FIGURE 4.

Effects of soluble OPN and P2X7 agonist BzATP on [Ca²⁺]_i in osteoclasts. Rat osteoclasts were plated on glass-bottomed culture dishes coated with bovine albumin. Cells were loaded with fura-2 and imaged using alternating excitation wavelengths of 345/380 nm. Test substances were applied by pressure ejection from a micropipette, at the times indicated by the vertical bars above the traces. A, trace of the fluorescence ratio at 345/380 nm for a single osteoclast stimulated repeatedly with soluble OPN (20 μg/ml in the micropipette). Changes in [Ca²⁺]_i were not observed. Trace is representative of 6 of 6 osteoclasts tested in 3 independent experiments. B, i, representative trace of the fluorescence ratio at 345/380 nm for a single osteoclast stimulated repeatedly with BzATP (100 μm in the micropipette). BzATP induced robust transient elevations in [Ca²⁺]_i. Ratio images of these global [Ca²⁺]_i elevations are available as supplemental Video S3. ii, fluorescence intensities at 345 and 380 nm for the cell illustrated in i are plotted as a function of time. Increases in the signal at 345 nm coincided with decreases in intensity at 380 nm, consistent with authentic changes in [Ca²⁺]_i. Data are representative of responses from 6 of 6 osteoclasts tested in 3 independent experiments.