Translocation of protein kinase C isoforms is involved in propofol-induced endothelial nitric oxide synthase activation.
Translocation of protein kinase C isoforms is involved in propofol-induced endothelial nitric oxide synthase activation.
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DOI：
10.1093/bja/aeq064
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发表时间：
2010-05
影响因子：
9.8
通讯作者：
L. Wang;B. Wu;Y. Sun;T. Xu;X. Zhang;M. Zhou;Wei Jiang
中科院分区：
文献类型：
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作者：
L. Wang;B. Wu;Y. Sun;T. Xu;X. Zhang;M. Zhou;Wei Jiang
研究背景：蛋白激酶C（PKC）可增强内皮型一氧化氮合酶（eNOS）的激活，但具体机制尚不清楚。在这项研究中，我们研究了PKC亚型在调节丙泊酚诱导的人脐静脉内皮细胞（HUVECs）eNOS激活中的作用。方法采用蛋白质印迹法（WB）检测丙泊酚对人脐静脉内皮细胞（HUVECs）中丝氨酸（1177）磷酸化依赖的eNOS活化的影响。亚硝酸盐（NO（2）（-））的积累使用Griess测定。WB法检测磷脂酰肌醇3激酶/Akt（PI 3 K/Akt）通路。用WB法分析了丙泊酚诱导的HUVECs亚细胞组分中单个PKC亚型的易位。结果在HUVEC中，方案处理（1-100 μ M）10分钟可诱导eNOS在Ser（1177）处磷酸化的浓度依赖性增加。NO产量也相应增加。PKC抑制剂，双吲哚马来酰亚胺I（0.1-1 μ M），和staurosporine（20和100 nM），有效地阻断了丙泊酚诱导的eNOS激活和NO的产生。对分级内皮细胞裂解物的进一步分析表明，短期丙泊酚处理（50 μ M）导致PKC-α、PKC-δ、PKC-zeta、PKC-eta和PKC-β从胞质组分易位到膜组分，这也可以被两种PKC抑制剂抑制。这些数据表明，这些同工酶的差异再分布是必不可少的丙泊酚诱导的eNOS激活。此外，Akt在Ser（473）或Thr（308）没有磷酸化。结论异丙酚通过诱导PKC异构体转位至HUVECs不同的胞内位点，诱导Ser（1177）磷酸化依赖的eNOS活化，该作用不依赖于PI 3 K/Akt非依赖性途径。
BACKGROUND Previous studies have indicated that protein kinase C (PKC) may enhance endothelial nitric oxide synthase (eNOS) activation, although the detailed mechanism(s) remains unclear. In this study, we investigated the roles of PKC isoforms in regulating propofol-induced eNOS activation in human umbilical vein endothelial cells (HUVECs). METHODS We applied western blot (WB) analysis to investigate the effects of propofol on Ser(1177) phosphorylation-dependent eNOS activation in HUVECs. Nitrite (NO(2)(-)) accumulation was measured using the Griess assay. The phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathway was examined by WB assay. Propofol-induced translocation of individual PKC isoforms in subcellular fractions in HUVECs was analysed using WB assay. RESULTS In HUVECs, protocol treatment (1-100 microM) for 10 min induced a concentration-dependent increase in phosphorylation of eNOS at Ser(1177). The NO production was also increased accordingly. PKC inhibitors, bisindolylmaleimide I (0.1-1 microM), and staurosporine (20 and 100 nM), effectively blocked propofol-induced eNOS activation and NO production. Further analyses in fractionated endothelial lysate showed that short-term propofol treatment (50 microM) led to translocation of PKC-alpha, PKC-delta, PKC-zeta, PKC-eta, and PKC-epsilon from cytosolic to membrane fractions, which could also be inhibited by both PKC inhibitors. These data revealed that the differential redistribution of these isozymes is indispensable for propofol-induced eNOS activation. In addition, Akt was not phosphorylated in response to propofol at Ser(473) or Thr(308). CONCLUSIONS Propofol induces the Ser(1177) phosphorylation-dependent eNOS activation through the drug-stimulated translocation of PKC isoforms to distinct intracellular sites in HUVECs, which is independent of PI3K/Akt-independent pathway.