Modulation of P-glycoprotein-mediated drug transport by alterations in lipid fluidity of rat liver canalicular membrane vesicles.
P-glycoprotein (P-gp) is believed to function as an ATP-dependent efflux pump for natural product anti-cancer drugs in multidrug-resistant (MDR) tumor cells and in certain normal tissues. P-gp has been localized to the apical plasma membrane of the bile canaliculus where it has been shown to transport [3H]daunomycin. In this study, we investigated whether alterations in membrane lipid fluidity of canalicular membrane vesicles (CMV) could modulate the P-gp-mediated accumulation of [3H]daunomycin and [3H]vinblastine. Accumulation of both cytotoxic agents was stimulated by ATP, exhibited temperature dependence and osmotic sensitivity, and followed Michaelis-Menten kinetics. Alterations in CMV lipid fluidity were induced by the known fluidizers, 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate (A2C) and benzyl alcohol, and were assessed by fluorescence polarization techniques using the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). Both A2C (2.5-5.0 microM) and benzyl alcohol (10-20 mM) produced a dose-dependent increase in CMV lipid fluidity. Moreover, both fluidizers, at the above doses, significantly inhibited (p < 0.05) the ATP-dependent accumulation of [3H]daunomycin. [3H]Vinblastine accumulation was also inhibited by A2C (p < 0.05). Lower doses of A2C (0.6 microM) and benzyl alcohol (1 mM) failed to influence either lipid fluidity or P-gp-mediated drug accumulation. Kinetic analysis revealed that A2C (5.0 microM) noncompetitively inhibited [3H]daunomycin accumulation and uncompetitively inhibited [3H]vinblastine accumulation with apparent Ki values of approximately 1.5 and approximately 1.2 microM, respectively. Verapamil competitively inhibited P-gp-mediated accumulation of [3H]daunomycin but failed to alter the fluidity of CMV. Taken together, the present results demonstrate that while increases in membrane fluidity of CMV are not necessarily required to inhibit P-gp-mediated drug accumulation, they can inhibit these processes, at least in CMV. Alterations in the physical state of CMV, therefore, appear to be at least one important modulator of P-gp function.
Department of Medicine, University of Chicago, Illinois 60637.
F A
FA
Sinicrope
P K
PK
Dudeja
B M
BM
Bissonnette
A R
AR
Safa
T A
TA
Brasitus
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D019905:Benzyl Alcohol; D001592:Benzyl Alcohols / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D003630:Daunorubicin / Q000378:metabolism*; D004351:Drug Resistance / Q000502:physiology*; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008297:Male; D008560:Membrane Fluidity / Q000187:drug effects; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013228:Stearates / Q000494:pharmacology; D014747:Vinblastine / Q000378:metabolism*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001592:Benzyl Alcohols; D008562:Membrane Glycoproteins; D013228:Stearates; C023714:A(2)C; D014747:Vinblastine; D000255:Adenosine Triphosphate; D019905:Benzyl Alcohol; D003630:Daunorubicin