Resorptive state and cell size influence intracellular pH regulation in rabbit osteoclasts cultured on collagen-hydroxyapatite films

Abstract

Diseases exhibiting excessive bone loss are often characterized by an increase in the size and number of osteoclasts in affected areas, suggesting that osteoclast size is associated with increased resorptive activity or efficiency. Because osteoclastic bone resorption depends on proton extrusion via a bafilomycin A 1-sensitive vacuolar type H + ATPase (V-ATPase), we investigated the relationship between osteoclast size and state of activity on the one hand, and proton-extruding mechanisms (bafilomycin A 1-sensitive V-ATPase and amiloride-sensitive Na +/H + exchange) on the other. In determining resorptive activities of individual osteoclasts, osteoclast-containing cell suspensions obtained from newborn rabbit long bones were cultured on apatite-collagen complex (ACC)-coated coverslips. Large osteoclasts resorbed 2.5 times more per cell than small osteoclasts, but the amount resorbed per nucleus was the same for the two categories. However, a much larger percentage of large osteoclasts was resorbing compared with small osteoclasts. To study pH regulatory mechanisms in individual large and small osteoclasts, the cells were loaded with the pH-sensitive indicator BCECF and analyzed by single-cell fluorescence. Small and large resorbing osteoclasts had significantly higher basal pH i than their nonresorbing counterparts. Also, small nonresorbing osteoclasts were insensitive to bafilomycin A 1 addition or Na + removal from the medium, large nonresorbing osteoclasts responded slightly, and all resorbing osteoclasts (small and large) responded strongly. Differences were also seen in the recovery from an acid load: both small and large nonresorbing osteoclasts were more sensitive to amiloride inhibition, while large resorbing cells were more sensitive to bafilomycin A 1 inhibition. Small resorbing cells were inhibited equally by bafilomycin A 1 and amiloride. These results clearly show that a greater proportion of large osteoclasts are active in resorption and that pH i regulation is associated with enhanced proton pump activity in actively resorbing osteoclasts. Thus, large and small osteoclasts differ in the proportion of cells that are resorbing, while pH regulatory mechanisms differ mainly between resorbing and nonresorbing cells.