Modulation of P-glycoprotein-mediated drug transport by alterations in lipid fluidity of rat liver canalicular membrane vesicles

Frank A Sinicrope, Pradeep K. Dudeja, Bruce M. Bissonnette, Ahmad R. Safa, Thomas A. Brasitus

Research output: Contribution to journalArticle

142 Citations (Scopus)

Abstract

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 Ki 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.

Original languageEnglish (US)
Pages (from-to)24995-25002
Number of pages8
JournalJournal of Biological Chemistry
Volume267
Issue number35
StatePublished - Dec 15 1992
Externally publishedYes

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Fluidity
P-Glycoprotein
Liver
Rats
Daunorubicin
Modulation
Membranes
Lipids
Benzyl Alcohol
Membrane Fluidity
Vinblastine
Pharmaceutical Preparations
Membrane Lipids
Adenosine Triphosphate
Bile Canaliculi
Diphenylhexatriene
Kinetics
Fluorescence Polarization
Cytotoxins
Cell membranes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Modulation of P-glycoprotein-mediated drug transport by alterations in lipid fluidity of rat liver canalicular membrane vesicles. / Sinicrope, Frank A; Dudeja, Pradeep K.; Bissonnette, Bruce M.; Safa, Ahmad R.; Brasitus, Thomas A.

In: Journal of Biological Chemistry, Vol. 267, No. 35, 15.12.1992, p. 24995-25002.

Research output: Contribution to journalArticle

Sinicrope, Frank A ; Dudeja, Pradeep K. ; Bissonnette, Bruce M. ; Safa, Ahmad R. ; Brasitus, Thomas A. / Modulation of P-glycoprotein-mediated drug transport by alterations in lipid fluidity of rat liver canalicular membrane vesicles. In: Journal of Biological Chemistry. 1992 ; Vol. 267, No. 35. pp. 24995-25002.
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abstract = "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 Ki 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.",
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AU - Safa, Ahmad R.

AU - Brasitus, Thomas A.

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N2 - 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 Ki 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.

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