Visualization of acidic pH compartments on cultured osteoclasts

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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.