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.