Roepe P D, Weisburg J H, Luz J G, Hoffman M M, Wei L Y
Program in Molecular Pharmacology & Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, New York.
Biochemistry. 1994 Sep 13;33(36):11008-15. doi: 10.1021/bi00202a021.
Previously [Luz et al. (1994) Biochemistry 33, 7239-7249], we determined that Cl(-)- and -HCO3-dependent pHi homeostasis was perturbed in multidrug resistant (MDR) cells created by transfecting LR73 Chinese hamster ovary fibroblasts with wild-type mu (murine) MDR 1 (Gros et al., 1991). Via single-cell photometry experiments performed under various conditions, we are now able to separate Na(+)-dependent and Na(+)-independent components of Cl-/-HCO3 exchange in the MDR transfectants and the parental LR73 cells. Cl(-)-dependent, Na(+)-independent reacidification of pHi, mediated by the anion exchanger 2 isoform in LR73 cells, is dramatically inhibited by mild overexpression of MDR protein. Analysis of H+ flux at different pHi shows that Cl(-)-dependent reacidification approaches 0.2 mM H+/s for LR73 cells at pHi = 8.0 but is at least 10-fold slower for MDR 1 transfectants that were never exposed to chemotherapeutics (EX4N7 cells). MDR 1 transfectants selected on the chemotherapeutic vinblastine (1-1 cells), which express approximately 10-fold more MDR protein relative to EX4N7 cells, exhibit similar behavior; however, alterations in Cl(-)-dependent pHi regulation are more severe. Hypotonic conditions, which have been shown to increase anomalous Cl- conductance in some cells overexpressing MDR protein (Valverde et al., 1992), are found to amplify the altered pHi homeostasis features in the primary transfectants that express lower levels of MDR protein such that they then mimic the behavior of the drug-selected cells that express substantially more MDR protein. Verapamil reverses the anomalous behavior.(ABSTRACT TRUNCATED AT 250 WORDS)
此前[卢兹等人（1994年），《生物化学》第33卷，7239 - 7249页]，我们确定，在用野生型μ（鼠）多药耐药1（MDR 1）（格罗斯等人，1991年）转染LR73中国仓鼠卵巢成纤维细胞所产生的多药耐药（MDR）细胞中，依赖Cl⁻和HCO₃⁻的细胞内pH稳态受到干扰。通过在各种条件下进行的单细胞光度测定实验，我们现在能够区分MDR转染细胞和亲本LR73细胞中Cl⁻/HCO₃交换的依赖Na⁺和不依赖Na⁺的成分。LR73细胞中由阴离子交换蛋白2亚型介导的依赖Cl⁻、不依赖Na⁺的细胞内pH再酸化，会因MDR蛋白的轻度过表达而受到显著抑制。对不同细胞内pH值下H⁺通量的分析表明，对于细胞内pH值为8.0的LR73细胞，依赖Cl⁻的再酸化接近0.2 mM H⁺/秒，但对于从未接触过化疗药物的MDR 1转染细胞（EX4N7细胞），其速度至少慢10倍。在化疗药物长春碱（1 - 1细胞）上筛选出的MDR 1转染细胞，其表达的MDR蛋白相对于EX4N7细胞约多10倍，表现出类似的行为；然而，依赖Cl⁻的细胞内pH调节的改变更为严重。低渗条件已被证明会增加一些过表达MDR蛋白的细胞中的异常Cl⁻电导（瓦尔韦德等人，1992年），结果发现它会放大表达较低水平MDR蛋白的原代转染细胞中改变的细胞内pH稳态特征，使其随后模仿表达大量更多MDR蛋白的药物筛选细胞的行为。维拉帕米可逆转这种异常行为。（摘要截短于250字）
