title: Real-time monitoring of P-glycoprotein activation in living cells.
abstract: 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.
authors.affiliation: Division of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, Switzerland.
authors.forename: Ewa
authors.initials: E
authors.lastname: Landwojtowicz
authors.forename: Pierluigi
authors.initials: P
authors.lastname: Nervi
authors.forename: Anna
authors.initials: A
authors.lastname: Seelig
mesh_terms: D016475:3T3 Cells; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000818:Animals; D015374:Biosensing Techniques; D027963:Chloride-Bicarbonate Antiporters / Q000378:metabolism; D016572:Cyclosporine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D004790:Enzyme Induction / Q000187:drug effects / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005110:Extracellular Space / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018374:LLC-PK1 Cells; D051379:Mice; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D013379:Substrate Specificity / Q000187:drug effects / Q000235:genetics; D013552:Swine; D014162:Transfection; D014268:Trifluoperazine / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D027963:Chloride-Bicarbonate Antiporters; D004791:Enzyme Inhibitors; D017923:Sodium-Hydrogen Exchangers; D014268:Trifluoperazine; D016572:Cyclosporine; D014700:Verapamil; D000251:Adenosine Triphosphatases