使用相同的针对NaPi-2 N端抗体的免疫荧光研究在离散的囊泡中检测到了该蛋白。在存在骨颗粒的情况下培养的破骨细胞分离出的膜组分中，该蛋白有所诱导。我们的初步研究表明，禽类破骨细胞中可能存在一种Na-Pi共转运体，在免疫上与NaPi-2家族相关，并且在有骨存在的情况下可能通过整合素介导的途径进行调节。我们还假设，在活跃的骨吸收过程中，含有Na-Pi共转运体的囊泡池可能会重新分布到极化破骨细胞上离散的质膜位点，以实现Pi的跨细胞转运。
