Pyne S, Pyne N J
Department of Physiology and Pharmacology, University of Strathclyde, Royal College, Glasgow, U.K.
Biochem Pharmacol. 1993 Feb 9;45(3):593-603. doi: 10.1016/0006-2952(93)90132-g.
Conditions were established for the primary culture of guinea-pig tracheal smooth muscle cells, the identity of which was confirmed by the presence of smooth muscle alpha-actin by western blotting. Cells were preincubated with [3H]palmitate which was incorporated, almost exclusively, into phosphatidylcholine. When these cells were stimulated by either bradykinin or phorbol 12-myristate 13-acetate (PMA), in the presence of butan-1-ol, the non-metabolizable product [3H]phosphatidylbutanol ([3H]PtdBut) accumulated by virtue of the phosphatidyltransferase activity of phospholipase D. The activation of phospholipase D by bradykinin was inhibited by 86 +/- 11% (N = 3 experiments) in the presence of the protein kinase C inhibitor, staurosporine (1 microM) and by 88 +/- 11% (N = 3 experiments) in cells that had been chronically treated with PMA to down-regulate their protein kinase C. PMA-stimulated phospholipase D was similarly affected (92 +/- 2% inhibited by staurosporine, 87 +/- 6% inhibited by protein kinase C down-regulation). Removal of extracellular Ca2+ markedly reduced the bradykinin-stimulated phospholipase D response (by 73 +/- 10%, N = 3 experiments) but had only a limited effect upon PMA-stimulated phospholipase D activity (by 23 +/- 6%, N = 3 experiments). AIF4-stimulation of the cells also resulted in the activation of phospholipase D, indicating the involvement of a G-protein. However, this was not Gi since pertussis-toxin pretreatment of the cells failed to abolish either bradykinin-stimulated inositol (1,4,5)trisphosphate formation or [3H]PtdBut accumulation. Western blotting revealed the presence of Gq/G11 which couples to the inositol lipid-directed phospholipase C. Indomethacin (10 microM) was without effect upon bradykinin-stimulated phospholipase D activity, suggesting that the bradykinin effects were not mediated indirectly by cyclooxygenase products. The role of phospholipase D activation in tracheal smooth muscle may be to, indirectly, produce diacylglycerol for the activation of protein kinase C which has been implicated in sustained contraction. However, the immediate product of phospholipase D, phosphatidate, has been proposed to have a number of second messenger roles and may itself, by an undefined mechanism, be involved in the sustained contraction of airway smooth muscle.
建立了豚鼠气管平滑肌细胞原代培养的条件，通过蛋白质免疫印迹法检测到平滑肌α -肌动蛋白，从而确认了细胞的身份。细胞先用[3H]棕榈酸进行预孵育，该物质几乎完全掺入磷脂酰胆碱中。当这些细胞在丁醇存在的情况下受到缓激肽或佛波酯12 -肉豆蔻酸13 -乙酸酯（PMA）刺激时，由于磷脂酶D的磷脂转移酶活性，不可代谢产物[3H]磷脂酰丁醇（[3H]PtdBut）会积累。在蛋白激酶C抑制剂星形孢菌素（1μM）存在的情况下，缓激肽对磷脂酶D的激活被抑制了86±11%（N = 3次实验），在长期用PMA处理以下调其蛋白激酶C的细胞中，抑制率为88±11%（N = 3次实验）。PMA刺激的磷脂酶D也受到类似影响（星形孢菌素抑制92±2%，蛋白激酶C下调抑制87±6%）。去除细胞外Ca2 +显著降低了缓激肽刺激的磷脂酶D反应（降低73±10%，N = 3次实验），但对PMA刺激的磷脂酶D活性影响有限（降低23±6%，N = 3次实验）。AIF4刺激细胞也导致磷脂酶D的激活，表明有G蛋白参与。然而，这不是Gi，因为用百日咳毒素预处理细胞未能消除缓激肽刺激的肌醇（1,4,5）三磷酸形成或[3H]PtdBut积累。蛋白质免疫印迹法显示存在与肌醇脂质定向磷脂酶C偶联的Gq/G11。吲哚美辛（10μM）对缓激肽刺激的磷脂酶D活性没有影响，这表明缓激肽的作用不是由环氧化酶产物间接介导的。磷脂酶D激活在气管平滑肌中的作用可能是间接产生二酰基甘油以激活蛋白激酶C，而蛋白激酶C与持续收缩有关。然而，磷脂酶D的直接产物磷脂酸已被提出具有多种第二信使作用，并且其本身可能通过一种未明确的机制参与气道平滑肌的持续收缩。
