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Fig. 1. Osteoclast motility is accelerated by cGMP. (A) Motility in human osteoclasts with cGMP activation. This is shown as superimposed frames at 30 minute intervals of osteoclast preparations on glass with the blocking cGMP analog Rp-cGMPS (50 µM, top) or with the cGMP agonist 8-pCPT-cGMP (100 µM, bottom). Two phase images were subtracted with the first image deleting the red channel and the second deleting the green channel, so that net motion can be seen as red and green double images (yellow where moving cells overlap). There was only minor movement when cGMP was blocked, mainly redistribution of nuclei and vesicles within the cells but also some slight variation of membrane spreading. The cGMP agonist in contrast causes net cell motion. The fields shown are 225 by 290 µm. The NO donor sodium nitroprusside (100 µM) produced motility at essentially the same rate, with a rapid time course. For phase-contrast microcinematography of these fields showing the comparison with the NO donor sodium nitroprusside, see supplementary material. (B) High-resolution measurements of motility in rat osteoclasts. This was measured using the attached area, summing new attachment and retracted area as a function of time as described (Zaidi et al., 1992) over 1 minute frames for 10 minutes before (left bar, mean±s.d. of eight frames) and after (right bar, mean±s.d. of 20 frames) addition of 250 µM 8-Br-cGMP. The difference in the rate of motility was statistically significant (P<0.01) even over this short time frame. Cellular motility by geometric center of attachment showed that motion was accelerated by cGMP activation (shown in supplementary material).
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