Cyclic nucleotide phosphodiesterase (PDE) activity was measured by a 3H cAMP method on the infranatant portion of the homogenate of adipocytes isolated from rat epididymal fat pads. DBcAMP (0.05 to 1.0 mM.) was found to progressively inhibit the PDE activity. The inhibition was not secondary to dilution of the 3H cAMP by cAMP derived from catabolism of the DBcAMP by deacylase activity in the homogenate. Adipocytes were prelabeled with radiocarbon adenine and the hydrolysis of radiocarbon cAMP was measured in the presence and absence of DBcAMP. Increasing concentrations of DBcAMP prevented the hydrolysis of the radiocarbon cAMP in a dosedependent manner with no breakdown occurring at 1.0 mM., correlating with the complete inhibition of the homogenate PDE activity at 1.0 mM. Kinetic analysis revealed the low Km of the PDE to be 0.8 μM., and this was shifted to 2.5 μM. by 0.05 mM. DBcAMP without a change in Vmax, indicating a competitive-type inhibition which was easily reversible upon removal of the DBcAMP.
The cAMP levels were measured in extracts of DBcAMP-treated adipocytes by the protein kinase technic. DBcAMP and O2′ MBcAMP did not affect the assay system until 200 pmoles were present, while N6 MBcAMP was indistinguishable from cAMP in the assay system set for a standard curve of 0.5 to 10 pmoles. A procedure was devised to separate the cAMP from DBcAMP and O2′ MBcAMP such that they would always be at levels too low to interfere with the assay. N6 MBcAMP was shown not to be present in the assayed material since the material was susceptible to PDE digestion and N6 MBcAMP was not. Cellular cAMP levels increased as DBcAMP increased, reaching over fifty times the control at 1.0 mM. DBcAMP. This cAMP was shown to be over 90 per cent of endogenous origin and less than 10 per cent from breakdown of DBcAMP. Lipolysis was stimulated proportional to the increase in cAMP levels but was not maximal until the fiftyfold elevation of cAMP. This suggested that DBcAMP might compete with cAMP in activating the enzymes involved in lipolysis and that the large excess of cAMP is required to overcome this antagonism by DBcAMP.
