Immunohistochemical Localization of Vacuolar H⁺-ATPase in Osteoclasts of Rat Tibiae

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The localization of vacuolar H⁺-ATPase (V-ATPase) in osteoclasts of rat tibiae was examined immunohistochemically using antibodies to 57kD and 72kD subunits of V-ATPase, with confocal laser scanning microscopy and transmission electron microscopy. The two antibodies showed similar immunoreactivities in osteoclasts. We distinguished three patterns of immunoreactivity in osteoclasts: The first pattern was that immunoreactivity was diffusely detected in cytoplasm of osteoclasts; The second one was that osteoclasts showed intense immunoreactivity in their regions contacting with bone surface; and the last was that little immunoreactivity was seen in osteoclasts. Post-embedding methods with protein A-gold complex revealed that the subunits of V-ATPase were localized not only on the membranes of the ruffled borders of osteoclasts, but also around the Golgi apparatus and accumulated tubular lysosomes.
These findings suggest that: 1) immunoreactive patterns for V-ATPase in osteoclasts reflect the polarity and activity of osteoclasts; and 2) V-ATPase on ruffled borders may be transported through tubular lysosomes.