The rate of P-glycoprotein activation depends on the metabolic state of the cell.
P-glycoprotein ATPase activity has been studied almost exclusively by measuring inorganic phosphate release from inside-out cellular vesicles. We have recently proposed a new method based on measurements of the extracellular acidification rate (ECAR) of living cells with a Cytosensor microphysiometer. This method allows for systematic investigation of the various factors influencing P-glycoprotein activation in living cells. Basal metabolic rates or ECARs of different MDR1-transfected cell lines were compared with those of the Mdr1a(-/-)1b(-/-) knockout, MRP1-transfected, and corresponding wild-type cell lines. Basal ECARs of all cells were on the order of 10(7) protons/cell/s, whereby those of genetically modified cells were on average (over all cell lines) slightly lower than those of wild-type cells. The expression level of P-glycoprotein in MDR1-transfected cells had no influence on basal ECARs. Verapamil-induced ECARs were specific for MDR1-transfected cells and increased with the expression level of P-glycoprotein. Moreover, ECARs were dependent on the metabolic state of the cell and were (2.8 +/- 1.2) x 10(6) and (8.0 +/- 1.5) x 10(6) protons/cell/s in glucose-deficient and glucose-fed NIH-MDR-G185 cells, respectively, after verapamil (10 muM) stimulation. The ECARs were practically identical to the rates of lactate extrusion and thus reflect the rates of ATP synthesis via glycolysis. Taking into account the number of P-glycoprotein molecules per cell, the rate of ATP hydrolysis in inside-out vesicles of the same cells was determined as (9.2 +/- 1.5) x 10(6) phosphates/cell/s, in good agreement with the rate of ATP synthesized in glucose-fed cells. The energy required for P-glycoprotein activation relative to the basal metabolic energy was twice as large in glucose-deficient as in glucose-fed cells, suggesting cellular protection by P-glycoprotein even under conditions of starvation.
Gatlik-Landwojtowicz
Ewa
E
Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.
Aänismaa
Päivi
P
Seelig
Anna
A
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D015374:Biosensing Techniques; D018929:Cell Culture Techniques; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D005110:Extracellular Space / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018374:LLC-PK1 Cells; D019344:Lactic Acid / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D041681:NIH 3T3 Cells; D013552:Swine; D014162:Transfection; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D017077:Culture Media, Conditioned; D019344:Lactic Acid; D014700:Verapamil; D000251:Adenosine Triphosphatases