Effects of the cAMP-elevating agents cilostamide, cilostazol and forskolin on the phosphorylation of Akt and GSK-3β in platelets
Effects of the cAMP-elevating agents cilostamide, cilostazol and forskolin on the phosphorylation of Akt and GSK-3β in platelets
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DOI：
10.1160/th08-12-0781
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发表时间：
2009-08-01
影响因子：
6.7
通讯作者：
Sudo, Toshiki
中科院分区：
文献类型：
--
作者：
Hayashi, Hideki;Sudo, Toshiki
升高细胞内 cAMP 已被证明可以抑制血小板功能。 CAMP 干扰血小板激活信号，导致聚集抑制，但确切机制尚不清楚。本研究通过免疫印迹检查了 cAMP 升高剂是否抑制大鼠血小板中的磷脂酰肌醇 3 激酶（PI3 激酶）信号传导。 Akt 是 PI3K 下游的关键分子之一，可通过胶原蛋白刺激而磷酸化。磷酸二酯酶 3 (PDE3) 抑制剂西洛酰胺和西洛他唑以及腺苷酸环化酶激活剂毛喉素可抑制胶原蛋白诱导的 Akt Ser473 磷酸化。在 PKA（环 AMP 依赖性蛋白激酶）抑制剂 H-89 存在的情况下，这些 cAMP 升高剂对 Akt 磷酸化的抑制作用没有改变。这些作用与抑制血小板聚集一致。已知抑制 Akt 磷酸化会导致抑制 Akt 效应子糖原合酶激酶 3-β (GSK-3 beta) 的磷酸化，但 cAMP 升高剂会刺激 GSK-3 beta Ser9 磷酸化。 PKA 抑制剂 H-89 减弱 GSK-3 β 磷酸化。 cAMP升高剂西洛酰胺、西洛他唑和毛喉素不直接影响PI 3-激酶的酶活性。这些结果表明，cAMP 升高剂对 PI3K 信号传导有两种作用：独立于 PKA 抑制 Akt 磷酸化；以及依赖于 PKA 的 GSK-3 β 磷酸化刺激。我们的结果为 cAMP 升高剂对血小板功能的抑制作用提供了新的见解。
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-3 beta),which is an effector of Akt,but cAMP-elevating agents stimulated GSK-3 beta phosphorylation at Ser9. The PKA inhibitor H-89 attenuated GSK-3 beta phosphorylation. The cAMP-elevating agents cilostamide,cilostazol and forskolin did not directly affect the enzyme activity of PI 3-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-3 beta phosphorylation dependent on PKA. Our results provide new insights into the inhibitory effect of cAMP-elevating agents on platelet function.