NOVEL (RP)-CAMPS ANALOGS AS TOOLS FOR INHIBITION OF CAMP-KINASE IN CELL-CULTURE - BASAL CAMP-KINASE ACTIVITY MODULATES INTERLEUKTIN-1-BETA ACTION

Novel (Rp)-cAMPS analogs differed widely in ability to antagonize cAMP activation of pure cAMP-dependent protein kinase I and II and to anta gonize actions of cAMP on gene expression, shape change, apoptosis, DN A replication, and protein phosphorylation in intact cells. These diff erences were related to different abilities of the analogs to stabiliz e the holoenzyme form relative to the dissociated form of cAMP kinase type I and II. (Rp)-8-Br-cAMPS and (Rp)-8-Cl-cAMPS were the most poten t cAMP antagonists for isolated type I kinase and for cells expressing mostly type I kinase, like IPC-81 leukemia cells, fibroblasts transfe cted with type I regulatory subunit (RI), and primary hepatocytes. It is proposed that (Rp)-8-Er-cAMPS or (Rp)-8-Cl-cAMPS should replace (Rp )-cAMPS as the first line cAMP antagonist, particularly for studies in cells expressing predominantly type I kinase. The phosphorylation of endogenous hepatocyte proteins was affected oppositely by (Rp)-8-Br-cA MPS and increased cAMP, indicating that (Rp)-8-Br-cAMPS inhibited basa l cAMP-kinase activity. The inhibition of basal kinase activity was ac companied by enhanced DNA replication, an effect which could be reprod uced by micro injected mutant cAMP subresponsive RI. It is concluded t hat the basal cAMP-kinase activity exerts a tonic inhibition of hepato cyte replication. (Rp)-8-Br-cAMPS and microinjected RI also desensitiz ed hepatocytes toward inhibition of DNA synthesis by interleukin-1 bet a. This indicates that basal cAMP-kinase activity can have a permissiv e role for the action of another (interleukin-1 beta) signaling pathwa y.