Effects of the cAMP-elevating agents cilostamide, cilostazol and forskolin on the phosphorylation of Akt and GSK-3beta in platelets.
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 effects were 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-3beta), which is an effector of Akt, but cAMP-elevating agents stimulated GSK-3beta phosphorylation at Ser9. The PKA inhibitor H-89 attenuated GSK-3beta 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-3beta phosphorylation dependent on PKA. Our results provide new insights into the inhibitory effect of cAMP-elevating agents on platelet function.
Hayashi
Hideki
H
First Institute of New Drug Discovery, Otsuka Pharmaceutical Co., Ltd., Kawauchi-cho, Tokushima, 771-0192, Japan.
Sudo
Toshiki
T
D000818:Animals; D001665:Binding Sites; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism*; D000077407:Cilostazol; D005576:Colforsin / Q000494:pharmacology*; D003094:Collagen / Q000494:pharmacology; D000242:Cyclic AMP / Q000097:blood*; D038362:Glycogen Synthase Kinase 3 / Q000097:blood* / Q000737:chemistry; D000071679:Glycogen Synthase Kinase 3 beta; D066298:In Vitro Techniques; D007546:Isoquinolines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000097:blood; D010766:Phosphorylation; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000097:blood* / Q000737:chemistry; D015363:Quinolones / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012694:Serine / Q000737:chemistry; D013449:Sulfonamides / Q000494:pharmacology; D013777:Tetrazoles / Q000494:pharmacology*
D007546:Isoquinolines; D010975:Platelet Aggregation Inhibitors; D047428:Protein Kinase Inhibitors; D015363:Quinolones; D013449:Sulfonamides; D013777:Tetrazoles; D005576:Colforsin; D012694:Serine; C021294:cilostamide; D003094:Collagen; D000242:Cyclic AMP; D019869:Phosphatidylinositol 3-Kinases; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D051057:Proto-Oncogene Proteins c-akt; D038362:Glycogen Synthase Kinase 3; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D000077407:Cilostazol