title: Role of Na-H exchangers and vacuolar H+ pumps in intracellular pH regulation in neonatal rat osteoclasts.
abstract: Osteoclasts resorb bone by pumping of H+ into a compartment between the cell and the bone surface. Intracellular pH (pHi) homeostasis requires that this acid extrusion, mediated by a vacuolar-type H+ ATPase, be complemented by other acid-base transporters. We investigated acid-extrusion mechanisms of single, freshly isolated, neonatal rat osteoclasts. Cells adherent to glass coverslips were studied in the nominal absence of CO2/HCO3-, using the pH-sensitive dye BCECF and a digital imaging system. Initial pHi averaged 7.31 and was uniform throughout individual cells. Intrinsic buffering power (beta 1) decreased curvilinearly from approximately 25 mM at pHi = 6.4 to approximately 6.0 mM at pHi = 7.4. In all polygonally shaped osteoclasts, and approximately 60% of round osteoclasts (approximately 20% of total), pHi recovery from acid loads was mediated exclusively by Na-H exchange. In these pattern-1 cells, pHi recovery was 95% complete within 200 s, and was blocked by removing Na+, or by applying 1 mM amiloride, 50 microM ethylisopropylamiloride (EIPA), or 50 microM hexamethyleneamiloride (HMA). The apparent K1/2 for HMA ([Na+]o = 150 mM) was 49 nM, and the apparent K1/2 for Na+ was 45 mM. Na-H exchange, corrected for amiloride-insensitive fluxes, was half maximal at pHi 6.73, with an apparent Hill coefficient for intracellular H+ of 2.9. Maximal Na-H exchange averaged 741 microM/s. In the remaining approximately 40% of round osteoclasts (pattern-2 cells), pHi recovery from acid loads was brisk even in the absence of Na+ or presence of amiloride. This Na(+)-independent pHi recovery was blocked by 7-chloro-4-nitrobenz-2-oxa-1,3-diazol (NBD-Cl), a vacuolar-type H+ pump inhibitor. Corrected for NBD-Cl insensitive fluxes, H+ pump fluxes decreased approximately linearly from 96 at pHi 6.8 to 11 microM/s at pHi 7.45. In approximately 45% of pattern-2 cells, Na+ readdition elicited a further pHi recovery, suggesting that H+ pumps and Na-H exchangers can exist simultaneously. We conclude that, under the conditions of our study, most neonatal rat osteoclasts express Na-H exchangers that are probably of the ubiquitous basolateral subtype. Some cells express vacuolar-type H+ pumps in their plasma membrane, as do active osteoclasts in situ.
authors.affiliation: Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
authors.forename: J H
authors.initials: JH
authors.lastname: Ravesloot
authors.forename: T
authors.initials: T
authors.lastname: Eisen
authors.forename: R
authors.initials: R
authors.lastname: Baron
authors.forename: W F
authors.initials: WF
authors.lastname: Boron
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology; D000136:Acid-Base Equilibrium / Q000502:physiology; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn / Q000502:physiology; D002021:Buffers; D002462:Cell Membrane / Q000648:ultrastructure; D005452:Fluoresceins; D006863:Hydrogen-Ion Concentration; D010010:Osteoclasts / Q000502:physiology* / Q000648:ultrastructure; D017494:Proton Pumps / Q000502:physiology*; D000644:Quaternary Ammonium Compounds / Q000009:adverse effects; D051381:Rats; D017208:Rats, Wistar; D012964:Sodium / Q000172:deficiency / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000502:physiology*; D014617:Vacuoles / Q000648:ultrastructure*
chemical_list: D002021:Buffers; D005452:Fluoresceins; D017494:Proton Pumps; D000644:Quaternary Ammonium Compounds; D017923:Sodium-Hydrogen Exchangers; C057360:5-(N,N-hexamethylene)amiloride; D000584:Amiloride; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D012964:Sodium; D009327:4-Chloro-7-nitrobenzofurazan; C039614:ethylisopropylamiloride