Horio M, Pastan I, Gottesman M M, Handler J S
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Biochim Biophys Acta. 1990 Aug 24;1027(2):116-22. doi: 10.1016/0005-2736(90)90074-x.
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 epithelia 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 Michaelis-Menten kinetics with an apparent Km = 1.1 microM. 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 greater than MDCK greater than OK 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 in MDCK cells was not affected by potency, were verapamil greater than vincristine greater than actinomycin D greater than daunomycin. The transport pattern we observed is that predicted to result from the function of the multidrug transporter in the apical plasma membrane.
我们提出了一种新的转运模型，该模型可能对多种跨上皮转运实验有用。该模型仅基于跨上皮示踪剂通量就能估算泵的米氏常数（Km）和泵活性。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转运上皮细胞（如肾近端小管细胞）的顶端质膜中。为了确定这种多药转运体在上皮细胞中的功能特性，我们研究了化疗药物长春碱在由肾细胞系MDCK、LLC-PK1和OK形成的上皮细胞中的跨上皮转运。我们之前已经表明，在MDCK上皮细胞中，100 nM长春碱从基底到顶端的通量比从顶端到基底的通量高约五倍，这表明长春碱在MDCK上皮细胞中存在净跨上皮转运。添加未标记的长春碱会以浓度依赖的方式降低示踪剂从基底到顶端的通量，并增加示踪剂从顶端到基底的通量，这种模式符合如果存在一个可饱和的泵在顶端质膜处排出长春碱的预期。该模型仅基于跨上皮示踪剂通量就能估算泵的Km和泵活性。根据转运模型，顶端膜泵具有米氏动力学，表观Km = 1.1 microM。在其他肾源性细胞系LLC-PK1细胞和OK细胞中也观察到了长春碱从基底到顶端的净转运。转运效力顺序为LLC-PK1大于MDCK大于OK细胞。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3 mM四甲基铵或四乙基铵的影响。MDCK细胞中长春碱转运抑制剂的效力顺序为维拉帕米大于长春新碱大于放线菌素D大于柔红霉素。我们观察到的转运模式是由顶端质膜中多药转运体的功能所预测的结果。
