Osteoclast cytosolic calcium, regulated by voltage-gated calcium channels and extracellular calcium, controls podosome assembly and bone resorption

The mechanisms of Ca²⁺ entry and their effects on cell function were investigated in cultured chicken osteoclasts and putative osteoclasts produced by fusion of mononuclear cell precursors. Voltage-gated Ca²⁺ channels (VGCC) were detected by the effects of membrane depolarization with K⁺, BAY K 8644, and dihydropyridine antagonists. K⁺ produced dose-dependent increases of cytosolic calcium ([Ca²⁺](i)) in osteoclasts on glass coverslips. Half-maximal effects were achieved at 70 mM K⁺. The effects of K⁺ were completely inhibited by dihydropyridine derivative Ca²⁺ channel blocking agents. BAY K 8644 (5 x l0^-6 M), a VGCC agonist, stimulated Ca²⁺ entry which was inhibited by nicardipine. VGCCs were inactivated by the attachment of osteoclasts to bone, indicating a rapid phenotypic change in Ca²⁺ entry mechanisms associated with adhesion of osteoclasts to their resorption substrate. Increasing extracellular Ca²⁺ ([Ca²⁺](e)) induced Ca²⁺ release from intracellular stores and Ca²⁺ influx. The Ca²⁺ release was blocked by dantrolene (10^-5 M), and the influx by La³⁺. The effects of [Ca²⁺](e) on [Ca²⁺](i) suggest the presence of a Ca²⁺ receptor on the osteoclast cell membrane that could be coupled to mechanisms regulating cell function. Expression of the [Ca²⁺](e) effect on [Ca²⁺](i) was similar in the presence or absence of bone matrix substrate. Each of the mechanisms producing increases in [Ca²⁺](i), (membrane depolarization, BAY K 8644, and [Ca²⁺](e)) reduced expression of the osteoclast-specific adhesion structure, the podosome. The decrease in podosome expression was mirrored by a 50% decrease in bone resorptive activity. Thus, stimulated increases of osteoclast [Ca²⁺](i) lead to cytoskeletal changes affecting cell adhesion and decreasing bone resorptive activity.