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

本研究的目的是研究活性破骨细胞中质子(H+)转运的机制。我们用荧光pH指示器测量玻璃玻片或骨片上分离的破骨细胞的细胞内pH(Phi)变化。破骨细胞通过酸负荷产生H~+，细胞的pH值迅速降至静息水平以下。降低的pH值在无Na+时不能恢复，但在富Na+时能恢复。用含有有效的钠质子交换(NHE)抑制剂阿米洛利的富Na+介质灌流时，未观察到phi的恢复。结果表明，破骨细胞通过NHE转运H~+，而载酸破骨细胞在阿米洛利和不加Na+的情况下表现出其PHI的恢复，说明活性破骨细胞有非NHE的H~+转运系统。这些结果表明，与玻片上的破骨细胞相比，破骨细胞至少有两种不同的H~+转运机制，提示破骨细胞与骨的附着激活了细胞内信号转导系统。另一方面，我们评价了双磷酸酯对低钙饮食大鼠骨细胞的影响。股骨和胫骨的骨形态计量学分析显示，双磷酸盐可抑制破骨细胞数量、破骨细胞核数量和成骨细胞数量的增加。双磷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，双磷酸盐抑制了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.