葡萄糖对内皮细胞蛋白激酶C活性、通透性及粘附性的影响

Effect of glucose level on protein kinase C,permeability and adhesion of endothelial cells

目的 研究不同浓度葡萄糖对血管内皮细胞的蛋白激酶C(PKC)活性、通透性及粘附性的影响。方法 用[γ32 P]ATP参入外源性底物的方法测定人脐静脉内皮细胞(HUVECs )PKC活性;用51 Cr标记的静止血小板与HUVECs保温,测定内皮细胞的粘附性;用HUVECs对白蛋白的体外通透性实验测定内皮细胞的通透性。结果 2 0mmol L的高浓度葡萄糖可最大程度活化HUVECs的PKC ,膜PKC活性为( 2 874±0 0 4 6 )mmol·mg- 1 ·min- 1 ,明显高于浆PKC活性[( 1 2 15±0 0 19)mmol·mg- 1 ·min- 1 ,P <0 0 1];HUVECs在2 0mmol L高浓度葡萄糖作用下的最大粘附率为( 6 2 84±3 2 4 ) % ,对白蛋白的最大通透率为( 0 32±0 0 0 2 )ng ml,明显高于对照组(P <0 0 1) ;HUVECs的PKC总活性与HUVECs的通透性及粘附率的相关系数r分别为0 84 6、0 734(P均<0 0 5 )。PKC抑制剂CalphostinC能抑制高浓度( 2 0mmol L)葡萄糖对HUVECs的PKC活化,并使HUVECs的通透性及粘附率明显下降。结论 高浓度葡萄糖( 2 0mmol L)可活化血管内皮细胞的PKC ,进而增加其通透性及粘附性;

Objective To study the effect of various glucose level on protein kinase C (PKC), permeability and adhesion of vascular endothelial cells. Methods The activity of PKC was determined by the incorporation of [γ 32 P] adenosine triphosphate (ATP) into exogenous substrate.The adhesion of vascular endothelial cells was measured by incubating human umbilical vein endothelial cells(HUVECs)with unactivated platelets labeled with 51 Cr.For the detection of endothelial permeability,albumin diffusion across HUVECs monolayers was measured. Results 20 mmol/L high concentration glucose (HCGS) could result in the largest activating level of PKC in HUVECs. Membranous PKC activity [(2.874±0.046) mmol·mg -1 ·min -1 ] was much higher than cytosol PKC activity [(1.215±0.019) mmol·mg -1 ·min -1 , P <0.01]. The largest rate of adhesion in HUVECs treated by 20 mmol/L high glucose level was (62.84±3.24)%, and the largest permeability rate of albumin across HUVECs was (0.32±0.002) ng/ml, which were all significantly higher than those of the control group ( P <0.01). The related coefficient between the total PKC activity and HUVECs adhesion rate was r =0.846, and the related coefficient between the total PKC activity and HUVECs permeability rate was r =0.734(all P <0.05). The PKC inhibitor, Calphostin C could inhibit PKC activation induced by HCGS and also markedly reduced the adhesion rate and permeability of HUVECs. Conclusion HCGS level may activate PKC, and increase the adhesion and permeability of vascular endothelial cells; while PKC inhibitor can markedly suppress the above effects.