【摘要】 目的:考察普萘洛尔诱导细胞内磷脂酶D的活化,分析该酶的活化与普萘洛尔刺激引起胞内磷脂酸水平升高的关系,研究蛋白激酶C在此过程中的作用。方法:[~3H]-豆蔻脂酸放射性标记细胞,通过转磷脂酰反应,抽提细胞脂类分子用薄层层析法分析磷脂酶D的产物。用蛋白激酶C的抑制剂来抑制蛋白激酶C的活性或通过佛波酯长时间处理细胞来下调蛋白激酶C的水平。以免疫印记法分析蛋白激酶C的含量。结果:在正丁醇的存在下,普萘洛尔处理A-549细胞引起磷脂酶D的活性快速增高,普萘洛尔诱导磷脂酰丁醇的生成导致磷脂酸生成水平的下降。用蛋白激酶C的抑制剂星形孢菌素(staurosporine),Ro-31-8220及粗糠柴毒素(rottlerin)预处理细胞或通过佛波酯长时间处理下调胞内蛋白激酶C的水平对普萘洛尔诱导的磷脂酶D的活性没有抑制作用,而有增强效应。结论:普萘洛尔引起细胞内磷脂酸水平的提高是由于普萘洛尔对磷脂酶D活性的强烈诱导作用而导致的,蛋白激酶C对该过程可起到负调节作用。更多还原

【Abstract】 AIM: To investigate the propranolol-induced phospholi-pase D (PLD) activity, its contribution to the increase in the level of phosphatidic acid, and the role of protein kinase C (PKC) in this event. METHODS: A combination of [3H]-myristate labeling, transphosphatidyl-ation reaction, lipid extraction, and thin layer chromato-graphy was used to measure the PLD activity. PKC inhibitors and prolonged phorbol-12-myristate-13-acetate (PMA) treatment were used to study the involvement of PKC in propranolol-induced PLD activation. Immuno-blotting was used to detect the intracellular levels of PKC. RESULTS: Treatment of A-549 cells with propranolol in the presence of butanol, resulted in the rapid activation of PLD. Propranolol induced the formation of phosphatidylbutanol ( PBut), a unique product of PLD, at the expense of phosphatidic acid (PA) formation. Pretreatment of cells with PKC inhibitors Ro-31-8220, staurosporine, and rottlerin increased the propranolol-induced PLD. Down-regulation of PKC by prolonged treatment of cells with PMA also potentiated the propranolol-induced PLD activity. CONCLUSION: Propranolol induces rapid activation of PLD activity, which results in the increase in intracellular level of PA. The data also indicate that propranolol-induced PLD activity could be negatively regulated byPKC.更多还原