Inoue M, Yoshida H, Akisaka T
Department of Anatomy, Asahi University School of Dentistry, Gifu, Japan.
Cell Tissue Res. 1999 Dec;298(3):527-37. doi: 10.1007/s004419900102.
Using the acidotrophic amine 3-(2,4-dinitroanillino)-3'-amino-N-methyldipropylamine (DAMP) as a marker for low pH and immunofluorescence cytochemistry, we examined acidic compartments of osteoclasts cultured on cover glasses or bone slices, where they could resorb the bone surface, forming resorptive lacunae. DAMP-positive structures were seen as vesicular and tubular forms in the cytoplasm, indicating lysosomes and endosomes. Not only the osteoclastic cytoplasm but also the extracellular area around the ruffled border and resorptive lacunae were stained with DAMP, suggesting acidic regions. Immunofluorescence was localized predominantly on the substratum side of actively resorbing osteoclasts, whereas an evenly distributed staining pattern was seen in the nonactive cell. The most intensive reaction was seen at the advancing front of resorptive lacunae within the actively resorbing osteoclasts. The distribution pattern of DAMP seemed to be correlated with the osteoclastic activity, since osteoclasts exhibit alternating resorption and migration phases during the bone-remodeling cycle. In this culture system, the resorptive lacunae were left behind after the osteoclasts had completed resorption and migrated along the bone surface. These exposed resorptive lacunae were also stained with DAMP, which were presumably kept at an acidic pH. The effect of treatment with monensin, chloroquine, ammonium chloride, or nigericin was varied in terms of the immunoreactivity for DAMP, but not complete abolition of the staining was obtained. Weak bases such as chloroquine or ammonium chloride inhibited both intra- and extracellular immunoreactivity. Immunoreactivity for the vacuolar type of proton ATPase (V-ATPase) was demonstrable in the cytoplasm of the osteoclasts but was weakened by the addition of bafilomycin. Immunofluorescence of the resorptive lacunae was still retained even after the treatment with bafilomycin and acetazolamide. Besides, both bafilomycin and acetazolamide reversibly inhibited cellular acidity as judged by DAMP immunocytochemistry, which agrees with the fact that ostoeclastic acidification results from the action of vacuolar proton-pump ATPase coupled with carbonic anhydrase.
我们使用酸营养胺3-(2,4-二硝基苯胺基)-3'-氨基-N-甲基二丙胺（DAMP）作为低pH的标志物，并采用免疫荧光细胞化学方法，研究了在盖玻片或骨切片上培养的破骨细胞的酸性区室，这些破骨细胞能够吸收骨表面，形成吸收陷窝。DAMP阳性结构在细胞质中表现为囊泡状和管状形式，表明是溶酶体和内体。不仅破骨细胞的细胞质，而且皱褶边缘和吸收陷窝周围的细胞外区域都被DAMP染色，提示存在酸性区域。免疫荧光主要定位于活跃吸收的破骨细胞的基质侧，而在非活跃细胞中则呈现均匀分布的染色模式。在活跃吸收的破骨细胞内，吸收陷窝的前沿观察到最强的反应。DAMP的分布模式似乎与破骨细胞活性相关，因为破骨细胞在骨重塑周期中表现出交替的吸收和迁移阶段。在这个培养系统中，破骨细胞完成吸收并沿骨表面迁移后，留下了吸收陷窝。这些暴露的吸收陷窝也被DAMP染色，推测其pH值保持在酸性。莫能菌素、氯喹、氯化铵或尼日利亚菌素处理对DAMP免疫反应性的影响各不相同，但并未完全消除染色。氯喹或氯化铵等弱碱抑制细胞内和细胞外的免疫反应性。在破骨细胞的细胞质中可检测到液泡型质子ATP酶（V-ATPase）的免疫反应性，但加入巴弗洛霉素后其免疫反应性减弱。即使在用巴弗洛霉素和乙酰唑胺处理后，吸收陷窝的免疫荧光仍然保留。此外，根据DAMP免疫细胞化学判断，巴弗洛霉素和乙酰唑胺均可可逆地抑制细胞酸性，这与破骨细胞酸化是由液泡质子泵ATP酶与碳酸酐酶的作用导致的事实相符。
