Scanning Electrochemical Microscopy at the Surface of Bone-Resorbing Osteoclasts: Evidence for Steady-State Disposal and Intracellular Functional Compartmentalization of Calcium

Osteoclast resorptive activity occurs despite the presence of extremely high levels of ionized calcium ([Ca2+]) within the osteoclast hemivacuole, which is generated as a by-product of its resorptive activity. Previous in vitro observations have shown that increases in extracellular [Ca2+] ([Ca2+]e) in the surrounding medium can inhibit the osteoclast resorptive activity. Therefore, it has been suggested that the osteoclast acts as a "sensor" for [Ca2+]e, and that high [Ca2+]e leads to an increase in intracellular [Ca2+] ([Ca2+]i), thereby inhibiting osteoclasts in a negative feedback manner. In this report we have carried out an experimental and theoretical analysis of calcium disposal during osteoclast activity to evaluate how in vitro models relate to in vivo osteoclast activity, where it is possible that high [Ca2+]e may be present in the hemivacuole but not over the nonresorbing surface of the cell. Scanning electrochemical microscopy (SECM) studies of [Ca2+] and superoxide anion (O2.-) generation by bone-resorbing osteoclasts on the surface of a bovine cortical bone slice were compared with microspectofluorometric measurements of the levels of [Ca2+]i in single osteoclasts and the effect of [Ca2+]i on various aspects of osteoclast function. The generation of O2.- by the osteoclasts has been shown to be positively correlated with osteoclast resorptive function and can therefore serve as an index of acute changes in osteoclast activity. The SECM of bone-resorbing osteoclasts at the surface of a bone slice revealed a continuous steady-state release of Ca2+. Even after prolonged incubation lasting 3 h the near-surface [Ca2+]e in the solution above the cell remained <2 mM. The SECM real-time measurement data were consistent with the osteoclast acting as a conduit for continuous Ca2+ disposal from the osteoclast-bone interface. We conclude that the osteoclast distinguishes [Ca2+]e in the hemivacuole and in the extracellular fluid above the cell which we denote [Ca2+]e. We found that an increase in [Ca2+]i may be associated with activation; inhibition; or be without effect on O2.- generation, bone-matrix, or bone resorption. Similarly, osteoclast adhesion and bone-resorbing activity was affected by [Ca2+]e' but showed no correlation with [Ca2+]i. The data suggest the existence of functional compartmentalization of [Ca2+]i within the osteoclast, where elevated calcium may have an inhibitory, excitatory, or no effect on the overall osteoclast activity while exerting a selective effect on different functional modalities. These observations lead to the conclusion that far from being inhibited by Ca2+ generated, the osteoclast by virtue of the observed functional compartmentalization is highly adapted at carrying out its activity even when the level of [Ca2+] in resorptive lacunae is elevated.