摘要
目的观察血管紧张素Ⅱ(AngⅡ)刺激心肌细胞后,与心肌细胞增殖密切相关的细胞内信号物质细胞外信号调节激酶(ERK)的变化情况,探讨ERK入核机制。方法以原代培养的心肌细胞为研究对象,实验分6组:AngⅡ组、AngⅡ加缬沙坦组、AngⅡ加AngⅡ2型受体(AT2R)拮抗剂CGP42112A组、AngⅡ加MEK(MAPK激酶)抑制剂PD98059组、AngⅡ加蛋白合成抑制剂亚胺环己酮(CHX)组和AngⅡ加蛋白酶体抑制剂硼替佐米(LLnL)组,每组又分为5～8个不同时相点。以细胞免疫荧光检测磷酸化ERK(pERK)核转位,Westernblot法检测pERK表达情况。结果 AngⅡ可以使pERK表达迅速增加,该作用可被缬沙坦、PD98059所抑制;CHX作用1h左右,可见有pERK入核现象,随作用时间延长,未出现明显pERK聚集胞核中的现象;LLnL作用后显示pERK核聚集现象,与AngⅡ作用相比,LLnL组pERK核聚集明显。结论 ERK主要以活化或磷酸化形式入核,AngⅡ可使ERK发生核聚集,用CHX抑制蛋白质的合成可阻断上述核聚集过程,而用LLnL抑制蛋白的降解则可增强AngⅡ引起的ERK核聚集。
Objective We studied the mechanism of extracelluar signal regulated kinase （ERK） nuclear translocation in the cardiomyocytes with angiotensin Ⅱ（Ang Ⅱ） stimulation.Methods Cardiomyocytes from neonatal rat were cultured and divided into six groups：Ang Ⅱ,Ang Ⅱ plus the valsartan,Ang Ⅱ plus the CGP42112A,Ang Ⅱ plus the PD98059,AngⅡ plus the CHX and Ang Ⅱ plus the LLnL.Each group was examined at different time points and we used immunocytofluorescence to observe the distributions of phosphated ERK and Western blot to semi-quantify the phosphated ERK for activity of ERK.Results ERK was expressed mainly in cytoplasm in unstimulated cardiac cells,but in nuclei when stimulated with Ang Ⅱ.ERK nuclear translocation was decreased by valsartan or PD98059.Gradually increased phosphated ERK（pERK） expression was showed after stimulating with Ang Ⅱ and a marked difference from control group.PD98059 induced an increasingly inhibition of phosphated ERK expression with the addition of CHX,an inhibitor of protein synthesis,or LLnL,an inhibitor of proteasome,there were no marked differences on pERK expression but CHX impeded ERK nuclear accumulation in response to Ang Ⅱ.In contrast,inhibition of targeted proteolysis by LLnL treatment markedly promoted nuclear accumulation of ERK after Ang Ⅱ stimulation.Conclusion ERK nuclear translocation mainly is in the type of phosphated ERK when stimulated with Ang Ⅱ.The nuclear accumulation of ERK decreased by CHX and increased by LLnL.The process may require the neosynthesis of some proteins.
出处
《中华高血压杂志》
CAS
CSCD
北大核心
2010年第10期946-950,共5页
Chinese Journal of Hypertension
基金
国家自然科学基金资助项目(30000196)
关键词
心肌细胞
血管紧张素Ⅱ
细胞外信号调节激酶
核转位
Cardiomyocyte
Angiotensin Ⅱ
Extracelluar signal regulated kinase
Nuclear translocation
