Differences in regulation of pHi in large (≥10 nuclei) and small (≤5 nuclei) osteoclasts

Osteoclasts are multinucleated cells that resorb bone by extrusion of protons and proteolytic enzymes. They display marked heterogeneity in cell size, shape, and resorptive activity. Because high resorptive activity in vivo is associated with an increase in the average size of osteoclasts in areas of greater resorption and because of the importance of proton extrusion in resorption, we investigated whether the activity of the bafilomycin A(1)-sensitive vacuolar-type H+-ATPase (V-ATPase) and amiloride-sensitive Na+/H+ exchanger differed between large and small osteoclasts. Osteoclasts were obtained from newborn rabbit bones, cultured on glass coverslips, and loaded with the pH-sensitive indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Intracellular pH (pH(i)) was recorded in single osteoclasts by monitoring fluorescence. Large (greater than or equal to 10 nuclei) and small (less than or equal to 5 nuclei) osteoclasts differed in that large osteoclasts had a higher basal pH(i), their pH(i) was decreased by bafilomycin A(1) addition or removal of extracellular Na+, and the realkalinization upon readdition of Na+ was bafilomycin A(1) sensitive. After acid loading, a subpopulation of large osteoclasts (40%) recovered by V-ATPase activity alone, whereas all small osteoclasts recovered by Na+/H+ exchanger activity. Interestingly, in 60% of the large osteoclasts, pH(i) recovery was mediated by both the Na+/H+ exchanger and V-ATPase activity. Our results show a striking difference between pH(i) regulatory mechanisms of large and small osteoclasts that we hypothesize may be associated with differences in the potential resorptive activity of these cells.