Effects of the cAMP-elevating agents cilostamide, cilostazol and forskolin on the phosphorylation of Akt and GSK-3β in platelets
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Hideki Hayashi; Toshiki Sudo
First Institute of New Drug Discovery, Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan
AKT, platelet aggregation, cAMP, phosphorylation, GSK
Elevating intracellular cAMP has been shown to inhibit platelet function. cAMP interferes with platelet-activating signals which lead to aggregation inhibition, but the precise mechanism is unclear. The present study examined if cAMP-elevating agents inhibited phosphatidylinositol 3-kinase (PI3-kinase) signaling in rat platelets by immunoblotting. Akt is one of the key molecules downstream of PI3K, and is phosphorylated by collagen stimulation. The phosphodiesterase-3 (PDE3) inhibitors cilostamide and cilostazol, and the adenylate cyclase activator forskolin, inhibited collagen-induced Akt phosphorylation at Ser473. The inhibitory effects of these cAMP-elevating agents on Akt phosphorylation were unchanged in the presence of the PKA (cyclic AMP-dependent protein kinase) inhibitor H-89. These effectswere consistent with inhibition of platelet aggregation. It is known that inhibition of Akt phosphorylation leads to inhibition of phosphorylation of glycogen synthase kinase 3-beta (GSK-3β), which is an effector of Akt, but cAMP-elevating agents stimulated GSK-3β phosphorylation at Ser9. The PKA inhibitor H-89 attenuated GSK-3β phosphorylation. The cAMP-elevating agents cilostamide, cilostazol and forskolin did not directly affect the enzyme activity of PI3-kinase. These results suggested that cAMP-elevating agents have two effects on PI3K signalling: inhibition of Akt phosphorylation independent of PKA; and stimulation of GSK-3β phosphorylation dependent on PKA. Our results provide new insights into the inhibitory effect of cAMPelevating agents on platelet function.
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