Visualization of acidic pH compartments on cultured osteoclasts

培养破骨细胞酸性 pH 区室的可视化

• 批准号: 07671996
• 负责人: AKISAKA Toshitaka
• 金额: $ 1.6万
• 依托单位: ASAHI UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07671996/
• 关键词: cultured osteoclast; acidic compartment; DAMP staining; pH probe; Laser scanning microscopy; 破骨細胞; 培養; 急速凍結; 酸性化; プロトンポンプ; DAMP; 蛍光顕微鏡

The use of acidic detecting probes such as acridine orange or neutral red have been widely used, but these are limited in living cells. In contrast DAMP (3-(2,4-dinitroanilino) 3'amino-N-methyl dipropylamine) introduced by Anderson et al., (1984) accumulated in acidic compartments of cell organelles and retained after chemical fixation. DAMP contained a dinitrophenol group (DNA) as a role of hapten enables us to detect with anti-DNP by immunocytochemical method under the microscope. Taking these advantages of DAMP,we have tried to study the intra and extracellular region of acidic compartment in cultured osteoclasts. DAMP was visualized for normal fluorescence and the laser scanning confocal microscopy with the use of fluorescen isothiocyanate (FITC) conjugate Ig-G.Not only in the Golgi complex, and tubulovesicular structures in osteoclasts but also the resorbed bone lanuna immunofluorescence were found, indicating that these regions appeared to be acidic compartments. Such extracellular acidic compartments may be responsible for the ruffled boder and bone resorptive lacuna. Immunofluorescence was localized along the ventral surface rather than the basolateral surface of the osteoclast cultured on bone slice. In osteoclasts cultured on glass palte, no extracellular fluoresence was detectable. The intensity of immunofluorescence in the osteoclasts were apparently diminished after the treatment of 1mum bafiromycin as an inhibitor of proton ATPase or 1mM acetoazolamide as an inhibitor of carbonic anhydrase. These results indicate an intimate relation betwen proton-pump ATPase and carbonic anhydrase in osteoclastic acidification.

使用酸性检测探针（例如灰烬橙色或中性红色）的使用已被广泛使用，但这些探针在活细胞中受到限制。相反，Anderson等人（1984年）引入的对比潮湿（3-（2,4-二硝基烷）3'amino-N-甲基二甲基胺），累积在细胞细胞器的酸性隔室中，并在化学固定后保留。潮湿包含二硝基苯酚基（DNA）作为触觉的作用，使我们能够通过显微镜下通过免疫细胞化学方法检测抗DNP。利用这些潮湿的优势，我们试图研究培养的破骨细胞中酸性区室的细胞内和细胞外区域。通过使用异硫氰酸荧光（FITC）共轭Ig-G.不在高尔基体复合物中，并在肾小管菌落中，在骨lanuna bone lanuna Impactormention中，可以看到潮湿的正常荧光和激光扫描共聚焦显微镜。这种细胞外酸性隔室可能是荷叶边的肋骨和骨骼吸收性腔。免疫荧光位于腹表面，而不是在骨切片上培养的破骨细胞的基底外侧表面。在玻璃帕尔特培养的破骨细胞中，未检测到细胞外荧光。在治疗1MUM Bafiromycin作为质子ATPase或1mm乙酰唑胺作为碳酸酐酶抑制剂的抑制剂后，破骨细胞中免疫荧光的强度显然会降低。这些结果表明，在整骨酸化中，质子 - 泵ATPase和碳酸酐酶之间的亲密关系。