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。
Objective To explore the effect of Ser^16 phosphorylation of phospholamban（PLB- Ser^16） 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 （hypoxiareoxygenation） and H-89 （treated with H-89）. The LDH of celluar supernatant was detected by automatic biochemistry analyzer. The fluorescence intensity of intracellular Ca^2＋ 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 Ca^2＋ 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 hypoxiareoxygenation injury may be related with upregulating PLB phosphorylation, which can be reduced by PKA inhibitor H-89.
出处
《江苏医药》
CAS
CSCD
北大核心
2012年第8期881-884,共4页
Jiangsu Medical Journal
基金
江苏省135工程医学重点人才课题资助基金(RC2002016)
