Andrews P A, Mann S C, Huynh H H, Albright K D
Cancer Center, University of California, San Diego, La Jolla 92093.
Cancer Res. 1991 Jul 15;51(14):3677-81.
We examined the importance of the Na+, K(+)-ATPase in cisplatin (DDP) accumulation in 2008 human ovarian carcinoma cells and describe changes in the Na+, K(+)-ATPase in DDP-resistant cells with DDP accumulation defects. Approximately 50% of DDP accumulation was inhibitable by ouabain. DDP accumulation into 2008 cells could be maximally inhibited when cells were preincubated with ouabain for 1 h prior to DDP exposure. The half-maximal inhibition was obtained with 0.13 microM ouabain. Similar inhibition of DDP accumulation was obtained when the Na+, K(+)-ATPase was blocked by ATP depletion or by incubating cells in K(+)-free medium. This same percentage of DDP accumulation was Na+ dependent and varied directly with Na+ concentration. These effects on DDP accumulation could be detected as early as 1 min after the imposition of 0-trans conditions, strongly suggesting that the inhibition was due to modulation of a drug influx step. The Na+, K(+)-ATPase in 2008/DDP cells had a similar KD for ouabain binding and 36% less Na+, K(+)-ATPase molecules/mg of protein than 2008 cells. 2008/DDP cells 2.3 +/- 0.2 (SE, n = 3) fold cross-resistant to ouabain in a continuous exposure clonogenic assay. Despite these changes in the Na+, K(+)-ATPase, the net basal Na+, K(+)-ATPase activity was the same in sensitive and DDP-resistant cells as determined by ouabain-inhibitable 86Rb+ influx. The basal Na+ levels were also similar in the sensitive and resistant cells. These data suggest that DDP accumulation is partially Na+ dependent and that, therefore, the Na+, K(+)-ATPase which maintains the Na+ gradient may play an important role in determining how much DDP enters cells. Whether there is a causal link between the changes in the Na+, K+-ATPase in DDP-resistant cells and their DDP accumulation defect is not yet known.
我们研究了钠钾ATP酶在2008人卵巢癌细胞顺铂（DDP）蓄积中的重要性，并描述了具有DDP蓄积缺陷的DDP耐药细胞中钠钾ATP酶的变化。约50%的DDP蓄积可被哇巴因抑制。在DDP暴露前，用哇巴因预孵育细胞1小时，可最大程度抑制2008细胞对DDP的蓄积。用0.13微摩尔哇巴因可获得半数最大抑制。当钠钾ATP酶被ATP耗竭或在无钾培养基中孵育细胞而被阻断时，也可获得类似的对DDP蓄积的抑制。相同比例的DDP蓄积依赖于钠离子，且与钠离子浓度直接相关。早在施加零转运条件后1分钟就能检测到这些对DDP蓄积的影响，这强烈表明抑制是由于药物流入步骤的调节。2008/DDP细胞中钠钾ATP酶对哇巴因结合的解离常数（KD）相似，且每毫克蛋白质中钠钾ATP酶分子比2008细胞少36%。在连续暴露克隆形成试验中，2008/DDP细胞对哇巴因有2.3±0.2（标准误，n = 3）倍的交叉耐药性。尽管钠钾ATP酶有这些变化，但通过哇巴因抑制的86Rb+流入测定，敏感细胞和DDP耐药细胞的基础钠钾ATP酶净活性相同。敏感细胞和耐药细胞的基础钠离子水平也相似。这些数据表明DDP蓄积部分依赖于钠离子，因此，维持钠离子梯度的钠钾ATP酶可能在决定有多少DDP进入细胞方面起重要作用。DDP耐药细胞中钠钾ATP酶的变化与其DDP蓄积缺陷之间是否存在因果联系尚不清楚。
