Different calcium sensitivity in osteoclasts on glass and on bone and maintenance of cytoskeletal structures on bone in the presence of high extracellular calcium

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Abstract
The sensitivity of rat osteoclasts to increased extracellular calcium concentrations ([Ca2+]e) was investigated by single cell measurements of free cytosolic calcium concentrations ([Ca2+]i), by changes in microfilament organization of resorbing osteoclasts, and by in vitro bone resorption assays. Osteoclasts cultured on glass and on bone showed clear differences in their responses, as in 44% and 52% of osteoclasts on glass but in only 21% and 25% of osteoclasts on bone [Ca2+]i increased when [Ca2+]e was increased from 2 mM to 6 or 10 mM via perfusion, respectively. Bone resorption was inhibited without changes in the osteoclast numbers only by 10 mM [Ca2+]e in 2 day cultures. Furthermore, there were no changes in the organization of microfilament structures in resorbing osteoclasts after increased [Ca2+]e (up to 20 mM [Ca2+]e, 30 min incubation). These results suggest that the sensitivity of osteoclasts to increased [Ca2+]e is dependent on their activation phase (resting/migrating vs. resorbing) and that resorbing osteoclasts are not sensitive to increased [Ca2+]e or that the sensing system cannot be reached in polarized resorbing osteoclasts. In contrast, increasing [Ca2+]i through the use of calcium ionophores dispersed specific microfilament structures at the sealing zone transiently in a few minutes. This shows that [Ca2+]i is used as a signaling mechanism to inactivate osteoclasts, with a similar end result on microfilament structures at the sealing zone as caused by increased concentration of cAMP and activation of protein kinase C.