Effects of PKA on isoproterenol (1 μM)-induced cAMP synthesis and hydrolysis in HEK-293 cells. (A) Time course of isoproterenol-induced stimulation of PDE4 activity in HEK-293 cell extracts. Cells were incubated with 1 μM isoproterenol for the indicated times. At the end of the incubation, cells were harvested and homogenates subjected to PDE activity assays using 1 μM cAMP as substrate. PDE activity was measured in the absence or presence of 10 μM rolipram. The rolipram-inhibited PDE4 activity is reported. It is clear that isoproterenol induced a two- to threefold increase in PDE4 activity. The increase in PDE4 activity was due to PKA-dependent phosphorylation of PDE4 and was prevented by pretreatment with PKA inhibitors (not depicted). (B) cAMP accumulation measured in HEK-293 cells using an enzyme immunoassay. Cells were stimulated with 10 μM isoproterenol for 5 min following pretreatment with vehicle (DMSO, 10 min), 10 μM H89 (10 min), 10 μM rolipram (5 min), or 10 μM H89 (10 min) and 10 μM rolipram (5 min). H89 had little or no effect on isoproterenol-induced cAMP accumulation in the presence of rolipram, indicating that PKA does not substantially stimulate receptor desensitization or inhibit the rate of cAMP synthesis following stimulation with saturating concentrations of isoproterenol. Data are the mean ± SEM of three (A) or four (B) separate experiments, each performed in triplicate.