Becker E M, Schmidt P, Schramm M, Schröder H, Walter U, Hoenicka M, Gerzer R, Stasch J P
Institute of Cardivascular and Arteriosclerosis Research, Bayer AG, Wuppertal, Germany.
J Cardiovasc Pharmacol. 2000 Mar;35(3):390-7. doi: 10.1097/00005344-200003000-00007.
The effects of the different types of soluble guanylate cyclase (sGC) stimulators on the phosphorylation status of vasodilator-stimulated phosphoprotein (VASP) in both human and rat platelets were studied under in vitro and in vivo conditions. sGC-dependent VASP phosphorylation (at Ser(239) and Ser(157)) both by the new direct sGC stimulator YC-1 and by NO donors was examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS/PAGE) with different antibodies. One antibody, which recognizes VASP independent of its phosphorylation state, was used to detect the mobility shift of VASP caused by Ser(157) phosphorylation. The other antibody was specifically directed against VASP phosphorylated at Ser(239), the cGMP-dependent protein kinase (PKG) preferred phosphorylation site of VASP. In vitro YC-1 increased both VASP phosphorylation and cyclic guanosine monophosphate (cGMP) levels as did the NO donors 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA/NO) and sodium nitroprusside (SNP). The combination of both types induced a synergistic effect in both VASP phosphorylation and cGMP increase. In rat platelets, similar effects could be shown in vitro. In vivo we observed a significant increase in cGMP and a distinct effect on VASP phosphorylation in rat platelets 1 h after oral administration of YC-1. These biochemical alterations are supported by a significant prolongation in rat-tail bleeding time. Direct stimulators of sGC like YC-1 are on the one hand direct potent stimulators of the cGMP/PKG/VASP pathway in platelets and on the other hand synergize with NO, the physiologic stimulator of sGC. Therefore YC-1-like substances are interesting tools for the development of new cardiovascular drugs with vasodilatory and antithrombotic properties.
在体外和体内条件下，研究了不同类型的可溶性鸟苷酸环化酶（sGC）刺激剂对人和大鼠血小板中血管舒张刺激磷蛋白（VASP）磷酸化状态的影响。通过使用不同抗体的十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS/PAGE），检测新型直接sGC刺激剂YC - 1和一氧化氮（NO）供体对sGC依赖性VASP磷酸化（Ser(239)和Ser(157)位点）的作用。一种可识别不依赖于其磷酸化状态的VASP的抗体，用于检测由Ser(157)磷酸化引起的VASP迁移率变化。另一种抗体特异性针对Ser(239)磷酸化的VASP，这是环磷酸鸟苷依赖性蛋白激酶（PKG）对VASP的优先磷酸化位点。在体外，YC - 1与NO供体2-(N,N - 二乙氨基) - 重氮酸酯 - 2 - 氧化物（DEA/NO）和硝普钠（SNP）一样，均可增加VASP磷酸化水平和环磷酸鸟苷（cGMP）水平。两种类型的刺激剂联合使用在VASP磷酸化和cGMP增加方面均产生协同作用。在大鼠血小板中，体外实验也显示出类似的效果。在体内，口服YC - 1 1小时后，我们观察到大鼠血小板中cGMP显著增加，并且对VASP磷酸化有明显影响。这些生化改变伴随着大鼠尾部出血时间的显著延长。像YC - 1这样的sGC直接刺激剂一方面是血小板中cGMP/PKG/VASP途径的直接强效刺激剂，另一方面与sGC的生理刺激剂NO协同作用。因此，类似YC - 1的物质是开发具有血管舒张和抗血栓特性的新型心血管药物的有趣工具。
