ACTIVITY OF THE MULTIDRUG TRANSPORTER RESULTS IN ALKALINIZATION OF THE CYTOSOL - MEASUREMENT OF CYTOSOLIC PH BY MICROINJECTION OF A PH-SENSITIVE DYE
ACTIVITY OF THE MULTIDRUG TRANSPORTER RESULTS IN ALKALINIZATION OF THE CYTOSOL - MEASUREMENT OF CYTOSOLIC PH BY MICROINJECTION OF A PH-SENSITIVE DYE
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DOI：
10.1177/38.5.1692055
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发表时间：
1990-05-01
影响因子：
3.2
通讯作者：
WILLINGHAM, MC
中科院分区：
文献类型：
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作者：
THIEBAUT, F;CURRIER, SJ;WILLINGHAM, MC
多药耐药细胞含有一个质膜外排泵，即多药转运蛋白，它能主动将某些药物从细胞质中排出到细胞外。这些药物通常是带正电的生理pH值。因为人们可能会预测，这种流出的带正电的分子smight耗尽质子的胞质溶胶，提高胞质pH值，我们检查了多药耐药细胞的胞质pH值直接使用pH敏感染料耦合到一个膜不可渗透的分子。将与10，000 MW葡聚糖共价偶联的染料（SNARF）机械显微注射到表达人多药转运蛋白的培养的多药耐药小鼠NIH 3T3细胞的胞质溶胶中。使用落射荧光显微镜在不同波长下测量活细胞中染料的荧光发射，以提供细胞溶质环境pH的测量。多药耐药细胞具有比药物敏感的正常亲本细胞更高的胞质pH。随着培养基pH值的增加，正常细胞的胞质pH值保持在7.0以下，而多药耐药细胞的胞质pH值上升。在外部培养基pH为8.2时，两种细胞类型之间的胞质pH差异超过0.2 pH单位。用抑制多药转运蛋白介导的外排的药物（如维拉帕米和长春碱）治疗，基本上消除了细胞溶质pH值的升高，可能是因为它们是泵的良好底物，而泵的能力压倒其他物质。这些结果表明，多药物转运体是间接的质子泵，细胞可能含有一个内源性底物或底物的情况下添加药物的转运。
Multidrug-resistant cells contains a plasma membrane efflux pump, the multidrug transporter, which actively expels certain hydrophogic drugs from the cytosol to the cell exterior. These drugs are usually positively charged at physiological pH. Because one might predict that this efflux of positively charged molecule smight deplete the cytosol of protons, raising the cytosolic pH, we examined the cytosolic pH of multidrug-resistant cells directly using a pH-sensitive dye coupled to a membrane-impermeable molecule. The dye (SNARF), covalently coupled to 10,000 MW dextran, was mechanically microinjected into the cytosol of cultured multidrug-resistant mouse NIH3T3 cells which express the human multidrug transporter. The fluorescence emission of the dye in living cells was measured using epifluorescence microscopy at different wavelengths to provide a measure of the pH of the cytosolic environment. Multidrug-resistant cells had a higher cytosolic pH than drug-sensitive normal parental cells. As the pH of the culture medium was increased, normal cells maintained their cytosolic pH below 7.0, whereas the cytosolic pH of multidrug resistant cells rose. The difference in cytosolic pH between the two cell types was more than 0.2 pH units at an external culture medium pH of 8.2. Treatment with agents that inhibit multidrug transporter-mediated efflux, such as verapamil and vinblastine, essentially eliminated the elevation of cytosolic pH, presumably because they are good substrates for the pump which overwhelm in capacity to pump other materials. These results suggest that the multidrug transporter is indirectly a proton pumps, and that cells may contain an endogenous substrate or substrates for this transporter in the absence of added drugs.