Portilla D, Morrissey J, Morrison A R
Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
J Clin Invest. 1988 Jun;81(6):1896-902. doi: 10.1172/JCI113536.
Previous studies have demonstrated that bradykinin stimulates the rapid release of inositol 1,4,5 trisphosphate (IP3) from membrane phosphatidylinositol 4,5 bisphosphate (PIP2) in Madin-Darby canine kidney (MDCK) cells. Since current evidence would suggest that the activation of phospholipase C (PLC) is mediated through a guanine nucleotide-binding protein in receptor-mediated activation of PLC, we evaluated the role of guanine nucleotide proteins in receptor-mediated (bradykinin-stimulated) activation of PLC in MDCK cells. Bradykinin at 10(-7) M produced a marked increase in IP3 formation within 10 s increasing from a basal level of 46.2 to 686.6 pmol/mg cell protein a 15-fold increase. Pretreatment of MDCK cells in culture with 200 ng/ml of pertussis toxin for 4 h reduced the bradykinin-stimulated response to 205.8 pmol/mg protein. A 41-kD protein substrate in MDCK membranes was ADP ribosylated in vitro in the presence of pertussis toxin. The ADP ribosylation in vitro was inhibited by pretreatment of the cells in culture with pertussis toxin. Membranes from MDCK cells incubated in the presence of [3H]PIP2/phosphatidyl ethanolamine liposomes demonstrated hydrolysis of [3H]PIP2 with release of [3H]IP3 when GTP 100 microM or GTP gamma S 10 microM was added. Bradykinin 10(-7) M added with GTP 100 microM markedly increased the rate of hydrolysis within 10 s, thus demonstrating a similar time course of PLC activation as intact cells. These results demonstrate that bradykinin binds to its receptor and activates a membrane-associated PLC through a pertussis toxin-sensitive, guanine nucleotide protein.
先前的研究表明，缓激肽可刺激麦迪逊-达比犬肾（MDCK）细胞中的膜磷脂酰肌醇4,5-二磷酸（PIP2）快速释放肌醇1,4,5-三磷酸（IP3）。由于目前的证据表明磷脂酶C（PLC）的激活是通过鸟嘌呤核苷酸结合蛋白介导的，在受体介导的PLC激活过程中，我们评估了鸟嘌呤核苷酸蛋白在MDCK细胞中受体介导的（缓激肽刺激的）PLC激活中的作用。10^(-7) M的缓激肽在10秒内使IP3形成显著增加，从基础水平的46.2皮摩尔/毫克细胞蛋白增加到686.6皮摩尔/毫克细胞蛋白，增加了15倍。用200纳克/毫升百日咳毒素对培养的MDCK细胞预处理4小时，可将缓激肽刺激的反应降低至205.8皮摩尔/毫克蛋白。在百日咳毒素存在的情况下，MDCK细胞膜中的一种41-kD蛋白底物在体外被ADP核糖基化。用百日咳毒素对培养的细胞进行预处理可抑制体外ADP核糖基化。当加入100微摩尔/升GTP或10微摩尔/升/升GTPγS时，在[3H]PIP2/磷脂酰乙醇胺脂质体存在下孵育的MDCK细胞膜显示[3H]PIP2水解并释放[3H]IP3。加入10^(-7) M缓激肽和100微摩尔/升GTP可在10秒内显著增加水解速率，从而证明PLC激活的时间进程与完整细胞相似。这些结果表明，缓激肽与其受体结合，并通过一种对百日咳毒素敏感的鸟嘌呤核苷酸蛋白激活膜相关的PLC。
