Luz J G, Wei L Y, Basu S, Roepe P D
Program in Molecular Pharmacology & Therapeutics, Memorial Sloan-Kettering Cancer Center, Cornell University, New York, New York 10021.
Biochemistry. 1994 Jun 14;33(23):7239-49. doi: 10.1021/bi00189a028.
We have used single-cell photometry to measure intracellular pH (pHi) for several MDR cell lines constructed by stably transfecting LR73 chinese hamster ovary fibroblasts with mutant and wild type murine MDR 1 genes. In addition, plasma membrane electrical potential (delta psi) has been measured for the same cells by the K+/valinomycin null point titration method using the ratiometric styryl probe di-4-ANEPPS. Both the untransfected, parental cell line and a cell line expressing substantial mutant MDR 1 protein (K432R/K1074R) that is unable to confer the MDR phenotype are found to have delta psi > or = -40 (+/- 5) mV and pHi < or = 7.16 (+/- 0.03) units. In contrast, MDR cell lines constructed by transfecting wild type mu MDR 1 cDNA are found to exhibit delta psi from 15 to 19 mV lower and pHi from 0.13 to 0.34 units higher. A cell line that overexpresses crippled MDR protein (S941F) that is not resistant to colchicine or doxorubicin, but which is resistant to vinblastine [Gros, P., Dhir, R., Croop, J., & Talbot, F. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 7289-7293], exhibits elevated pHi and slightly elevated delta psi, relative to LR73. Northern and western blot analyses confirm the substantial overexpression of the mu MDR genes and proteins in these lines, as well as the mild overexpression of endogenous hamster p-GP mRNA in some lines. In general agreement with previous studies that examined myeloma cells overexpressing hu MDR 1 protein [Roepe, P.D., Wei, L.-Y., Cruz, J., & Carlson, D. (1993) Biochemistry 32, 11042-11056] we find that overexpression of wild type mu MDR 1 protein inhibits Cl(-)- and -HCO3-dependent pHi homeostasis. Via single-cell photometry studies we now conclude that this is due to inhibition of Na(+)-independent Cl-/-HCO3 exchange (strict anion exchange or AE). As concluded previously for other MDR cells, decreased AE activity is not due to decreased expression of the exchanger; in fact, again similar to previous work [Roepe et al. (1993) Biochemistry 32, 11042-11056], we find increased levels of AE mRNA in some MDR cell lines. Models that may explain these data that are also consistent with the known physiology of cells that endogenously express MDR protein are suggested. These data are consistent with a model for MDR protein function wherein overexpression of the protein decreases delta psi and/or elevates pHi via Cl(-)- and -HCO3-dependent mechanisms.
我们利用单细胞光度法测量了几种多药耐药（MDR）细胞系的细胞内pH值（pHi），这些细胞系是通过用突变型和野生型鼠MDR 1基因稳定转染LR73中国仓鼠卵巢成纤维细胞构建而成的。此外，使用比率型苯乙烯基探针二-4-ANEPPS，通过K⁺/缬氨霉素零电位滴定法测量了相同细胞的质膜电位（δψ）。未转染的亲本细胞系以及表达大量无法赋予MDR表型的突变型MDR 1蛋白（K432R/K1074R）的细胞系，均被发现其δψ≥ -40（±5）mV且pHi≤7.16（±0.03）单位。相比之下，通过转染野生型μMDR 1 cDNA构建的MDR细胞系，其δψ降低了15至19 mV，pHi升高了0.13至0.34单位。一个过表达无功能MDR蛋白（S941F）的细胞系，它对秋水仙碱或阿霉素不耐药，但对长春碱耐药[格罗斯，P.，迪尔，R.，克鲁普，J.，& 塔尔博特，F.（1991年）美国国家科学院院刊88，7289 - 7293]，相对于LR73，其pHi升高且δψ略有升高。Northern和western印迹分析证实了这些细胞系中μMDR基因和蛋白大量过表达，以及某些细胞系中内源性仓鼠p - GP mRNA轻度过表达。与先前研究过表达人MDR 1蛋白的骨髓瘤细胞的研究[罗普，P.D.，魏，L.-Y.，克鲁兹，J.，& 卡尔森，D.（1993年）生物化学32，11042 - 11056]总体一致，我们发现野生型μMDR 1蛋白的过表达抑制了Cl⁻和 - HCO₃依赖的pHi稳态。通过单细胞光度法研究，我们现在得出结论，这是由于抑制了不依赖Na⁺的Cl⁻/ - HCO₃交换（严格阴离子交换或AE）。正如之前对其他MDR细胞得出的结论，AE活性降低并非由于交换体表达减少；事实上，再次与先前的研究[罗普等人（1993年）生物化学32，11042 - 11056]相似，我们发现在一些MDR细胞系中AE mRNA水平升高。提出了一些可能解释这些数据且也与内源性表达MDR蛋白的细胞的已知生理学一致的模型。这些数据与MDR蛋白功能模型一致，即该蛋白的过表达通过Cl⁻和 - HCO₃依赖的机制降低δψ和/或升高pHi。
