title: Cilostazol inhibits cytokine-induced tetrahydrobiopterin biosynthesis in human umbilical vein endothelial cells.
abstract: OBJECTIVE
  Cilostazol, a type III phosphodiesterase inhibitor, is utilized for the treatment of intermittent claudication and is considered to have the beneficial effects against the atherogenic process. In the present study, we examined the effects of cilostazol on BH(4) biosynthesis in HUVEC treated with a mixture of the pro-inflammatory cytokines IFN-γ and TNF-α.
  
  
  METHODS
  Isolated HUVECs were grown to confluence and treated with IFN-γ (300 units/mL) and TNF-α (300 units/mL) for 16 h in order to stimulate BH(4) biosynthesis. The BH(4) levels were measured by HPLC. The mRNA expression of GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme of BH(4) biosynthesis, and GTPCH feedback regulatory protein (GFRP) were quantified by real-time PCR. The GTPCH protein expression was assessed by western blot analysis.
  
  
  RESULTS
  Cilostazol significantly reduced the BH(4) levels in cytokine-stimulated HUVEC. Cilostazol produced a concomitant increase in the cAMP levels in HUVEC. Cilostazol decreased the GTPCH activity as well as the expression of GTPCH mRNA and protein. 8-bromo-cAMP (8Br-cAMP), a cell-permeable cAMP analogue, did not reproduce the effects of cilostazol. Cilostazol did not affect the cytokine-induced inhibition of GFRP mRNA expression.
  
  
  CONCLUSIONS
  We conclude that cilostazol inhibited cytokine-stimulated BH(4) biosynthesis via a cAMP-independent mechanism in HUVEC. Our data indicate that cilostazol reduced GTPCH activity and did so by suppressing the GTPCH protein levels.
authors.affiliation: Department of Pharmacology, Fujita Health University School of Medicine, Toyoake, Aichi, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Shiraishi
authors.forename: Kazuhisa
authors.initials: K
authors.lastname: Ikemoto
authors.forename: Shin
authors.initials: S
authors.lastname: Tada
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Udagawa
authors.forename: Masatsugu
authors.initials: M
authors.lastname: Ohtsuki
authors.forename: Chiho
authors.initials: C
authors.lastname: Sumi-Ichinose
authors.forename: Kazunao
authors.initials: K
authors.lastname: Kondo
authors.forename: Takahide
authors.initials: T
authors.lastname: Nomura
mesh_terms: D001708:Biopterins / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D002478:Cells, Cultured; D000077407:Cilostazol; D000242:Cyclic AMP; D016207:Cytokines / Q000494:pharmacology*; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D005343:Fibrinolytic Agents; D006136:GTP Cyclohydrolase / Q000032:analysis; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D013777:Tetrazoles / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014471:Umbilical Veins / Q000166:cytology
chemical_list: D016207:Cytokines; D005343:Fibrinolytic Agents; D013777:Tetrazoles; D014409:Tumor Necrosis Factor-alpha; D001708:Biopterins; D007371:Interferon-gamma; D000242:Cyclic AMP; D006136:GTP Cyclohydrolase; C003402:sapropterin; D000077407:Cilostazol