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Visualization of acidic pH compartments on cultured osteoclasts

Research Project

Project/Area Number 07671996
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Morphological basic dentistry
Research InstitutionASAHI UNIVERSITY

Principal Investigator

AKISAKA Toshitaka  Asahi Univ.Sch.Dent., Professor, 歯学部, 教授 (70116523)

Co-Investigator(Kenkyū-buntansha) YOSHIDA Hisaho  Asahi Univ.Sch.Dent., Assistant Prof., 歯学部, 助手 (80102119)
Project Period (FY) 1995 – 1996
Project Status Completed (Fiscal Year 1996)
Budget Amount *help
¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1996: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1995: ¥1,400,000 (Direct Cost: ¥1,400,000)
Keywordscultured 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
Research Abstract

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.

Report

(3 results)
  • 1996 Annual Research Report   Final Research Report Summary
  • 1995 Annual Research Report

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Published: 1995-04-01   Modified: 2016-04-21  

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