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 μM) 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 μM) and benzyl alcohol (1 mM) failed to influence either lipid fluidity or P-gp-mediated drug accumulation. Kinetic analysis revealed that A₂C (5.0 μM) noncompetitively inhibited [³H]daunomycin accumulation and uncompetitively inhibited [³H]vinblastine accumulation with apparent K_i values of ≈1.5 and ≈1.2 μM, 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.