Nickel W, Helms J B, Kneusel R E, Wieland F T
Institut für Biochemie I, Ruprecht-Karls-Universität Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany.
J Biol Chem. 1996 Jul 5;271(27):15870-3. doi: 10.1074/jbc.271.27.15870.
Forskolin has been shown to prevent the effects brefeldin A (BFA) exerts on many mammalian cells with respect to the disassembly of the Golgi apparatus as well as an increase of sphingomyelin synthesis (Lippincott, S. J., Glickman, J., Donaldson, J. G., Robbins, J., Kreis, T. E., Seamon, K. B., Sheetz, M. P., and Klausner, R. D. (1991) J. Cell Biol. 112, 567-577). It has been speculated that forskolin interferes with the action of BFA by competition for the binding of BFA to its target protein, which is most likely the Golgi-localized nucleotide exchange factor specific for ADP-ribosylation factor 1. Here we show that in vitro forskolin does not prevent inhibition of Golgi-catalyzed nucleotide exchange by BFA. Therefore it appears unlikely that forskolin and BFA bind to the same target protein. Using [3H]BFA we have measured detoxification of BFA by Chinese hamster ovary (CHO) cells. BFA is secreted from CHO cells as cysteine and glutathione conjugates (Brüning, A., Ishikawa, T., Kneusel, R. E., Matern, U., Lottspeich, F., and Wieland, F. T. (1992) J. Biol. Chem. 267, 7726-7732). We present evidence that forskolin treatment of CHO cells results in increased levels of Cys-BFA, the major BFA conjugate secreted by CHO cells, in the medium. Elevated levels of Cys-BFA are also found intracellularly. The effect of forskolin is shown to be independent of its ability to raise the intracellular concentration of cyclic AMP. Therefore, we suggest that the effect of forskolin on BFA-induced disassembly of the Golgi apparatus might be due to an enhanced detoxification of the drug.
已证明福斯高林可防止布雷菲德菌素A（BFA）对许多哺乳动物细胞在高尔基体解体以及鞘磷脂合成增加方面产生的影响（利平科特，S. J.，格利克曼，J.，唐纳森，J. G.，罗宾斯，J.，克雷斯，T. E.，西蒙，K. B.，希茨，M. P.，和克劳斯纳，R. D.（1991年）《细胞生物学杂志》112卷，567 - 577页）。据推测，福斯高林通过竞争BFA与其靶蛋白的结合来干扰BFA的作用，该靶蛋白很可能是高尔基体定位的、对ADP - 核糖基化因子1特异的核苷酸交换因子。在此我们表明，在体外福斯高林并不能防止BFA对高尔基体催化的核苷酸交换的抑制作用。因此，福斯高林和BFA似乎不太可能结合到同一靶蛋白上。我们使用[³H]BFA测定了中国仓鼠卵巢（CHO）细胞对BFA的解毒作用。BFA以半胱氨酸和谷胱甘肽共轭物的形式从CHO细胞中分泌出来（布吕宁，A.，石川，T.，克内塞尔，R. E.，马特恩，U.，洛茨皮希，F.，和维兰德，F. T.（1992年）《生物化学杂志》267卷，7726 - 7732页）。我们提供的证据表明，用福斯高林处理CHO细胞会导致培养基中CHO细胞分泌的主要BFA共轭物半胱氨酸 - BFA（Cys - BFA）水平升高。细胞内也发现Cys - BFA水平升高。已证明福斯高林的作用与其提高细胞内环磷酸腺苷浓度的能力无关。因此，我们认为福斯高林对BFA诱导的高尔基体解体的作用可能是由于该药物解毒作用增强所致。
