CAMP-dependent inhibition is dominant in regulating superoxide production in the bone-resorbing osteoclasts

Calciotropic hormones such as parathyroid hormone (PTH) and calcitonin have been shown to have stimulatory and inhibitory effects respectively on superoxide anion (O-2(-)) generation by osteoclasts, but the exact intracellular signalling mediating these pathways has not been investigated. In order to elucidate the intracellular pathways controlling O-2(-) generation, we have carried out a systematic study of the effect of different agents on O-2(-) production in osteoclasts cultured on bovine cortical bone. Dibutyryl cAMP and cholera toxin, while having no effect on the basal level of O-2(-) production in bone-resorbing osteoclasts, were, however, found to completely block the stimulation of free radical production by PTH, pertussis toxin and ionomycin. The stimulation of O-2(-) production was found to be independent of protein kinase C-dependent pathways since the presence of bisindolyl-maleimide (GF109203X) (1 mu M) did not block stimulation by PTH and pertussis toxin. Interestingly, while exposure to bisindolylmaleimide at this concentration did not have any effect on the basal level of O-2(-) production, exposure to a higher concentration (10 mu M), which is known to inhibit both protein kinase C and A, produced significant stimulation. These in vitro findings suggest that in the bone-resorbing cells, cAMP-dependent protein kinases prevent further stimulation of NADPH oxidase by agents such as PTH and pertussis toxin. The increase in cAMP has also been recently demonstrated to be associated with down-regulation of the oxidative burst in adherent neutrophils; and the findings reported here suggest a similar role for cAMP in O-2(-) generation in osteoclasts cultured on bone.