DEVELOPMENT OF DRUG WHICH REGULATES BONE RESORPTION ACTIVITY IN OSTEOCLASTS VIA CYTOSKELTON AND SIGNAL TRANSDUCTION SYSTEM

本研究旨在探讨质子（H+）在活性破骨细胞中的转运机制。我们用荧光pH指示剂测量了玻璃罩或骨片上分离的破骨细胞的细胞内pH （pHi）变化。酸负荷使破骨细胞产生H^+，细胞pHi迅速下降到静息水平以下。降低的pHi在无Na^+培养基中没有恢复，但在富Na^+培养基中恢复。在含有amiloride（一种有效的钠质子交换剂（NHE）抑制剂）的富Na+介质中，未观察到pHi的恢复。结果表明，玻璃上的破骨细胞具有通过NHE转运的H^+系统。相比之下，骨上的酸负载破骨细胞在添加阿米洛林和不添加Na+的情况下表现出pHi的恢复，这表明活性破骨细胞有除NHE以外的H+转运系统。从这些结果来看，与玻璃盖上的破骨细胞相比，破骨细胞至少有两种不同的H+转运机制，这表明破骨细胞附着在骨上激活了细胞内信号转导系统。另一方面，我们评估了双膦酸盐对低钙饮食大鼠骨细胞的影响。股骨和胫骨骨形态学分析显示，双膦酸盐抑制破骨细胞数量、破骨细胞核数量和成骨细胞数量的增加。双膦酸盐还能抑制矿化结节面积和抗酒石酸酸性磷酸酶阳性多核细胞数的增加。这些结果表明，双膦酸盐抑制低钙饮食激活的骨代谢的转换。

The aim of this study was to examine mechanisms of proton (H+ ) transport in active osteoclasts. We measured intracellular pH (pHi) changes in osteoclasts isolated on glasscoverslips or bone slices with a fluorescent pH indicator. H^+ was produced in osteoclasts by acid loading, and the pHi of the cells rapidly fell below the resting level. The decreased pHi did not recover in Na^+ free medium, but recovered in Na^+ rich medium. The pHi recovery was not observed on superfusion with Na+ rich medium containing amiloride, a potent sodium proton exchanger (NHE) inhibitor. The results reveal that osteoclasts on glass had H^+ transport system via NHE.In contrast, acid-loaded osteoclasts on bone showed their pHi recovery with amiloride and without Na+, presenting that active osteoclasts have H+ transport system other than NHE.From these results, osteoclasts have at least two different mechanisms of H+ transport compared to osteoclasts on glasscoverslips, suggesting that attachment to bone by osteoclasts activate intracellular signal transduction system.On the other hand, we evaluated the effects of bisphpsphonates on bone cells in rats fed with low calcium diet. Bone morphometrical analysis in femur and tibiae revealed that bisphpsphonates inhibited the increase of osteoclasts number, osteoclastic nucleus number, and osteoblasts number. Bisphpsphonates also inhibited the increment of mineralized nodule area and tartrate-resistant acid phosphatase positive-multi nuclear cells number. These results suggest that bisphpsphonates inhibit turnover in bone metabolism activated by low calcium diet.