Real-time monitoring of P-glycoprotein activation in living cells
Real-time monitoring of P-glycoprotein activation in living cells
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DOI：
10.1021/bi025720s
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发表时间：
2002-06-25
期刊：
影响因子：
2.9
通讯作者：
Seelig, A
中科院分区：
文献类型：
--
作者：
Landwojtowicz, E;Nervi, P;Seelig, A
用细胞传感器微生理仪实时测定了8种不同药物激活或抑制P-糖蛋白(Pgp)ATPase后的胞外酸化率(ECARs)。在转基因细胞中，ECARs呈钟形依赖于药物浓度(对数比例)，但在野生型细胞中，ECARs可以忽略不计。根据一个假设PGP-ATPase被一个药物分子激活，两个药物分子结合而被抑制的模型，分析了激活曲线(ECARs与浓度的关系)。PGP-ATPase激活的动力学常数[半最大激活(抑制)浓度K(I)和最大(最小)转运蛋白活性V(I)]分别与先前测定的PGP-ATPase激活的定性和定量结果一致。此外，在两种不同的细胞系中，ECARs与P-p的表达水平相关，并且被PGP-ATPase的有效抑制剂环孢素A：以浓度依赖的方式降低。相反，用Na(+)/H(+)或Cl-/HCO(3)(-)交换器的抑制剂处理细胞并不降低ECARs。微pH测量首次提供了细胞外质子排出速率和细胞内磷释放对P-p-ATPase激活的紧密耦合的直接证据。它们支持PGP介导的质子从ATP水解点到细胞表面的运输。因此，ECARs的测定可以构成一种新的方法来方便地分析活细胞中PGP-ATPase的激活动力学。
Extracellular acidification rates (ECARs) in response to eight different drugs activating or inhibiting the ATPase of P-glycoprotein (Pgp) were measured in real time by means of a Cytosensor microphysiometer in MDR1-transfected and corresponding wild-type cell lines, i.e., pig kidney cells (LLC-MDR1 and LLC-PK1) and mouse embryo fibroblasts (NIH-MIDR-G185 and NIH3T3). The ECARs showed a bell-shaped dependence on drug concentration (log scale) in transfected cells but were negligibly small in wild-type cells. The activation profiles (ECARs vs concentration) were analyzed in terms of a model assuming activation of Pgp-ATPase with one and inhibition with two drug molecules bound. The kinetic constants [concentration of half-maximum activation (inhibition), K(i), and the maximum (minimum) transporter activity, V(i)] were in qualitative and quantitative agreement with those determined earlier for Pgp-ATPase activation monitored by phosphate release in inside-out cellular vesicles and in purified reconstituted systems, respectively. Furthermore, the ECARs correlated with the expression level of P-p in the two different cell lines and were reduced in a concentration-dependent manner by cyclosporin A:, a potent inhibitor of the Pgp-ATPase. In contrast, treatment of cells with inhibitors of the Na(+)/H(+) or the Cl-/HCO(3)(-) exchanger did not reduce the ECARs. The micro-pH measurements provide for the first time direct evidence for a tight coupling between the rate of extracellular proton extrusion and intracellular phosphate release upon P-p-ATPase activation. They support a Pgp-mediated transport of protons from the site of ATP hydrolysis to the cell surface. Measurement of the ECARs could thus constitute a new method to conveniently analyze the kinetics of Pgp-ATPase activation in living cells.