Fluctuation of Adenosine 3',5'-Monophosphate-Binding Site Occupancy as an Index of Hormone-Dependent Adenosine 3',5'-Monophosphate Formation in Bone Cells*

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Abstract
We studied binding of [3H]cAMP to bone cell cytosol. Scatchard analysis of the data demonstrated specific binding (Kd = 5.9 ×10-8 M). In the basal state, 60% of total cAMP-binding sites were unoccupied. Incubation of cells with parathyroid hormone (PTH) or prostaglandin Ei (PGEi) led to the depletion of unoccupied binding sites by 50–80%. Maximal depletion was achieved at submaximal levels of cAMP and was more pronounced after PGE1 than after PTH treatment. In combination, PTH and PGE1 caused additive cAMP accumulation, but binding site depletion did not exceed that in response to PGE1 alone. During prolonged incubation, unoccupied binding site depletion mirrored changes in cAMP concentration, with peak effect at 10 min and return to basal conditions by 60 min. Subsequent elevation of cAMP led promptly to further depletion of unoccupied binding sites. Restoration of basal state levels was not impaired by cycloheximide. Changes in unoccupied binding sites were not accompanied by changes in total cAMP-binding site content. These results are compatible with published evidence for bone cell heterogeneity and suggest that physiological regulation of bone cell metabolism may require only modest changes in cAMP concentration. Analysis of cAMP-binding site occupancy did not prove to be more sensitive an index of PTH or PGE1 action on bone cells than direct measurement of cAMP. cAMP receptor occupancy appears to fluctuate as a direct consequence of changes in ambient cAMP concentration and is not independently regulated.