Bartholomé Kilian, Rius Maria, Letschert Katrin, Keller Daniela, Timmer Jens, Keppler Dietrich
Institute for Physics, University of Freiburg, Freiburg, Germany.
Drug Metab Dispos. 2007 Sep;35(9):1476-81. doi: 10.1124/dmd.107.015636. Epub 2007 Jun 4.
Vectorial transport of endogenous small molecules, toxins, and drugs across polarized epithelial cells contributes to their half-life in the organism and to detoxification. To study vectorial transport in a quantitative manner, an in vitro model was used that includes polarized MDCKII cells stably expressing the recombinant human uptake transporter OATP1B3 in their basolateral membrane and the recombinant ATP-driven efflux pump ABCC2 in their apical membrane. These double-transfected cells enabled mathematical modeling of the vectorial transport of the anionic prototype substance bromosulfophthalein (BSP) that has frequently been used to examine hepatobiliary transport. Time-dependent analyses of (3)H-labeled BSP in the basolateral, intracellular, and apical compartments of cells cultured on filter membranes and efflux experiments in cells preloaded with BSP were performed. A mathematical model was fitted to the experimental data. Data-based modeling was optimized by including endogenous transport processes in addition to the recombinant transport proteins. The predominant contributions to the overall vectorial transport of BSP were mediated by OATP1B3 (44%) and ABCC2 (28%). Model comparison predicted a previously unrecognized endogenous basolateral efflux process as a negative contribution to total vectorial transport, amounting to 19%, which is in line with the detection of the basolateral efflux pump Abcc4 in MDCKII cells. Rate-determining steps in the vectorial transport were identified by calculating control coefficients. Data-based mathematical modeling of vectorial transport of BSP as a model substance resulted in a quantitative description of this process and its components. The same systems biology approach may be applied to other cellular systems and to different substances.
内源性小分子、毒素和药物跨极化上皮细胞的矢量转运有助于它们在生物体内的半衰期和解毒作用。为了以定量方式研究矢量转运，使用了一种体外模型，该模型包括在其基底外侧膜稳定表达重组人摄取转运体OATP1B3且在其顶端膜稳定表达重组ATP驱动的外排泵ABCC2的极化MDCKII细胞。这些双转染细胞能够对阴离子原型物质溴磺酞（BSP）的矢量转运进行数学建模，BSP常用于检查肝胆转运。对滤膜上培养的细胞的基底外侧、细胞内和顶端区室中（3）H标记的BSP进行了时间依赖性分析，并对预先加载BSP的细胞进行了外排实验。将一个数学模型拟合到实验数据。通过除重组转运蛋白外还纳入内源性转运过程，对基于数据的建模进行了优化。对BSP总体矢量转运的主要贡献由OATP1B3（44%）和ABCC2（28%）介导。模型比较预测了一种先前未被认识的内源性基底外侧外排过程，该过程对总矢量转运有负贡献，达19%，这与在MDCKII细胞中检测到基底外侧外排泵Abcc4一致。通过计算控制系数确定了矢量转运中的速率决定步骤。以BSP作为模型物质对矢量转运进行基于数据的数学建模，得到了该过程及其组成部分的定量描述。相同的系统生物学方法可应用于其他细胞系统和不同物质。
