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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 investigate the regulation of intracellular calcium concentration ([Ca2 +] i) by protein kinase A (PKA) -activated phosphosphosphorylation site 16-serine (PLB-Ser16) during hypoxia preconditioning. Methods The neonatal rat cardiomyocytes cultured in vitro were randomly divided into four groups: normal group (C), hypoxia-reoxygenation injury (HR) group, hypoxic preconditioning group (HP) and H-89 intervention group . Lactate dehydrogenase (LDH), [Ca2 +] i and apoptosis rate were detected in each group. PLB protein expression and phosphorylation levels were determined by Western blotting and autoradiography. Results The LDH, [Ca2 +] i and apoptosis index in HR group were significantly higher than those in C group and HP group (P <0.05). The protein expression and phosphorylation of PLB in HR group were lower than those in C group (P <0.05) (P <0.05). The LDH value, intracellular calcium fluorescence intensity and apoptosis index in H-89 group were significantly higher than those in HP group (P <0.05), while those in PLB protein The expression and phosphorylation levels were significantly down-regulated (P <0.05). Conclusion The protective effects of hypoxic preconditioning on cardiomyocyte hypoxia-reoxygenation injury may be related to the upregulation of PLB phosphorylation; PKA inhibitor H-89 may reduce this cardioprotection.