Use of forskolin to study the relationship between cyclic AMP formation and bone resorption in vitro

Abstract

The effect of the adenylate cyclase activator forskolin on bone resorption and cyclic AMP accumulation was studied in an organ-culture system by using calvarial bones from 6-7-day-old mice. Forskolin caused a rapid and fully reversible incerase of cyclic AMP, which was maximal after 20-30 min. The phosphodiesterase inhibitor rolipram (30 .mu.mol/l), enhanced the cyclic AMP response to forskolin (50 .mu.mol/l) from a net cyclic AMP response of 1234 .+-. 154 pmol/bone to 2854 .+-. 193 pmol/bone (mean .+-. S.E.M., n = 4). The cyclic AMP level in bones treated with forskolin (30 .mu.mol/l) was significantly increased after 24 h of culture. Forskolin, at and above 0.3 .mu.mol/l, in the absence and the presence of rolipram (30 .mu.mol/l), caused a dose-dependent cyclic AMP accumulation wth an calculated EC50 (concentration producing half-maximal stimulation) value at 8.3 .mu.mol/l. In 24 h cultures forskolin inhibited spontaneous and PTH (parathyroid hormone)-stimulated 45Ca release with calculated IC50 (concentation producing half-maximal inhibiton) values at 1.6 and 0.6 .mu.mol/l respectively. Forskolin significantly inhibited the release of 3H from [3H]proline-labelled bones stimulated by PTH (10 nmol/l). The inhibitory effect by forskolin on PTH-stimulated 45Ca release was significant already after 3 h of culture. In 24 h cultures forskolin (3 .mu.mol/l) significantly inhibited 45Ca release also from bones stimulated by prostaglandin E2 (1 .mu.mol/l) and 1.alpha.-hydroxycholecalciferol (0.1 .mu.mol/l). The inhibitory effect of forskolin on spontaneous and PTH-stimulated 45Ca release was transient. A dose-dependent stimulation of basal 45Ca release was seen in 120 h cultures, at and above 3 nmol of forskolin/l, with a calculated EC50 value at 16 nmol/l. The stimulatory effect of forskolin (1 .mu.mol/l) could be inhibited by calcitonin (0.1 unit/ml), but was insensitive to indomethacin (1 .mu.mol/l). Forskolin increased the release of 3H from [3H]proline-labelled bones cultured for 120 h and decreased the amount of hydroxyproline in bones after culture. Forskolin inhibited PTH-stimulated release of Ca2+, Pi, .beta.-glucuronidase and .beta.-N-acetylglucosaminidase in 24 h cultures. In 120 h cultures forskolin stimulated the basal release of minerals and lysosomal enzymes. In osteolast-like cells, isolated by enzyme digestion from 2-3-day-old mice, forskolin caused a rapid cyclic AMP response, which was maximal at 15 min. These data show that an increase of cyclic AMP levels in bone by forskolin causes a transient inhibition of bone resorption and support the view that cyclic AMP may be a mediator of the action of calcitonin. The results indicate that the rapid stimulation by PTH on bone resorption is not mediated by cyclic AMP. Finally the data suggest that the PTH-induced increase of cyclic AMP may be involved in a delayed action of the hormone, presumably related to recruitment of new osteoclasts.