【摘要】 目的探讨由蛋白激酶A(PKA)介导的受磷蛋白磷酸化位点16-丝氨酸(PLB-Ser16)在缺氧预适应中对心肌细胞内钙离子浓度([Ca2+]i)的调控作用。方法体外培养的乳鼠心肌细胞随机分成四组:正常培养(C)组,缺氧-复氧损伤(H-R)组、缺氧预适应(HP)组和H-89干预(H-89)组。检测各组细胞上清液乳酸脱氢酶(LDH)、[Ca2+]i和细胞凋亡率。用免疫印迹法和放射自显影法测定PLB的蛋白表达和磷酸化水平。结果 H-R组的LDH值、[Ca2+]i及细胞凋亡指数均显著高于C组和HP组(P<0.05);H-R组PLB的蛋白表达和磷酸化水平较C组均降低(P<0.05),HP组较C组增加(P<0.05);H-89组的LDH值、胞内钙荧光强度值及细胞凋亡指数均较HP组明显升高(P<0.05),而PLB的蛋白表达和磷酸化水平显著下调(P<0.05)。结论缺氧预适应对心肌细胞缺氧-复氧损伤的保护作用可能与PLB磷酸化水平上调有关;PKA抑制剂H-89可降低这种心肌保护作用。更多还原

【Abstract】 Objective To explore the effect of Ser16 phosphorylation of phospholamban(PLB-Ser16) on intracellular calcium in hypoxia preconditioning in cardiomyocytes treated with protein kinase A(PKA) inhibitor H-89.Methods The neonatal rat myocardiocytes cultured in vetro were randomly divided into 4 groups of C(normal control),HP(hypoxia preconditioning),HR(hypoxia-reoxygenation) and H-89(treated with H-89).The LDH of celluar supernatant was detected by automatic biochemistry analyzer.The fluorescence intensity of intracellular Ca2+ and apoptosis rate were detected with Fluo-3/AM and Annexin-V FITC loading by flow cytometer.The level of PLB protein expression and phosphorylation were measured by Western blot and radioautograph technique.Results LDH,fluorescence intensity of intracellular Ca2+ and apoptosis index were significantly higher in group HR than those in groups of C and HP(P<0.01).The levels of PLB protein expression and phosphorylation were lower in group H-R than those in group C,which were higher in group HP than those in group C(P<0.01).Compared with group HP,the values of 3 parameteres in group H-89 were all higher,but PLB protein expression and phosphorylation were down-regulated(P<0.01).Conclusion The cardioprotective effect of hypoxia preconditioning against hypoxia-reoxygenation injury may be related with upregulating PLB phosphorylation,which can be reduced by PKA inhibitor H-89.更多还原