血管内皮生长因子（VEGF）介导的内皮细胞一氧化氮（NO）的即时和延迟合成：磷脂酰肌醇-3-激酶（PI3K）、蛋白激酶C（PKC）及磷脂酶C（PLC）信号通路的作用

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

Gélinas David S, Bernatchez Pascal N, Rollin Simon, Bazan Nicolas G, Sirois Martin G

Department of Pharmacology, University of Montreal, Montreal (Qc), Canada.

Br J Pharmacol. 2002 Dec;137(7):1021-30. doi: 10.1038/sj.bjp.0704956.

Abstract

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(1177)-phosphorylation and NO synthesis measured by cGMP production. 3. Pretreatment with phosphatidylinositol 3-kinase (PI3K) (Wortmannin, LY294002) or protein kinase C (PKC) (GF109203X, Ro318220) inhibitors attenuated eNOS Ser(1177)-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.

摘要

血管内皮生长因子（VEGF）诱导内皮型一氧化氮合酶（eNOS）激活的机制在一定程度上仍不清楚。因此，我们试图通过测量环磷酸鸟苷（cGMP）的产生来评估多种途径在VEGF诱导的一氧化氮（NO）合成中的作用。此外，由于VEGF诱导NO和血小板活化因子（PAF）的合成，我们想评估PAF和NO的诱导是否相互促进合成。2. 在此，我们表明用磷脂酶C（PLC）抑制剂（U73122）、钙调蛋白拮抗剂（W-7）或细胞内钙螯合剂（乙二醇双四乙酸/乙酰甲酯，1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸/乙酰甲酯）处理内皮细胞可阻止VEGF介导的eNOS丝氨酸（Ser）1177磷酸化以及通过cGMP产生测量的NO合成。3. 用磷脂酰肌醇3-激酶（PI3K）（渥曼青霉素，LY294002）或蛋白激酶C（PKC）（GF109203X，Ro318220）抑制剂预处理可减弱VEGF介导的eNOS Ser1177磷酸化，但不改变VEGF诱导的即时（0 - 10分钟）cGMP合成，但可将延迟（10 - 30分钟）cGMP合成消除高达84%。4. 用PAF合成抑制剂或PAF受体拮抗剂预处理既不消除VEGF介导的eNOS Ser1177磷酸化也不消除cGMP合成。5. 总之，VEGF通过PLC - Ca²⁺/钙调蛋白（CaM）途径诱导即时cGMP合成，而延迟cGMP合成的诱导意味着蛋白激酶B（Akt）和PKC活性。