Amadori糖化白蛋白对牛视网膜血管二酯酰甘油——蛋白激酶C级联的影响

The effect of Amadori glycated serum albumin on diacylglycerol-protein kinase C cascade in retinal blood vessels of bovine

目的 :探讨Amadori糖化血清白蛋白对牛视网膜血管二酯酰甘油 (DAG)———蛋白激酶C(PKC)信号级联的影响及d-α-生育酚的干预作用。方法 :体外制备的Amadori糖化的牛血清白蛋白 (AGSA)分别在生理浓度葡萄糖 (5 .5mmol/L)和高浓度葡萄糖 (2 0mmol/L)培养液中作用于新鲜牛视网膜血管。采用薄层层析和放射自显影法测定DAG含量和PKC活性 ;并检测d -α-生育酚预处理后DAG、PKC改变。结果 :在生理葡萄糖浓度下 ,牛视网膜血管暴露于AGSA2 4h或 72h后细胞内DAG含量、PKC活性均较对照组显著增加 ,当AGSA和高葡萄糖同时作用于牛视网膜血管时 ,DAG -PKC级联显著激活 ,分别为正常葡萄糖组的 3.4 7倍和 4 .5 4倍 ;在正常血清培养液中 ,与 5 .5mmol/L的葡萄糖相比 ,2 0mmol/L高糖在 2 4h时并不刺激DAG含量增加 (P >0 .0 5 ) ,而 72h时则较对照组增加 4 5 % ,PKC活性较对照增加 5 6 % ,而d -α -生育酚可逆转上述生化改变。结论 :Amadori糖化血清白蛋白可在生理葡萄糖浓度下刺激DAG -PKC途径活化 ,从而影响血管一系列生理功能 ,而d -α-生育酚对血管生化改变有保护作用。

Objective:To investigate the effect of Amadori glycated serum albumin (AGSA) on diacylglycerol-protein kinase C (PKC) cascade in cultured retinal blood vessels of bovine. Methods: Glycated serum albumin was prepared by incubating sterile-filtered normal serum albumin for five days at 37℃ with 25mmol/L glucose in phosphated buffered saline(PBS).Albumin incubated for the same period in PBS without glucose was used as control(normal albumin,NA).Diacyglycerol level and PKC activity were determined in blood vessels incubated in media containing 10% nonglycated serum albumin (NSA) or AGSA,ant 5.5mmol/L glucose versus 20mmol/L glucose for 24 or 72 hours with or without 50 mmol/L d-α-tocopherol. Results: In blood vessels cultured for 24 hours in 5.5 mmol/L glucose,the relative ratio for diacyglycerol level in vessels supplemented with AGSA compared with the level exposed to NSA was l.76,and PKC activity in vessels supplemented with AGSA was also higher than the level exposed to NSA.The relative ratio for diacylglycerol level and PKC activity in vessels cultured in 20mmol/L glucose for 72 hours compared with that in vessels cultured in 5.5mmol/L glucose was 2.44,and 2.62 respectively.20mmol/L glucose also augmented the AGSA induced increase in diacylglycerol level and PKC activity relative to that observed in 5.5mmol/L glucose concentration.(Relative ratio of diacylglycerol level and PKC activity in cell cultured for 72 hours in 20mmol/L glucose supplemented with AGSA versus 5.5mmol/L glucose was 3.47,4.54 respectively).D-α-tocopherol can prevent activation of diacyglycerol-PKC cascade induced by high glucose and/or AGSA. Conclusion: AGSA activate diacyglycerol-PKC pathway by mechanisms that are operative under normoglycemic condition,which may contribute to the development of diabetic retinopathy.