Hashizume Y, Araki S, Sawada K, Yamada K, Katayama K
Department of Biochemistry, Eisai Tsukuba Research Laboratories, Ibaraki, Japan.
Exp Cell Res. 1995 Jun;218(2):452-9. doi: 10.1006/excr.1995.1178.
Since acid secretion of polarized osteoclasts on the bone surface has an important role in bone resorption, we examined the acid-productive activities of osteoclasts by measuring the pH of acidic organelles such as endosomes, lysosomes, and vacuoles in cultured rabbit osteoclasts with FITC-dextran as a fluorescent pH probe. The average pH value of acidic organelles of osteoclasts cultured on plain glass coverslips was 5.3 +/- 0.2. The pH of the acidic organelles correlated well with the size and amount of the vacuoles in the cells. Using this FITC-dextran method, we also examined the effects of adhesive substrates, such as type I collagen, vitronectin, and dentine, on the acid-productive activities of osteoclasts and found that the pH value of acidic organelles of osteoclasts cultured on dentine slices was significantly lower than that of osteoclasts cultured on plain glass coverslips. Likewise, in the case of the osteoclasts cultured on type I collagen- or vitronectin-coated glass coverslips, the pH values of acidic organelles were slightly lower and the proportion of osteoclasts having large vacuoles was increased compared with the cells cultured on the plain glass coverslips. These results indicate that osteoclasts containing large vacuoles have high acid-productive activities, and adhesive substrates such as type I collagen and vitronectin influence the formation of large vacuoles in cultured osteoclasts.
由于骨表面极化破骨细胞的酸分泌在骨吸收中起重要作用，我们通过用异硫氰酸荧光素葡聚糖作为荧光pH探针测量培养的兔破骨细胞中内体、溶酶体和液泡等酸性细胞器的pH值，来检测破骨细胞的产酸活性。在普通玻璃盖玻片上培养的破骨细胞酸性细胞器的平均pH值为5.3±0.2。酸性细胞器的pH值与细胞中液泡的大小和数量密切相关。使用这种异硫氰酸荧光素葡聚糖方法，我们还检测了I型胶原、玻连蛋白和牙本质等黏附底物对破骨细胞产酸活性的影响，发现牙本质切片上培养的破骨细胞酸性细胞器的pH值显著低于普通玻璃盖玻片上培养的破骨细胞。同样，在I型胶原或玻连蛋白包被的玻璃盖玻片上培养的破骨细胞中，与普通玻璃盖玻片上培养的细胞相比，酸性细胞器的pH值略低，且有大液泡的破骨细胞比例增加。这些结果表明，含有大液泡的破骨细胞具有高产酸活性，I型胶原和玻连蛋白等黏附底物会影响培养的破骨细胞中大液泡的形成。
