尼古丁对脐静脉内皮细胞蛋白激酶C和Ras蛋白活性及胞浆钙浓度的影响

Effects of nicotine on cytoplasma calcium level and activities of PKC and Ras protein in HUVEC

为研究尼古丁对血管内皮细胞胞内游离钙、蛋白激酶C(PKC)和Ras蛋白的作用 ,用激光共聚焦显微镜观察细胞内游离钙离子浓度的变化 ,用γ 32 P ATP掺入法检测PKC活性 ,用GST RBD沉淀并纯化活化的Ras蛋白和Western印迹杂交法检测Ras蛋白含量。观察到 (1～ 5 ) 10 - 4 mol L尼古丁使人脐静脉内皮细胞 [Ca2 + ]i 迅速增加 ,峰值约超过基础值 5 0 %～ 2 0 0 % ,钙通道阻滞剂尼莫地平可拮抗尼古丁的此种效应 ;10 - 4 mol L尼古丁作用 1～ 12h后胞浆PKC活性明显降低 ;而胞膜PKC活性则升高。 10 - 4 ～ 10 - 3 mol L尼古丁作用于人脐静脉内皮细胞 10～ 30min可使活化的Ras蛋白含量增加。以上结果提示尼古丁可使人脐静脉内皮细胞 [Ca2 + ]i

To study the effects of nicotine on intracellular free calcium concentration ([Ca 2+ ] i) and Protein Kinase C (PKC)and Ras protein in ECs.[Ca 2+ ] i in single HUVEC was observed with Laser Confocal Microscope. The PKC activity was examined by the method of γ 32 P ATP incorporation assay.The activated Ras was detected with Activated Ras interaction Assay(ARIA), a new method in which GST RBD was used as an affinity reagent to precipitate Ras GTP from cell and affinity precipitated Ras was detected by Western blotting with anti Ras antibodies. It was observed that after treatment by 10 -4 ～5×10 -4 mol/L of nicotine [Ca 2+ ] i in single HUVEC increased by 50%～200% over base line value, which could be inhibited by Nimodipine. The PKC activity was induced by 10 -4 mol/L of nicotine from 1～12h; Ras protein activity was induced by 10 -4 ～10 -3 mol/L of nicotine from 10～30min.Nicotine was suggested to increase [Ca 2+ ] i,activate PKC and Ras protein in ECs.