ROLE OF NA-H EXCHANGERS AND VACUOLAR H+ PUMPS IN INTRACELLULAR PH REGULATION IN NEONATAL RAT OSTEOCLASTS

Osteoclasts resorb bone by pumping of H+ into a compartment between the cell and the bone surface. Intracellular pH (pH(i)) 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 pH(i) averaged 7.31 and was uniform throughout individual cells. Intrinsic buffering power (beta(I)) decreased curvilinearly from similar to 25 mM at pH(i) = 6.4 to similar to 6.0 mM at pH(i) = 7.4. In all polygonally shaped osteoclasts, and similar to 60% of round osteoclasts (similar to 20% of total), pH(i) recovery from acid loads was mediated exclusively by Na-H exchange. In these pattern-1 cells, pH(i) recovery was 95% complete within 200 s, and was blocked by removing Na+, or by applying 1 mM amiloride, 50 mu M ethylisopropylamiloride (EIPA), or 50 mu M hexamethyleneamiloride (HMA). The apparent K-1/2 for HMA ([Na+](o) = 150 mM) was 49 nM, and the apparent K-1/2 for Na+ was 45 mM. Na-H exchange, corrected for amiloride-insensitive fluxes, was half maximal at pH(i) 6.73, with an apparent Hill coefficient for intracellular H+ of 2.9. Maximal Na-H exchange averaged 741 mu M/s. In the remaining similar to 40% of round osteoclasts (pattern-2 cells), pH(i) recovery from acid loads was brisk even in the absence of Na+ or presence of amiloride. This Na+-independent pH(i) 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 pH(i) 6.8 to 11 mu M/s at pH(i) 7.45. In similar to 45% of pattern-2 cells, Na+ readdition elicited a further pH(i) 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.