缺氧后适应中蛋白激酶C_ε与钙敏感受体相互作用对乳鼠心肌细胞的保护作用

Interaction of PKC_ε with the CaR in hypoxic post-conditioning protects the cardiomyocytes in neonatal rat

目的:研究缺氧后适应中蛋白激酶Cε(PKCε)与钙敏感受体(CaR)相互作用对乳鼠心肌细胞的保护作用。方法:Wistar乳鼠(出生后3-7d)心肌细胞培养3-5d,随机分为7组:正常对照组(N组),缺氧/复氧组(H/Re组),缺氧后适应组(HPC组),GdCl3组(GdCl3,NiCl2,CdCl2组),caffeine组(caffeine,GdCl3,NiCl2,CdCl2组),PKCε抑制剂组(PKCI组)和PKCI+GdCl3组(PKCI,GdCl3,NiCl2,CdCl2组)。采用饱和氮气pH6.8的D-Hanks液和含20%新生牛血清的DMEM液复制心肌缺氧/复氧模型。检测各组细胞存活率及各组乳酸脱氢酶(LDH)活力和丙二醛(MDA)含量变化,Westernblotting检测心肌细胞膜CaR和PKCε蛋白表达水平,免疫共沉淀检测细胞膜上CaR和PKCε的相互作用,激光扫描共聚焦显微镜(LSCM)测定心肌细胞内游离钙([Ca2+]i)的变化,TUNEL染色观察细胞凋亡。结果:H/Re、GdCl3和PKCI组的细胞存活率低于对照组和HPC组;反之,LDH活力和MDA含量高于对照组和HPC组。同时,在GdCl3、caffeine和PKCI+GdCl3组CaR的蛋白表达水平较HPC和PKCI组增高,且[Ca2+]i和细胞凋亡率也高于后2组;而PKCI和PKCI+GdCl3组PKCε蛋白表达水平低于H/Re、HPC、GdCl3和caffeine组。免疫共沉淀检测到CaR和PKCε在细胞膜上发生相互作用。结论:在乳鼠心肌细胞缺氧后适应中,PKCε已转位到细胞膜和CaR相互作用,此相互作用能减少[Ca2+]i,对缺氧的乳鼠心肌细胞在进行复氧时产生保护作用。

AIM： To study the interaction of PKCε with the CaR in hypoxic post - conditioning for protecting the cardiomyocyte of neonatal rat. METHODS ： The ventricular cardiomyocytes of Wistar neonatal rat （3 - 7 d after birth） were incubated for about 3 - 5 d, then randomly divided them into 7 groups ： （ 1 ） Sham control group; （2） Hypoxic/re - oxygenation group （ H/Re group） ; （ 3 ） Hypoxic post - conditioning group （ HPC group） ; （4） HPC ＋ GdCl3, NiCl2, CdCl2 group; （5） HPC ＋ caffeine, GdCl3, NiCl2, CdCl2 group; （6） HPC ＋ PKCε inhibitor group （PKCI group） ; （7） HPC ＋ PKCI ＋ GdCl3, NiCl2, CdCl2 group. The neonatal cells were incubated in the D - Hanks solution （ pH = 6. 8） which was saturated with N2 gas for 1 h at least and then re - incubated in the DMEM solution containing 20% new - born calf serum to establish a model of H/Re. The viability of cardiomyocytes was assayed by MTT, the activity of LDH and the content of MDA were determined, the expression of CaR and PKCε of the membrane in each group was analyzed by Western blotting, PKCε interaction with CaR in the membrane was detected by immunoprecipitation, and the concentration of intra- cellular calcium （ [ Ca^2＋] i ） was measured by laser confocal scanning microscope （ LCSM）, apoptotic cells were measured by TUNEL assay. RESULTS ： The viability of cardiomyocytes in H/Re, GdCl3 and PKCI groups was lower than that in N and HPC groups, while the activity of LDH and the content of MDA were significantly higher than those in N and HPC groups. Meanwhile, the quantitative expression of CaR in GdCl3 , caffeine and PKCI ＋ GdCl3 groups was higher than that in HPC and PKCI groups, and so were the [ Ca^2＋] i and the apoptosis index. The quantitative expression of PKCε in PKCI and PKCI ＋ GdCl3 groups was lower than that in H/Re, HPC, GdCl3 and caffeine groups. Immunoprecipitation of cell membrane PKCε revealed the interaction of PKCε with CaR. CONCLUSION： In the cardiomyocytes of HPC, PKC, translocates to the membrane and interacts with CaR to reduce [ Ca^2＋] i, which protects the cardiomyocytes of neonatal rat during hypoxic/oxygenation.