Extracellular Phosphates Enhance Activities of Voltage-gated Proton Channels and Production of Reactive Oxygen Species in Murine Osteoclast-like Cells

Osteoclasts are highly differentiated bone-resorbing cells and play a significant role in bone remodelling. In the resorption pit, inorganic phosphate (Pi) concentrations increase because of degradation of hydroxyapatite. We studied effects of extracellular Pi on voltage-gated H channels in osteoclast-like cells derived from a macrophage cell line (RAW264). Extracellular Pi (1.25-20 mM) increased the H channel currents dose dependently and reversibly. The Pi-induced increases were attenuated by removal of extracellular Na and by phosphonoformic acid, a blocker of Na-dependent Pi transporters. Pi increased the maximal conductance, decreased activation time constant, increased deactivation time constant, and shifted the conductance-voltage relationship to more negative voltages. The most marked change was enhanced gating which was mainly caused by elevation of intracellular Pi levels. The Pi-induced enhanced gating was partially inhibited by protein kinase C (PKC) inhibitors, GF109203X and staurosporine, indicating that PKC-mediated phosphorylation was involved in part. The increase in the maximal conductance was mainly due to accompanying decrease in intracellular pH. These effects of Pi were not affected by intracellular Mg, bafilomycin A (V-ATPase inhibitor) and removal of intracellular ATP. Extracellular Pi also upregulated reactive oxygen species (ROS). Diphenyleneiodonium chloride, an inhibitor of NADPH oxidases, decreased ROS production and partially attenuated the enhanced gating. In the cells during later passages where osteoclastogenesis declined, H channel activities and ROS production were both modest. These results suggest that, in osteoclasts, ambient Pi is a common enhancer for H channels and ROS production and that potentiation of H channels may help ROS production.