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

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.

A new signaling mechanism of hepatocyte growth factor-induced endothelial proliferation.

BACKGROUND: The hepatocyte growth factor (HGF) has been shown to promote endothelial cell proliferation. In this study, the signaling cascade responsible for the HGF-induced proliferation was examined. METHODS: The proliferation of human umbilical cord vein endothelial cells (HUCVEC) was determined using cell counts. Changes of the membrane potential were analyzed using the fluorescence dye DiBAC. Intracellular cGMP-levels were measured by means of [3H]-cGMP-radioimmunoassay. Phosphorylation of the p42/p44 MAP-kinase (MAPK) and the endothelial nitric oxide synthase (eNOS) was analyzed by immunocytochemistry. RESULTS: A dose-dependent (1-30 ng mL(-1)) increase of HUCVEC proliferation with a maximum at a concentration of 15 ng mL(-1) was induced by HGF. This effect was significantly reduced by the addition of the K+ channel blocker iberiotoxin (100 nmol L(-1)), eNOS inhibitor L-NMMA (300 micromol L(-1)), or the MEK inhibitor PD 98059 (20 micromol L(-1)). A HGF-induced hyperpolarization that was blocked by iberiotoxin was observed. In addition, HGF-induced activation of the eNOS was blocked by the K+ channel inhibitor. An increase of +101% MAPK phosphorylation was induced by HGF, which was blocked, if the cells were treated with L-NMMA (n = 20; P < 0.05), whereas HGF-induced phosphorylation of the eNOS was not affected by MEK inhibition. CONCLUSIONS: Hepatocyte growth factor modulates endothelial K+ channels causing an activation of the eNOS; the increase of nitric oxide is necessary for the phosphorylation of the MAPK inducing the proliferation of HUCVEC.