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 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 μ m. 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 [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. © 2007 International Society on Thrombosis and Haemostasis.