High glucose concentrations increase endothelial cell permeability via activation of protein kinase C alpha
High glucose concentrations increase endothelial cell permeability via activation of protein kinase C alpha
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DOI：
10.1161/01.res.81.3.363
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发表时间：
1997-09-01
影响因子：
20.1
通讯作者：
Haller, H
中科院分区：
文献类型：
--
作者：
Hempel, A;Maasch, C;Haller, H
糖尿病患者内皮细胞通透性受损，细胞外高浓度葡萄糖可增加内皮细胞通透性。高葡萄糖激活蛋白激酶C（PKC），一个对细胞内信号传导至关重要的激酶家族。我们测试了高葡萄糖浓度激活内皮细胞中的PKC并通过不同的PKC亚型导致内皮细胞通透性增加的假设。使用猪主动脉内皮细胞，并在这些细胞中鉴定了PKC亚型α、δ、β、ζ和θ。葡萄糖引起内皮细胞通透性的快速剂量依赖性增加，EC 50为17.5 mmol/L。佛波醇12-肉豆蔻酸酯13-乙酸酯（TPA）诱导的渗透性增加非常类似于葡萄糖引起的。用PKC抑制剂staurosporine（10（-8）mol/L）和Goe 6976（10（-8）mol/L）预孵育细胞可完全逆转葡萄糖和TPA的作用。通过与TPA预孵育24小时下调PKC也消除了葡萄糖和TPA对内皮细胞通透性的影响。高糖（20 mmol/L）可引起2、10和30 min时PKC活性升高。细胞分级分离和蛋白质印迹分析显示葡萄糖诱导的PKC α和PKC β易位。共聚焦显微镜证实了易位，并显示了PKC α和PKC β与核结构和细胞膜的关联。针对PKC α的特异性反义寡脱氧核苷酸（ODNs）降低了PKC α亚型的表达，完全消除了葡萄糖对内皮细胞通透性的影响，并显著降低了TPA的作用。相反，针对PKC β的特异性反义ODNs对葡萄糖诱导的渗透性没有影响，对TPA诱导的渗透性增加只有轻微的影响。我们得出结论，细胞外葡萄糖的增加导致通过激活PKC的内皮细胞通透性的快速剂量依赖性增加，这种作用是由PKC亚型α介导的。
Endothelial cell permeability is impaired in diabetes mellitus and may be increased by high extracellular glucose concentrations. High glucose activates protein kinase C (PKC), a family of kinases vital to intracellular signaling. We tested the hypothesis that high glucose concentration activates PKC in endothelial cells and leads to an increase in endothelial cell permeability via distinct PKC isoforms, Porcine aortic endothelial cells were used, and the PKC isoforms alpha, delta, epsilon, zeta, and theta were identified in these cells. Glucose caused a rapid dose-dependent increase in endothelial cell permeability, with an EC50 of 17.5 mmol/L. Phorbol 12-myristate 13-acetate (TPA) induced an increase in permeability very similar to that elicited by glucose. The effect of glucose and TPA was totally reversed by preincubating the cells with the PKC inhibitors staurosporine (10(-8) mol/L) and Goe 6976 (10(-8) mol/L). Downregulation of PKC by preincubation with TPA for 24 hours also abolished the effect of glucose and TPA on endothelial cell permeability. High glucose (20 mmol/L) caused an increase in PKC activity at 2, 10, and 30 minutes. Cell fractionation and Western blot analysis showed a glucose-induced translocation of PKC alpha and PKC epsilon. Confocal microscopy confirmed the translocation and showed an association of PKC alpha and PKC epsilon with nuclear structures and the cell membrane. Specific antisense oligodesoxynucleotides (ODNs) against PKC alpha reduced the expression of the isoform, abolished the effects of glucose on endothelial cell permeability completely, and reduced the TPA effect significantly. In contrast, specific antisense ODNs against PKC epsilon had no effect on glucose-induced permeability and only a minor effect on the TPA-induced increase in permeability. We conclude that an increase in extracellular glucose leads to a rapid dose-dependent increase in endothelial cell permeability via the activation of PKC and that this effect is mediated by the PKC isoform alpha.