6533b858fe1ef96bd12b5aa6

RESEARCH PRODUCT

Apigenin‐induced nitric oxide production involves calcium‐activated potassium channels and is responsible for antiangiogenic effects

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Summary. 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 Ca2+-activated K+ channels (KCa) are involved in endothelial nitric oxide (NO) production and antiangiogenic effects.Methods: Endothelial NO generation was monitored using a cyclic guanosine monophosphate radioimmunoassay. KCa activity and changes of the intracellular Ca2+ concentration [Ca2+]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, [3H]-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 μm. Api-induced hyperpolarization was blocked by the small and large conductance KCa inhibitors apamin and iberiotoxin, respectively. Furthermore, apamin and iberiotoxin blocked the late, long-lasting plateau phase of the Api-induced biphasic increase of [Ca2+]i. Inhibition of Ca2+ signaling and the KCa blockade both blocked NO production. Prevention of all three (NO, Ca2+, and KCa 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 KCa, leading to a hyperpolarization that is followed by a Ca2+ influx. The increase of [Ca2+]i is responsible for an increased NO production that mediates the antiangiogenic effects of Api via Akt dephosphorylation.