title: Apigenin-induced nitric oxide production involves calcium-activated potassium channels and is responsible for antiangiogenic effects.
abstract: BACKGROUND
  The dietary flavonoid apigenin (Api) has been demonstrated to exert multiple beneficial effects upon the vascular endothelium. The aim of this study was to examine whether Ca(2+)-activated K(+) channels (K(Ca)) are involved in endothelial nitric oxide (NO) production and antiangiogenic effects.
  
  
  METHODS
  Endothelial NO generation was monitored using a cyclic guanosine monophosphate radioimmunoassay. K(Ca) activity and changes of the intracellular Ca(2+) concentration [Ca(2+)](i) were analyzed using the fluorescent dyes bis-barbituric acid oxonol, potassium-binding benzofuran isophthalate, and fluo-3. The endothelial angiogenic parameters measured were cell proliferation, [(3)H]-thymidine incorporation, and cell migration (scratch assay). Akt phosphorylation was examined using immunohistochemistry.
  
  
  RESULTS
  Api caused a concentration-dependent increase in cyclic guanosine monophosphate levels, with a maximum effect at a concentration of 1 mum. Api-induced hyperpolarization was blocked by the small and large conductance K(Ca) inhibitors apamin and iberiotoxin, respectively. Furthermore, apamin and iberiotoxin blocked the late, long-lasting plateau phase of the Api-induced biphasic increase of [Ca(2+)](i). Inhibition of Ca(2+) signaling and the K(Ca) blockade both blocked NO production. Prevention of all three (NO, Ca(2+), and K(Ca) signaling) reversed the antiangiogenic effects of Api under both basal and basic fibroblast growth factor-induced culture conditions. Basic fibroblast growth factor-induced Akt phosphorylation was also reduced by Api.
  
  
  CONCLUSIONS
  Based on our experimental results we propose the following signaling cascade for the effects of Api on endothelial cell signaling. Api activates small and large conductance K(Ca), leading to a hyperpolarization that is followed by a Ca(2+) influx. The increase of [Ca(2+)](i) is responsible for an increased NO production that mediates the antiangiogenic effects of Api via Akt dephosphorylation.
authors.affiliation: Department of Cardiology and Angiology, Justus-Liebig-University of Giessen, Giessen, Germany. ali.erdogan@innere.med.uni-giessen.de
authors.forename: A
authors.initials: A
authors.lastname: Erdogan
authors.forename: A K
authors.initials: AK
authors.lastname: Most
authors.forename: B
authors.initials: B
authors.lastname: Wienecke
authors.forename: A
authors.initials: A
authors.lastname: Fehsecke
authors.forename: C
authors.initials: C
authors.lastname: Leckband
authors.forename: R
authors.initials: R
authors.lastname: Voss
authors.forename: M T
authors.initials: MT
authors.lastname: Grebe
authors.forename: H
authors.initials: H
authors.lastname: Tillmanns
authors.forename: C A
authors.initials: CA
authors.lastname: Schaefer
authors.forename: C R W
authors.initials: CR
authors.lastname: Kuhlmann
mesh_terms: D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D047310:Apigenin / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002465:Cell Movement; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D009569:Nitric Oxide / Q000378:metabolism*; D010766:Phosphorylation; D015221:Potassium Channels / Q000737:chemistry / Q000378:metabolism*; D011863:Radioimmunoassay; D015398:Signal Transduction; D013997:Time Factors; D014471:Umbilical Veins / Q000378:metabolism
chemical_list: D020533:Angiogenesis Inhibitors; D015221:Potassium Channels; D009569:Nitric Oxide; D047310:Apigenin; D002118:Calcium