Department of Cellular Physiological Chemistry, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8549, Japan,
J Bone Miner Metab. 2014 Jan;32(1):17-28. doi: 10.1007/s00774-013-0462-9. Epub 2013 Jul 16.
We established a system to separately analyze the role of protons and bicarbonate ions in vitro in which the pH of the medium was controlled by HEPES at various concentrations of sodium bicarbonate (NaHCO3) in the absence of carbon dioxide (CO2). Using this system, we demonstrated that acidosis promoted osteoclast formation independently of extracellular NaHCO3 in a short-term culture. Protons and bicarbonate ions acted on osteoclast differentiation with opposite effects, the former positively and the latter negatively. The HEPES-based system maintained pH in the absence of extracellular NaHCO3 without CO2. Therefore, we could demonstrate that osteoblast differentiation was promoted at higher pH in a long-term culture system without NaHCO3 in which ALP activity and nodule mineralization were enhanced. This finding indicates that protons negatively control osteoblast differentiation independently of extracellular bicarbonate ions. However, the difference in the concentration of NaHCO3 did not have any influence on nodule mineralization. The opposite effects of protons, the promotion of osteoclast formation and the inhibition of osteoblast differentiation, were suppressed in the presence of 5 mM N-acetyl cysteine, a reagent activating the scavenging of reactive oxygen species (ROS), implying that ROS act on both systems, the promotion of large osteoclast formation and the deterioration of osteoblast formation under acidosis.
我们建立了一个系统，在没有二氧化碳（CO2）的情况下，通过 HEPES 在不同浓度的碳酸氢钠（NaHCO3）中控制介质的 pH 值，从而分别分析质子和碳酸氢根离子在体外的作用。使用该系统，我们证明了在短期培养中，酸中毒可独立于细胞外 NaHCO3 促进破骨细胞形成。质子和碳酸氢根离子对破骨细胞分化的作用相反，前者促进，后者抑制。基于 HEPES 的系统在没有 CO2 的情况下维持 pH 值，而无需细胞外 NaHCO3。因此，我们可以证明在没有 NaHCO3 的长期培养系统中，碱性磷酸酶（ALP）活性和矿化结节增强，较高的 pH 值促进成骨细胞分化。这一发现表明，质子独立于细胞外碳酸氢根离子负调控成骨细胞分化。然而，NaHCO3 浓度的差异对矿化结节没有任何影响。在 5mM N-乙酰半胱氨酸（一种激活活性氧（ROS）清除的试剂）存在下，质子的相反作用（促进破骨细胞形成和抑制成骨细胞分化）被抑制，这意味着 ROS 作用于这两个系统，在酸中毒下促进大型破骨细胞形成和恶化成骨细胞形成。
