Visualization of acidic pH compartments on cultured osteoclasts

酸性检测探针如吖啶橙子或中性红的使用已被广泛使用，但这些仅限于活细胞。相反，安德森等人介绍的DAMP（3-（2，4-二硝基苯胺基）3 ′氨基-N-甲基二丙胺），（1984）在细胞器的酸性隔室中积累，并在化学固定后保留。DAMP含有二硝基酚基团（DNA）作为半抗原的作用，使我们能够在显微镜下用免疫细胞化学方法与抗DNP进行检测。利用DAMP的这些优点，我们尝试对体外培养的破骨细胞酸性区室的胞内和胞外区进行研究。DAMP被可视化为正常的荧光和激光扫描共聚焦显微镜与使用异硫氰酸荧光素（FITC）共轭Ig-G。不仅在高尔基复合体，和tubulovesicular结构中的破骨细胞，但也被发现的吸收骨lanuna免疫荧光，表明这些地区似乎是酸性车厢。这种细胞外酸性区室可能是造成皱褶边界和骨吸收陷窝的原因。免疫荧光定位于破骨细胞的沿着腹侧表面，而不是在骨片上培养的基底外侧表面。在玻璃板上培养的破骨细胞中，未检测到细胞外荧光。用质子ATP酶抑制剂1 mM巴非罗霉素或碳酸酐酶抑制剂1 mM乙酰唑胺处理后，破骨细胞的免疫荧光强度明显减弱。这些结果表明质子泵ATP酶和碳酸酐酶在骨细胞酸化中有密切的关系。

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.