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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。
OBJECTIVE: To investigate the effect of propranolol on the phospholipase D activation induced by propranolol, and to investigate the relationship between the activation of phospholipase D and the increase of intracellular phosphatidic acid level induced by propranolol stimulation and the role of protein kinase C in this process. Methods: Radiolabelled cells were labeled with [~ 3H] -myristoleic acid and the phospholipase D product was analyzed by thin-layer chromatography by transphosphatidic reaction. Inhibition of protein kinase C activity with inhibitors of protein kinase C or downregulation of protein kinase C levels by phorbol ester prolonged treatment of cells. Protein kinase C levels were analyzed by immunoblotting. Results: In the presence of n-butanol, propranolol treatment of A-549 cells caused a rapid increase in phospholipase D activity, and propranolol-induced phosphatidyl-butanol production resulted in a decrease in phosphatidic acid production. Pretreatment of cells with inhibitors of protein kinase C, staurosporine, Ro-31-8220, and rottlerin, or downregulation of intracellular protein kinase C levels by phorbol ester over time Propranolol-induced phospholipase D activity has no inhibitory effect but a potentiating effect. CONCLUSIONS: Propranolol causes an increase in intracellular phosphatidic acid levels due to the strong induction of propranolol for phospholipase D activity, which may play a negative regulatory role in this process.