Landwojtowicz Ewa, Nervi Pierluigi, Seelig Anna
Division of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, Switzerland.
Biochemistry. 2002 Jun 25;41(25):8050-7. doi: 10.1021/bi025720s.
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-MDR-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 Pgp 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 Pgp-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.
通过细胞传感器微生理仪实时测量了在MDR1转染的和相应的野生型细胞系（即猪肾细胞LLC-MDR1和LLC-PK1以及小鼠胚胎成纤维细胞NIH-MDR-G185和NIH3T3）中，八种不同药物激活或抑制P-糖蛋白（Pgp）的ATP酶时的细胞外酸化率（ECAR）。在转染细胞中，ECAR对药物浓度（对数标度）呈钟形依赖性，但在野生型细胞中可忽略不计。根据一个假设，即一个药物分子结合激活Pgp-ATP酶，两个药物分子结合抑制Pgp-ATP酶的模型，分析了激活曲线（ECAR与浓度的关系）。动力学常数[半最大激活（抑制）浓度、K(i)以及最大（最小）转运活性V(i)]在定性和定量上与之前分别通过内翻细胞膜囊泡中的磷酸盐释放和纯化的重组系统监测的Pgp-ATP酶激活所确定的结果一致。此外，ECAR与两种不同细胞系中Pgp的表达水平相关，并且被Pgp-ATP酶的强效抑制剂环孢素A以浓度依赖性方式降低。相比之下，用Na(+)/H(+)或Cl(-)/HCO(3)(-)交换体抑制剂处理细胞并没有降低ECAR。微pH测量首次直接证明了Pgp-ATP酶激活时细胞外质子挤出速率与细胞内磷酸盐释放之间的紧密耦合。它们支持Pgp介导质子从ATP水解位点转运到细胞表面。因此，测量ECAR可能构成一种方便地分析活细胞中Pgp-ATP酶激活动力学的新方法。
