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Fig. 4. Ca²⁺ signals during movement of cGMP- or NO-activated osteoclasts. (A) Elevated Ca²⁺ in a moving osteoclast after cGMP activation. Four false-color Ca²⁺ images at 5-minute intervals are shown. Each frame depicts an average Fluo 3 signal from a confocal image exposed for 200 mseconds, showing Ca²⁺ activity as fluorescence at 525 nm. Sixty minutes before imaging, cells were treated with 100 µM 8-pCPT-cGMP, which induces motility in a large fraction of cells. Two cells are shown. One shows minimal changes in Ca²⁺ and did not move (right cell in each frame). The other (left cell in each frame) moved during the period shown. Movement occurred with large, localized, changes in Ca²⁺ (first and second frames) and was largely complete after 20 minutes. False color scale: red 1 µM > violet 100 nM > blue; black, no signal. (B) Large Ca²⁺ transients and motility were much less common in cells when the the cGMP pathway was inhibited. The number of moving cells and a Fluo3 signal two-fold above background was determined in four experiments, at 3-minute intervals over 1 hour in each experiment. Ca²⁺ fluxes with motility were uncommon in cells treated with the cGMP antagonist Rp-cGMPS (50 µM; left bar, n=43 cells) but frequent in cells treated with the cGMP analog 8-pCPT-cGMP (100 µM; right bar, n=77 cells) 30 minutes prior to analysis. (C) Mean intracellular Ca²⁺ activity from ratio imaging of osteoclasts with Oregon-Green-BAPTA and Fura Red. The average intracellular Ca²⁺ levels in unactivated cells was 74 nM; 15-30 minutes after treatment with 100 µM SNP the average intracellular Ca²⁺ level increased to 5 µM, although some measured values were indistinguishable from the maximum G:R ratio so, probably, some cells with higher intracellular Ca²⁺ levels occur (mean ± s.e.m.; n=10).

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