title: Mechanisms of proton transport in isolated rat osteoclasts attached to bone.
abstract: Osteoclasts resorb bone by transporting protons (H+) into the space between the cell and the bone. To investigate the roles of a sodium-hydrogen exchanger (NHE) and a vacuolar type H+-ATPase (V-ATPase) in H+ extrusion of osteoclasts attached to bone, changes in intracellular pH (pHi) were monitored in osteoclasts isolated on glass coverslips and on bone slices using a fluorescent pHi indicator. Acid-loaded osteoclasts on glass coverslips recovered their pHi in the presence of Na+. The pHi recovery was inhibited by 5-(N, N-hexamethylene) amiloride (HMA), an inhibitor of NHE, or by removal of Na+. 7-chloro-4-nitrobenz-2-oxa-1, 3-diazol (NBD-CI), a blocker of V-ATPase, did not block the H+ transport in osteoclasts on glass coverslips. Acid-loaded osteoclasts on bone slices recovered their pHi in the absence of Na+, and this recovery was blocked by NBD-Cl. In addition, neither HMA nor NBD-Cl inhibited the pH, recovery in the presence of Na+. These results indicated that osteoclasts attached to bone extrude H+ by both V-ATPase and NHE and that osteoclasts attached to glass mainly extrude H+ by NHE.
authors.affiliation: Department of Hard Tissue Engineering, Graduate School, Tokyo Medical and Dental University, Japan. shiba.hpha@tmd.ac.jp
authors.forename: T
authors.initials: T
authors.lastname: Shibata
authors.forename: H
authors.initials: H
authors.lastname: Amano
authors.forename: S
authors.initials: S
authors.lastname: Yamada
authors.forename: K
authors.initials: K
authors.lastname: Ohya
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology; D000584:Amiloride / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001842:Bone and Bones / Q000378:metabolism*; D002448:Cell Adhesion; D018929:Cell Culture Techniques; D003470:Culture Media; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D005898:Glass; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport; D010010:Osteoclasts / Q000378:metabolism*; D011510:Proteolipids / Q000378:metabolism; D011522:Protons; D051381:Rats; D017208:Rats, Wistar; D012964:Sodium / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D003470:Culture Media; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D011510:Proteolipids; D011522:Protons; D017923:Sodium-Hydrogen Exchangers; C057360:5-(N,N-hexamethylene)amiloride; D000584:Amiloride; D012964:Sodium; D025262:Vacuolar Proton-Translocating ATPases; D009327:4-Chloro-7-nitrobenzofurazan