TRANSEPITHELIAL TRANSPORT OF VINBLASTINE BY KIDNEY-DERIVED CELL-LINES - APPLICATION OF A NEW KINETIC-MODEL TO ESTIMATE INSITU KM OF THE PUMP
TRANSEPITHELIAL TRANSPORT OF VINBLASTINE BY KIDNEY-DERIVED CELL-LINES - APPLICATION OF A NEW KINETIC-MODEL TO ESTIMATE INSITU KM OF THE PUMP
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DOI：
10.1016/0005-2736(90)90074-x
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发表时间：
1990-08-24
期刊：
影响因子：
--
通讯作者：
HANDLER, JS
中科院分区：
文献类型：
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作者：
HORIO, M;PASTAN, I;HANDLER, JS
我们提出了一种新的转运模型，可用于多种经上皮转运实验。该模型允许仅根据经上皮示踪剂通量估计泵的Km和泵的活性。我们将该模型应用于多药外排泵p -糖蛋白的研究，p -糖蛋白通常位于某些运输上皮细胞（如肾近端小管细胞）的顶质膜上。为了确定这种多药转运体在上皮中的功能特性，我们研究了化疗药物长春花碱在肾细胞系MDCK、LLC-PK1和OK形成的上皮中的经上皮转运。我们之前的研究表明，在MDCK上皮中，100 nM长春碱的基底到根尖通量大约是根尖到基底通量的5倍，这表明长春碱在MDCK上皮中存在净的跨上皮运输。未标记长春碱的加入降低了示踪剂的基底到根尖的通量，并以浓度依赖的方式增加了示踪剂的根尖到根底的通量，如果在根尖质膜上有一个可饱和泵挤出长春碱，则可以预期这种模式。该模型允许仅根据经上皮示踪剂通量估计泵的Km和泵的活性。根据输运模型，顶膜泵具有表观Km = 1.1 μ n的Michalis-Menten动力学。在lc - pk1细胞和OK细胞（其他肾源性细胞系）中也观察到长春碱从基底到顶端的净转运。转运的效价顺序为OC细胞lc - pk1 > MDCK >。有机阳离子转运体不参与这种长春碱转运，因为MDCK细胞中的长春碱转运不受3mm四甲基或四乙基铵的影响。长春新碱跨MDCK上皮转运抑制剂的效价顺序为维拉帕米b>长春新碱>放线菌素D >道诺霉素。我们观察到的转运模式与预测的顶端质膜中多药转运体的功能有关。
We present a new transport model that may be useful for many kinds of transepithelial transport experiments. The model permits estimation of a pump Km and pump activity solely on the basis of transepithelial tracer fluxes. We apply the model to studies of a multidrug efflux pump, P-glycoprotein, which is normally located in the apical plasma membrane of certain transporting epithelial such as kidney proximal tubule cells. To determine the functional properties of this multidrug transporter in an epithelium, we studied the transepithelial transport of the chemotherapeutic drug, vinblastine, in epithelia formed by the kidney cell lines MDCK, LLC-PK1, and OK. We have previously shown that basal to apical flux of 100 nM vinblastine was about five times higher than apical to basal flux in MDCK epithelia, indicating that there is a net transepithelial transport of vinblastine across MDCK epithelia. Addition of unlabeled vinblastine reduced basal to apical flux of tracer and increased apical to basal flux of tracer in a concentration-dependent manner, a pattern expected if there is a saturable pump that extrudes vinblastine at the apical plasma membrane. The model permits estimation of a pump Km and pump activity solely on the basis of transepithelial tracer fluxes. According to the transport model the apical membrane pump has Michalis-Menten kinetics with an apparent Km = 1.1 .mu.N. Net basal to apical transport of vinblastine was also observed in LLC-PK1 cells and OK cells which are other kidney-derived cell lines. The order of potency of the transport is LLC-PK1 > MDCK > OC cells. The organic cation transporter is not involved in this vinblastine transport because vinblastine transport in MDCK cells was not affected by 3 mM tetramethyl- or tetraethylammonium. Inhibitors of vinblastine transport across MDCK epithelia, in order of potency, were verapamil > vincristine > actinomycin D > daunomycin. The transport pattern we observed is that predicted to result from the function of the multidrug transporter in the apical plasma membrane.