The effects of stimulators of intracellular cyclic AMP on rat and chick osteoclasts in vitro: validation of a simplified light microscope assay of bone resorption.

The aim of this study was to investigate whether a cyclic AMP-mediated inhibitory mechanism is present in embryonic chick osteoclasts and to extend data implicating cyclic AMP in the inhibition of neonatal rat osteoclasts. Dibutyryl cyclic AMP ((Bu)2cAMP) (5 x 10(-4) M and above) and isobutylmethylxanthine (IBMX) (10(-4) M and above) reduced the number of pits made in slices of devitalized bovine cortical bone by chick osteoclasts over 24 h. The effect of forskolin (FSK) on chick osteoclasts was biphasic, 10(-5) M producing a weak and variable reduction in pit number while 10(-6) M and 10(-7) M stimulated resorption. Doses of FSK (10(-5) M) and (Bu)2cAMP (3 x 10(-4) M), which individually produced no consistent significant effect, produced a synergistic and highly significant reduction in pit number when used in combination, implying that these agents were acting through a common mechanism, presumably cyclic AMP. Stimulatory doses of FSK were associated with increased osteoclast numbers, implicating cyclic AMP in the formation of osteoclasts. In comparative experiments using neonatal rat osteoclasts, (Bu)2cAMP (10(-4) M and above), IBMX (10(-3) M) and FSK (10(-7) M and above) all reduced the number of pits excavated. Strongly inhibitory doses of these agents caused contraction of chick osteoclasts into a hemispherical shape; contraction of rat osteoclasts into a stellate shape occurred with (Bu)2cAMP and FSK, but not with IBMX. Our results implicate cyclic AMP in the inhibition of both rat and chick osteoclasts, and show that pit counting in the light microscope is a valid method of analyzing the disaggregated osteoclast resorption assay.