香烟提取物活化大鼠支气管平滑肌细胞蛋白激酶C亚型及下调钾通道BK_(Ca)、Kv1.5表达
叶红, 马万里, 金肆, 刘声远, 王迪浔, 胡清华
华中科技大学同济医学院病理生理学系,卫生部呼吸系疾病重点实验室.湖北,武汉 430030;华中科技大学同济医学院附属协和医院呼吸内科.湖北,武汉 430030
摘要
大电导的钙活化钾通道(large--conductance calcium--activated potassium channel,BK_(Ca))和电压依赖性钾通道Kv1.5在气道高反应性的发生机制中具有重要作用。已知吸烟可致气道高反应,但钾通道的变化在其发病中的作用尚需进一步阐明。该文旨在研究香烟提取物(cigarette smoke extract,CSE)对培养的大鼠支气管平滑肌细胞(bronchial smooth muscle cells,BSMCs)钾通道BK_(Ca)和Kv1.5表达的直接作用,以及蛋白激酶C(protein kinase C,PKC)在其中的作用。实验采用原代培养大鼠BSMCs,用5%CSE刺激,免疫印迹检测PKC亚型的表达和转位,半定量RT--PCR、免疫印迹实验检测BK_(Ca)和Kv1.5的mRNA和蛋白表达,然后用PKC抑制剂BIM和Göe6983与CSE共作用,检测其对BKCa和Kv1.5的mRNA和蛋白表达的影响。结果显示,5%CSE使PKC#epsilon#、#eta#、#thet#发生明显的膜转位,并使BK_(Ca)和Kv1.5的蛋白和mRNA表达明显降低;选择性PKC抑制剂BIM或Göe6983与CSE共同作用,均可使BK_(Ca)和Kv1.5的蛋白和mRNA表达部分恢复。上述结果提示,CSE可引起BSMCs的BKCa和Kv1.5表达下调,PKC#epsilon#、#eta#、#thet#参与其信号转导。
关键词: 支气管; 平滑肌; 香烟提取物; 钾通道; 蛋白激酶C
Cigarette smoke extract activates PKC isoforms and down--regulates the expressions of potassium channels BK_(Ca) and Kv1.5 in rat bronchial smooth muscle cells
Ye Hong, Ma Wanli, Jin Si, Liu Shengyuan, Wang Dixun, Hu Qinghua
Department of Pathophysiology,Key Laboratory of Pulmonary Diseases,Ministry of Health of China,Tongji Medical College,Huazhong University of Science and Technology.Wuhan 430030,Hubei;China
Abstract
Large-conductance calcium-activated potassium channel(BK_(Ca)) and voltage-gated potassium channel Kv1.5 play an impor-tant role in the pathogenesis of bronchial hyperresponsiveness(BHR).It is known that cigarette smoke can induce BHR,however,the role of BK_(Ca) and Kv1.5 expression in it remains to be further elucidated.The purpose of the present study was to investigate the direct effects of cigarette smoke extract(CSE) on BK_(Ca) and Kv1.5 expression,and the role of protein kinase C(PKC) isoforms activation in primary cultured rat bronchial smooth muscle cells(BSMCs).Primarily cultured rat BSMCs were treated with 5% CSE,the expression and translocation of PKC isoforms were measured by Western blot,and the mRNA and protein levels of BK_(Ca) and Kv1.5 #alpha#-subunits were determined by semi-quantitative RT-PCR and Western blot,respectively.The results showed that 5% CSE induced the translo-cation of PKC#epsilon#,PKC#eta#,PKC#thet# from soluble fraction to particulate fraction,and reduced mRNA and protein expressions of BK_(Ca) and Kv1.5 #alpha#-subunits.The decreased expression of potassium channels was partly restored by PKC inhibitor,BIM or Göe6983.In summary,CSE may activate PKC isoforms #epsilon#,#eta#,#thet#,thereby down-regulate the expressions of BK_(Ca) and Kv1.5 in BSMCs.
Key words: bronchus;myocyte;cigarette smoke extract;Potassium channel;Protein kinase C
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引用本文:
叶红, 马万里, 金肆, 刘声远, 王迪浔, 胡清华. 香烟提取物活化大鼠支气管平滑肌细胞蛋白激酶C亚型及下调钾通道BK_(Ca)、Kv1.5表达[J]. 生理学报 2008; 60 (6): 709-714.
Ye Hong, Ma Wanli, Jin Si, Liu Shengyuan, Wang Dixun, Hu Qinghua. Cigarette smoke extract activates PKC isoforms and down--regulates the expressions of potassium channels BK_(Ca) and Kv1.5 in rat bronchial smooth muscle cells. Acta Physiol Sin 2008; 60 (6): 709-714 (in Chinese with English abstract).