REGULATION OF CYCLIC NUCLEOTIDE PHOSPHODIESTERASE ACTIVITY IN PLATELETS BY PHOSPHORYLATION

Cyclic nucleotide phosphodiesterases (PDE) provide the only known pathway for the hydrolysis of cyclic nucleotides in cells and thus have the potential for modulating the effects of cAMP and cGMP on cells. In platelets a rise in intracellular cAMP levels inhibits platelet aggregation and secretion. Since cAMP exerts many of its effects through a cAMP-dependent kinase we questioned whether phosphorylation of cAMP PDE might be a mode for regulation of PDE activity in platelets. When platelets were incubated for 10 min with forskolin (100 μM) the level of cAMP rose at least 10-fold.When the low Km cyclic nucleotide PDE was isolated from freeze-thaw lysates of forskolin treated platelets by chromatography on blue dextran-Sepharose, the specific activity of this enzyme was increased 3 to 13-fold over similarly processed control platelets. The specific activity of a second PDE, the cGMP-stimulated cAMP PDE, was increased 1.5 to 3-fold by forskolin treatment of platelets. Forskolin had no direct effect on either purified PDE. The stimulation of the low Km cAMP PDE activity by exposure of platelets to forskolin was blocked when the platelets were simultaneously treated with the protein kinase inhibitor H-8 (100 μM) which is most potent toward cAMP dependent protein kinase indicating that this kinase may be responsible for the stimulation. When platelets which had been prelabeled with 32P inorganic phosphate were treated with forskolin and the low Km cAMP PDE isolated by blue dextran-Sepharose chromatography, a protein migrating in SDS gels at Mr=110,000, the molecular weight of the low Km cAMP PDE, was labeled indicating that phosphorylation of the PDE occurred coincident with stimulation of activity. These results suggest that phosphorylation of the low Km cAMP PDE by protein kinase may be an important regulatory mechanism for cAMP PDE activity and cyclic nucleotide levels in platelets.