Activation of endothelial nitric oxide synthase by cilostazol via a cAMP/protein kinase A- and phosphatidylinositol 3-kinase/Akt-dependent mechanism.
We investigated the effect of cilostazol on nitric oxide (NO) production in human aortic endothelial cells (HAEC). Cilostazol increased NO production in a concentration-dependent manner, and NO production was also increased by other cyclic-AMP (cAMP)-elevating agents (forskolin, cilostamide, and rolipram). Cilostazol increased intracellular cAMP level, and that effect was enhanced in the presence of forskolin. In Western blot analysis, cilostazol increased phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser(1177) and of Akt at Ser(473) and dephosphorylation of eNOS at Thr(495). Cilostazol's regulation of eNOS phosphorylation was reversed by protein kinase A inhibitor peptide (PKAI) and by LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. Moreover, the cilostazol-induced increase in NO production was inhibited by PKAI, LY294002, and N(G)-nitro-l-arginine methyl ester hydrochloride (l-NAME), a NOS inhibitor. In an in vitro model of angiogenesis, cilostazol-enhanced endothelial tube formation, an effect that was completely attenuated by inhibitors of PKA, PI3K, and NOS. These results suggest that cilostazol induces NO production by eNOS activation via a cAMP/PKA- and PI3K/Akt-dependent mechanism and that this effect is involved in capillary-like tube formation in HAEC.
Research Institute of Pharmacological & Therapeutical Development, Otsuka Pharmaceutical Co. Ltd., 463-10 Kagasuno, Kawauchi-cho, Tokushima 771-0192, Japan.
Ayako
A
Hashimoto
Goro
G
Miyakoda
Yoshimi
Y
Hirose
Toyoki
T
Mori
D001011:Aorta / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D000077407:Cilostazol; D017868:Cyclic AMP-Dependent Protein Kinases / Q000187:drug effects / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D009025:Morpholines / Q000494:pharmacology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000502:physiology; D052250:Nitric Oxide Synthase Type III / Q000187:drug effects / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000187:drug effects / Q000378:metabolism*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D013777:Tetrazoles / Q000494:pharmacology*
D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D010726:Phosphodiesterase Inhibitors; D013777:Tetrazoles; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D000077407:Cilostazol; D019331:NG-Nitroarginine Methyl Ester