Role of the plasma membrane leaflets in drug uptake and multidrug resistance.

The present study aimed to investigate the role played by the leaflets of the plasma membrane in the uptake of drugs into cells and in their extrusion by P-glycoprotein and multidrug resistance-associated protein 1. Drug accumulation was monitored by fluorescence resonance energy transfer from trimethylammonium-diphenyl-hexatriene (TMA-DPH) located at the outer leaflet to a rhodamine analog. Uptake of dye into cells whose mitochondria had been inactivated was displayed as two phases of TMA-DPH fluorescence quenching.

Competition between innate multidrug resistance and intracellular binding of rhodamine dyes.

The present study aimed to elucidate the contribution of the intracellular binding of drugs to multidrug resistance. For this purpose, uptake of rhodamines was studied in cells whose mitochondria had been uncoupled with carbonyl cyanide m-chlorophenylhydrazone. Surprisingly, in a variety of drug-untreated cells, presumed to be sensitive to multidrug resistance-type drugs, rhodamines were excluded from entering the cells.

Modulation of P-glycoprotein-mediated multidrug resistance by acceleration of passive drug permeation across the plasma membrane.

The drug concentration inside multidrug-resistant cells is the outcome of competition between the active export of drugs by drug efflux pumps, such as P-glycoprotein (Pgp), and the passive permeation of drugs across the plasma membrane. Thus, reversal of multidrug resistance (MDR) can occur either by inhibition of the efflux pumps or by acceleration of the drug permeation. Among the hundreds of established modulators of Pgp-mediated MDR, there are numerous surface-active agents potentially capable of accelerating drug transbilayer movement.

The tyrosine kinase inhibitors imatinib and AG957 reverse multidrug resistance in a chronic myelogenous leukemia cell line.

The K562 cell line derived from a chronic myelogenous leukemia (CML) patient exhibits ATP-dependent exclusion of the multidrug resistance (MDR)-type drugs. The protein tyrosine kinases inhibitors, imatinib mesylate and AG957 allowed for increased doxorubicin and calcein-AM accumulation in these cells. Maximal modulation was achieved at 3 and 10 microM imatinib and AG957, respectively.

Transport of anthracyclines and mitoxantrone across membranes by a flip-flop mechanism.

The objectives of the present work are to characterize the transport of mitoxantrone and three anthracyclines in terms of binding to the membrane surface, flip-flop across the lipid core of the membrane, and release into the medium. Mitoxantrone and anthracyclines are positively charged amphipathic molecules, and as such are located at the surface of membranes among the headgroups of the phospholipids. Therefore, their transport across membranes occurs by a flip-flop mechanism, rather than by diffusion down a continuous concentration gradient located in the lipid core of the membrane.

Mechanism of multidrug resistance in relation to passive membrane permeation.

Passive uptake of drugs into cells is described in terms of the following steps: (1) massive immediate binding of the drugs to the outer leaflet of the plasma membrane resulting in practical equilibrium between extremely high drug concentrations at the cell surface compared to the drug concentration in the medium. (2) Due to their amphipathic nature, anticancer drugs are practically excluded from the lipid core of the membrane. They cross the lipid core by distinct flip-flop events that occur in the case of doxorubicin and daunorubicin after an average period of 0.

Cells from chronic myelogenous leukaemia patients at presentation exhibit multidrug resistance not mediated by either MDR1 or MRP1.

Tetramethylrosamine (TMR) is excluded from P-glycoprotein (MDR1)-enriched cell lines, but it stains efficiently MDR1-poor parent lines. Application of the TMR resistance assay to cells obtained from chronic myelogenous leukaemia (CML) patients revealed, in all individuals, a significant resistance compared with healthy donors (P < 0.001).

A storage Dewar near-field scanning optical microscope.

A near-field scanning optical microscope for operation within a storage Dewar is described. It was designed for studies of opaque samples and operates in the collection mode. Illumination can be either through the tip or from the side via a separate fiber.

Mechanism of action of P-glycoprotein in relation to passive membrane permeation.

This review presents a survey of studies of the movement of chemotherapeutic drugs into cells, their extrusion from multidrug-resistant (MDR) cells overexpressing P-glycoprotein (Pgp), and the mode of sensitization of MDR cells to anticancer drugs by Pgp modulators. The consistent features of the kinetics from studies of the operation of Pgp in cells were combined in a computer model that enables the simulation of experimental scenarios. MDR-type drugs are hydrophobic and positively charged and as such bind readily to negatively charged phospholipid head groups of the membrane.

Membrane fluidization by ether, other anesthetics, and certain agents abolishes P-glycoprotein ATPase activity and modulates efflux from multidrug-resistant cells.

The anesthetics benzyl alcohol and the nonaromatic chloroform and diethyl ether, abolish P-glycoprotein (Pgp) ATPase activity in a mode that does not fit classical competitive, noncompetitive, or uncompetitive inhibition. At concentrations similar to those required for inhibition of ATPase activity, these anesthetics fluidize membranes leading to twofold acceleration of doxorubicin flip-flop across lipid membranes and prevent photoaffinity labeling of Pgp with [125I]-iodoarylazidoprazosin. Similar concentrations of ether proved nontoxic and modulated efflux from Pgp-overexpressing cells.

Flip-flop of doxorubicin across erythrocyte and lipid membranes.

Doxorubicin, an anticancer drug, is extruded from multidrug resistant (MDR) cells and from the brain by P-glycoprotein located in the plasma membrane and the blood-brain barrier, respectively. MDR-type drugs are hydrophobic and, as such, enter cells by diffusion through the membrane without the requirement for a specific transporter. The apparent contradiction between the presumably free influx of MDR-type drugs into MDR cells and the efficient removal of the drugs by P-glycoprotein, an enzyme with a limited ATPase activity, prompted us to examine the mechanism of passive transport within the membrane.

Efficiency of P-glycoprotein-mediated exclusion of rhodamine dyes from multidrug-resistant cells is determined by their passive transmembrane movement rate.

The aim of the present study was to examine the relationship between the rate of the passive transmembrane movement of multidrug resistance (MDR)-type substrates and the ability of P-glycoprotein to extrude them from MDR cells. For this purpose, seven rhodamine dyes were examined for their P-glycoprotein-mediated exclusion from MDR cells, their localization in wild-type drug-sensitive cells, their capacity to stimulate the ATPase activity of P-glycoprotein reconstituted in proteoliposomes, and their transmembrane movement rate in artificial liposomes. All these rhodamine dyes were accumulated in wild-type drug-sensitive cells and were localized mainly in the mitochondria.

The role of passive transbilayer drug movement in multidrug resistance and its modulation.

The successful lowering of the intracellular concentration of multidrug resistance (MDR)-type drugs by P-glycoprotein (Pgp) relies on its ability to overcome the passive influx rate of each MDR-type drug. Thus, the aim of the present work was to study the effect of passive transbilayer drug movement on the multidrug resistance and its modulation. Fluorescence quenching studies indicated that whereas the Pgp substrate rhodamine 123 traverses an artificial lipid membrane with a lifetime of 3 min, the transbilayer movement rate of the MDR modulators, quinidine and quinine, was too fast to be detected with present methods.

Functional reconstitution of P-glycoprotein reveals an apparent near stoichiometric drug transport to ATP hydrolysis.

We have recently described an ATP-driven, valinomycin-dependent 86Rb+uptake into proteoliposomes reconstituted with mammalian P-glycoprotein (Eytan, G. D., Borgnia, M.

Competition of hydrophobic peptides, cytotoxic drugs, and chemosensitizers on a common P-glycoprotein pharmacophore as revealed by its ATPase activity.

The aim of the present study was to demonstrate that the modulation of P-glycoprotein (Pgp) ATPase activity by peptides, drugs, and chemosensitizers takes place on a common drug pharmacophore. To this end, a highly emetine-resistant Chinese hamster ovary cell line was established, in which Pgp constituted 18% of plasma membrane protein. Reconstituted proteoliposomes, the Pgp content of which was up to 40%, displayed a basal activity of 2.

Alteration of mitochondrial gene expression and disruption of respiratory function by the lipophilic antifolate pyrimethamine in mammalian cells.

To clone the mammalian gene(s) associated with a novel lipophilic antifolate resistance provoked by the antiparasitic drug pyrimethamine (Assaraf, Y. G., and Slotky, J.

Potentiation of anticancer-drug cytotoxicity by multidrug-resistance chemosensitizers involves alterations in membrane fluidity leading to increased membrane permeability.

We are studying the mechanism underlying chemosensitization of anticancer-drug cytotoxicity in wild-type and multidrug-resistant (MDR) mammalian cells. We show here that the chemosensitizers, reserpine and verapamil, display a dramatic potentiation of taxol, anthracycline and Vinca alkaloids cytotoxicity in P-glycoprotein-(P-gp)-deficient hamster and human nasopharyngeal carcinoma cells. We have therefore utilized this phenomenon to probe for the putative P-gp-independent component of cytotoxicity chemosensitization.

Transport of polypeptide ionophores into proteoliposomes reconstituted with rat liver P-glycoprotein.

The aim of this study was to examine the peptide transport activity of a naturally occurring P-glycoprotein such as that present in rat liver canalicular membrane vesicles. The peptide ionophores valinomycin and gramicidin D, which are known substrates of P-glycoprotein, served to monitor the P-glycoprotein activity indirectly as the ATP-dependent uptake of 86Rb+ mediated by these ionophores. Canalicular membrane vesicles proved inherently permeable to K+ ions, which prevented assay of transport ionophore activity.

Energy-linked transhydrogenase. Effects of valinomycin and nigericin on the ATP-driven transhydrogenase reaction catalyzed by reconstituted transhydrogenase-ATPase vesicles.

Reconstituted transhydrogenase-ATPase vesicles obtained with purified beef heart transhydrogenase and oligomycin-sensitive ATPase were investigated with respect to the mode of interaction between the two proton pumps, with special reference to the relative contributions of the membrane potential and proton gradient using valinomycin and nigericin in the presence of potassium. In the absence of ionophores and at low ATP concentrations, below 20 microM, the ATPase generated a proton motive force which was predominantly due to a membrane potential, whereas at saturating concentrations of ATP the proton gradient was the predominant component. The ATP-dependence of the rate of the ATP-driven transhydrogenase reaction showed apparent Km values in the low and high ATP concentration range of about 3 and 56 microM, respectively, with a corresponding difference in Vmax of about 3-fold.

Gramicidin S and dodecylamine induce leakage and fusion of membranes at micromolar concentrations.

The effect of the antibiotic gramicidin S and the synthetic cationic amphipath dodecylamine on membranes was studied with large unilamellar vesicles containing phosphatidylcholine and varying concentrations of cardiolipin. Fusion of vesicles composed of equal amounts of the two phospholipids occurred with both drugs at concentrations lower than 10 microM. Fusion was accompanied by leakage of the contents, while higher drug concentrations caused complete loss of vesicle contents.

Energy-linked nicotinamide-nucleotide transhydrogenase. Light-driven transhydrogenase catalyzed by transhydrogenase from beef heart mitochondria reconstituted with bacteriorhodopsin.

Purified nicotinamide-nucleotide transhydrogenase from beef heart mitochondria was co-reconstituted with bacteriorhodopsin to from transhydrogenase-bacteriorhodopsin vesicles that catalyze a 20-fold light-dependent and uncoupler-sensitive stimulation of the reduction of NADP+ and NADP+ analogs by NADH and a 50-fold shift of the nicotinamide nucleotide ratio. In the presence of light, the transhydrogenase-bacteriorhodopsin vesicles catalyzed a pronounced light intensity-dependent inward proton pumping as indicated by a pH shift of the medium. As indicated by pH shifts, proton pumping by the bacteriorhodopsin essentially paralleled the light-driven transhydrogenase.

Energy-linked nicotinamide-nucleotide transhydrogenase. Characterization of reconstituted ATP-driven transhydrogenase from beef heart mitochondria.

The interaction between pure transhydrogenase and ATPase (Complex V) from beef heart mitochondria was investigated with transhydrogenase-ATPase vesicles in which the two proteins were co-reconstituted by dialysis or dilution procedures. In addition to phosphatidylcholine and phosphatidylethanolamine, reconstitution required phosphatidylserine and lysophosphatidylcholine. Transhydrogenase-ATPase vesicles catalyzed a 20-30-fold stimulation of the reduction of NADP+ or thio-NADP+ by NADH and a 70-fold shift of the apparent equilibrium expressed as the nicotinamide nucleotide ratio [NADPH][NAD+]/[NADP+][NADH].

Fusion between Sendai virus envelopes and biological membranes as monitored by energy transfer methods.

Chlorophyll a and chlorophyll b have been inserted into reconstituted envelopes of Sendai virus particles. Fluorescence measurements indicated a high efficiency of energy transfer between the two chlorophyll molecules due to their close proximity in the viral envelope. Fusion of reconstituted, pigmented virus envelopes with various biological cell membranes at 37 degrees C resulted in a significant decrease in the yield of energy transfer.

Melittin-induced fusion of acidic liposomes.

Melittin-induced fusion of acidic liposomes. Fusion was observed in the electron-microscope and assayed as intermixing of both liposomes' contents and membranes. The melittin concentrations required for fusion induction were in the microM range compared to over 10 mM Ca2+ required for a comparable effect.