The Mode of Action of Dibutyryl Adenosine 3′,5′-Monophosphate on Bone Tissue in Vitro

Fetal rat calvaria were incubated in Krebs-Ringer-bicarbonate medium, containing glucose (2 mg per ml) and bovine serum albumin (1 mg per ml). Incubation in a medium with 0.3 or 0.6 mm dibutyryl adenosine 3',5'-monophosphate (DBcAMP) for 15 min caused a 2- to 3-fold increase in the tissue concentration of adenosine 3',5'-monophosphate (3',5'-AMP). This rise could not be explained by degradation of dibutyryl adenosine 3',5'-monophosphate during the incubation period or the isolation procedure since labeled DBcAMP was not altered in the tissue or medium in incubations carried out for either 15 or 60 min. The intracellular concentration of dibutyryl adenosine 3',5'-monophosphate was 0.12 mm after 15 min of incubation and increased to about 0.26 mm at 2 hours. Dibutyryl adenosine 3',5'-monophosphate was not a substrate for bone cyclic 3',5'-nucleotide phosphodiesterase. Bone phosphodiesterase activity was progressively inhibited by increasing concentrations of dibutyryl adenosine 3',5'-monophosphate which was a more potent inhibitor than theophylline. The intracellular dibutyryl adenosine 3',5'-monophosphate concentration in the calvaria appears to be high enough to inhibit intracellular phosphodiesterase activity. When 3',5'-[3H]AMP was added to the media, the radioactivity of the calvaria increased continuously for up to 2 hours. After 15 and 60 min of incubation nearly all of the radioactivity was present in the tissue in the form of [3H]adenosine, suggesting either rapid breakdown of 3',5'-[3H]AMP entering the cell or uptake of [3H]adenosine formed extracellularly by breakdown of 3',5'-[3H]AMP. Adenosine could be detected in the media after 60 min of incubation but not after 15 min. These findings indicate that the biological effects of dibutyryl 3',5'-AMP can be accounted for by an increase in the intracellular concentration of endogenous 3',5'-AMP secondary to inhibition of intracellular phosphodiesterase activity.