Phosphorylation of blood vessel vasodilator-stimulated phosphoprotein at serine 239 as a functional biochemical marker of endothelial nitric oxide/cyclic GMP signaling
Phosphorylation of blood vessel vasodilator-stimulated phosphoprotein at serine 239 as a functional biochemical marker of endothelial nitric oxide/cyclic GMP signaling
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DOI：
10.1124/mol.61.2.312
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发表时间：
2002-02-01
影响因子：
3.6
通讯作者：
Schmidt, HHHW
中科院分区：
文献类型：
--
作者：
Ibarra-Alvarado, C;Galle, J;Schmidt, HHHW
内皮源性松弛因子一氧化氮(NO)和前列环素(PGI(2))是重要的抗血栓、舒张剂和抗血管壁增殖剂，主要通过cGMP和cAMP依赖的蛋白激酶(CGK、CAK)发挥细胞内效应。然而，除了cGMP和cAMP浓度的一过性升高外，在完整的血管中没有反映它们活性的生化标记物。利用Western印迹分析和特异性抗体，我们发现兔主动脉中血管扩张剂刺激的磷酸蛋白(Vasp)Ser239(P-Ser239-Vasp)的磷酸化只在内皮完整的节段中检测到，尽管至少三分之一的Vasp包含在剩余的血管壁中。在去内皮的主动脉中，NO供体硝普钠(SNP)可增加VASP的磷酸化。P-Ser239-Vasp在内皮细胞、SNP或8-溴-cAMP存在时水平最高。CGK抑制剂RP-8-BR-PET-cGMPS可显著抑制8-溴-cAMP诱导的VASP磷酸化。刺激的P-Ser239-Vasp的形成是完全可逆的，在重复洗涤10分钟后达到基础水平。与NO/cGMP途径在兔主动脉P-Ser239-Vasp形成中的重要作用一致，N-omega-硝基-L-精氨酸甲酯(1 MM)抑制NO合成酶或1H-[1，2，4]恶二唑并[3，4-a]喹恶啉-1-酮(ODQ；50 um)抑制可溶性鸟苷酸环化酶几乎完全消除内皮完整血管P-Ser239-VASP的形成。这些数据表明，血管P-Ser239-VASP主要受NO/cGMP途径的调节，因此可以作为这一重要途径在内皮功能中活动状态的生化标志物。
The endothelium-derived relaxing factors nitric oxide (NO) and prostacyclin (PGI(2)) are important antithrombotic, relaxant, and anti proliferative agents of the blood vessel wall that exert their intracellular effects primarily via cGMP- and cAMP-dependent protein kinases (cGK, cAK). However, no biochemical marker for their activity in the intact blood vessel is available except for transient increases in the concentration of cGMP and cAMP. Using Western blot analysis and specific antibodies, we show here that phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) at Ser239 (P-Ser239-VASP) in rabbit aorta was detectable only in segments with an intact endothelium, although at least one third of VASP is contained in the remaining vascular wall. In endothelium-denuded aorta, VASP phosphorylation was increased by the NO donor sodium nitroprusside (SNP). Levels Of P-Ser239-VASP, in the presence of endothelium and either SNP or 8-bromo-cAMP, were maximal. VASP phosphorylation elicited by 8-bromo-cAMP was inhibited significantly by the cGK inhibitor Rp-8-Br-PET-cGMPS. Stimulated P-Ser239-VASP formation was fully reversible, reaching basal levels after 10 min of repeated washouts. Consistent with the important role that the NO/cGMP pathway plays in the formation of P-Ser239-VASP in rabbit aorta, inhibition of NO synthase by N-omega-nitro-L-arginine methyl ester (L-NAME; 1 mM) or of soluble guanylyl cyclase by 1H-[1,2,4]oxadiazolo[3,4-a]quinoxalin-1-one (ODQ; 50 muM) almost completely abolished P-Ser239-VASP formation in endothelium intact blood vessels. These data suggest that vascular P-Ser239-VASP is primarily regulated by the NO/cGMP pathway and may thus serve as a biochemical marker for the activity state of this essential pathway in endothelial function.