title: Cilostazol Induces PGI2 Production via Activation of the Downstream Epac-1/Rap1 Signaling Cascade to Increase Intracellular Calcium by PLCε and to Activate p44/42 MAPK in Human Aortic Endothelial Cells.
abstract: BACKGROUND
  Cilostazol, a selective phosphodiesterase 3 (PDE3) inhibitor, is known as an anti-platelet drug and acts directly on platelets. Cilostazol has been shown to exhibit vascular protection in ischemic diseases. Although vascular endothelium-derived prostaglandin I2 (PGI2) plays an important role in vascular protection, it is unknown whether cilostazol directly stimulates PGI2 synthesis in endothelial cells. Here, we elucidate the mechanism of cilostazol-induced PGI2 stimulation in endothelial cells.
  
  
  RESULTS
  Human aortic endothelial cells (HAECs) were stimulated with cilostazol and PGI2 accumulation in the culture media was measured. Cilostazol increased PGI2 synthesis via the arachidonic acid pathway. Cilostazol-induced intracellular calcium also promoted PGI2 synthesis via the inositol 1,4,5-trisphosphate receptor. Using RNAi, silencing of PDE3B abolished the induction effect of cilostazol on PGI2 synthesis and intracellular cAMP accumulation. Inhibition of the exchange protein, which was directly activated by cyclic AMP 1 (Epac-1) and its downstream signal the Ras-like small GTPase (Rap-1), abolished cilostazol-induced PGI2 synthesis, but this did not take place via protein kinase A (PKA). Inhibition of downstream signaling, such as mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K) γ, and phospholipase C (PLC) ε, suppressed cilostazol-induced PGI2 synthesis.
  
  
  CONCLUSIONS
  The PDE3/Epac-1/Rap-1 signaling pathway plays an important role in cilostazol-induced PGI2 synthesis. Namely, stimulation of HAECs with cilostazol induces intracellular calcium elevation via the Rap-1/PLCε/IP3 pathway, along with MAPK activation via direct activation by Epac-1/Rap-1 and indirect activation by Epac-1/Rap-1/PI3Kγ, resulting in synergistically induced PGI2 synthesis.
authors.affiliation: First Institute of New Drug Discovery, Otsuka Pharmaceutical Co., Ltd., Tokushima-shi, Tokushima, Japan.
authors.forename: Ayako
authors.initials: A
authors.lastname: Hashimoto
authors.affiliation: Medical Chemistry Research Institute, Otsuka Pharmaceutical Co., Ltd. Tokushima-shi, Tokushima, Japan.
authors.forename: Michinori
authors.initials: M
authors.lastname: Tanaka
authors.affiliation: Qs'Research Institute, Otsuka Pharmaceutical Co., Ltd. Tokushima-shi, Tokushima, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Takeda
authors.affiliation: First Institute of New Drug Discovery, Otsuka Pharmaceutical Co., Ltd., Tokushima-shi, Tokushima, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Ito
authors.affiliation: First Institute of New Drug Discovery, Otsuka Pharmaceutical Co., Ltd., Tokushima-shi, Tokushima, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Nagano
mesh_terms: D001011:Aorta / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D016718:Arachidonic Acid / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D000077407:Cilostazol; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3 / Q000235:genetics / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D011464:Epoprostenol / Q000819:agonists* / Q000096:biosynthesis; D005786:Gene Expression Regulation; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D058987:Phosphodiesterase 3 Inhibitors / Q000494:pharmacology*; D054801:Phosphoinositide Phospholipase C / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D000089804:Shelterin Complex; D015398:Signal Transduction; D034501:Telomere-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D013777:Tetrazoles / Q000494:pharmacology*
chemical_list: D016718:Arachidonic Acid; D002118:Calcium; D000077407:Cilostazol; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D011464:Epoprostenol; D020662:Guanine Nucleotide Exchange Factors; D053496:Inositol 1,4,5-Trisphosphate Receptors; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D019869:Phosphatidylinositol 3-Kinases; D058987:Phosphodiesterase 3 Inhibitors; D054801:Phosphoinositide Phospholipase C; D034741:RNA, Small Interfering; D000089804:Shelterin Complex; D034501:Telomere-Binding Proteins; D013777:Tetrazoles; C116122:RAPGEF3 protein, human; C440809:TERF2IP protein, human; C423320:phospholipase C epsilon; C517291:PDE3B protein, human