Immediate and delayed VEGF-mediated NO synthesis in endothelial cells:: Role of PI3K, PKC and PLC pathways

1 The mechanism(s) by which vascular endothelial growth factor (VEGF) induces endothelial nitric oxide synthase (eNOS) activation remain(s) unclear up to a certain extent. Therefore, we sought to evaluate the contribution of numerous pathways in VEGF-induced nitric oxide (NO) synthesis by measuring cGMP production. In addition, as VEGF induces the synthesis of NO and platelet-activating factor (PAF), we wanted to assess if the induction of PAF and NO is contributing to the synthesis of each other. 2 Herein, we show that a treatment of endothelial cells with a phospholipase C (PLC) inhibitor (U73122), a calmodulin antagonist (W-7) or with intracellular calcium chelators (EGTA/AM, BAPTA/AM) prevented VEGF-mediated eNOS Ser(1117)-phosphorylation and NO synthesis measured by cGMP production. 3 Pretreatment with phosphatidylinositol 3-kinase (PI3K) (Wortmannin, LY294002) or protein kinase C (PKC) (GF109203)(, Ro318220) inhibitors attenuated eNOS Ser(1117)-phosphorylation mediated by VEGF, but did not alter immediate (0 - 10 min) cGMP synthesis induced by VEGF, but abrogated by up to 84% the delayed (10-30 min) cGMP synthesis. 4 Pretreatment with PAF synthesis inhibitors or with PAF receptor antagonists did not abrogate neither eNOS Ser(1177)-phosphorylation nor cGMP synthesis mediated by VEGF. 5 In conclusion, VEGF induces an immediate cGMP synthesis through the PLC-Ca2+/CaM pathway, and that the induction of delayed cGMP synthesis implies Akt and PKC activity.