Gera D. Eytan

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 2.6 ± 0.45 μmol of P/min/mg of protein, suggesting the presence of an endogenous Pgp substrate. This basal ATPase activity was stimulated (up to 5.2 μmol of P/min/mg of protein) by valinomycin and various Pgp substrates, whereas, to our surprise, gramicidin D, an established Pgp substrate, was inhibitory. Taking advantage of this novel inhibition of Pgp ATPase activity by gramicidin D, a drug competition assay was devised in which gramicidin D-inhibited Pgp ATPase was coincubated with increasing concentrations of various substrates that stimulate its ATPase activity. Gramicidin D inhibition of Pgp ATPase was reversed by Pgp substrates, including various cytotoxic agents and chemosensitizers. The inhibition of the basal ATPase activity and the reversal of gramicidin D inhibition of Pgp ATPase by its various substrates conformed to classical Michaelis-Menten competition. This competition involved an endogenous substrate, the inhibitory drug gramicidin D, and a stimulatory substrate. We conclude that the various MDR type substrates and chemosensitizers compete on a common drug binding site present in Pgp.

Polycation-induced fusion of negatively-charged vesicles

Sonicated vesicles of 20-50 nm in diameter consisting of neutral phospholipids and a variety of acidic phospholipids were interacted with polylysine, cytochrome c, Ca2+ and Mg2+. The addition of polycations caused massive aggregation accompanied by an increase of membrane permeability as determined by leakage of fluorescent dye. Aggregation was followed by fusion of the vesicles into structures that in some cases exceeded 1 micron in diameter. Polylysine induced aggregation and appreciable fusion at charge ratios (polylysine/phospholipid) of 0.5-2, while divalent cations did so only at charge ratios (cation/phospholipid) greater than 10. Aggregation and fusion induced by polylysine were dependent also on the size of the polycation, i.e., the longer the molecule the less needed to induce similar aggregation. It appears that, due to the concentration of charges on a single molecule, polylysine is at least an order of magnitude more effective than divalent cations at inducing fusion of membranes. Cytochrome c induced fusion of similar vesicles at moderately acidic pH (pH 4.2).

Functional Reconstitution of P-glycoprotein Reveals an Apparent Near Stoichiometric Drug Transport to ATP Hydrolysis

We have recently described an ATP-driven, valinomycin-dependent Rbuptake into proteoliposomes reconstituted with mammalian P-glycoprotein (Eytan, G. D., Borgnia, M. J., Regev, R., and Assaraf, Y. G.(1994) J. Biol. Chem. 269, 26058-26065). P-glycoprotein mediated the ATP-dependent uptake of Rb-ionophore complex into the proteoliposomes, where the radioactive cation was accumulated, thus, circumventing the obstacle posed by the hydrophobicity of P-glycoprotein substrates in transport studies. Taking advantage of this assay and of the high levels of P-glycoprotein expression in multidrug-resistant Chinese hamster ovary cells, we measured simultaneously both the ATPase and transport activities of P-glycoprotein under identical conditions and observed 0.5-0.8 ionophore molecules transported/ATP molecule hydrolyzed. The amount of Rb ions transported within 1 min via the ATP- and valinomycin-dependent P-glycoprotein was equivalent to an intravesicular cation concentration of 8 mM. Thus, this stoichiometry and transport capacity of P-glycoprotein resemble various ion-translocating ATPases, that handle millimolar substrate concentrations. This constitutes the first demonstration of comparable rates of P-glycoprotein-catalyzed substrate transport and ATP hydrolysis.

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. Transmembrane movement of MDR-type drugs, such as doxorubicin, occurs by a flip-flop mechanism with a lifetime of about 1 min rather than by diffusion down a gradient present in the lipid core. A long residence time of a drug in the membrane leaflet increases the probability that P-glycoprotein will remove it from the cell. In a manner similar to ion-transporting ATPases, such as Na+,K(+)-ATPase, Pgp transports close to one drug molecule per ATP molecule hydrolyzed. Computer simulation of cellular pharmacokinetics, based on partial reactions measured in vitro, show that the efficiency of Pgp, in conferring MDR on cells, depends on the pumping capacity of Pgp and its affinity toward the specific drug, the transmembrane movement rate of the drug, the affinity of the drug toward its pharmacological cellular target, and the affinity of the drug toward intracellular trapping sites. Pgp activities present in MDR cells allow for the efficient removal of drugs, whether directly from the cytoplasm or from the inner leaflet of the plasma membrane. A prerequisite for a successful modulator, capable of overcoming cellular Pgp, is the rapid passive transbilayer movement, allowing it to reenter the cell immediately and thus successfully occupy the Pgp active site(s).

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. The kinetics of doxorubicin transport demonstrated the presence of two similar sized drug pools located in the two leaflets of the membrane. The transbilayer movement of doxorubicin occurred by a flip-flop mechanism of the drug between the two membrane leaflets. At 37 degrees, the flip-flop exhibited a half-life of 0.7 min, in both erythrocyte membranes and cholesterol-containing lipid membranes. The flip-flop was inhibited by cholesterol and accelerated by high temperatures and the fluidizer benzyl alcohol. The rate of doxorubicin flux across membranes is determined by both the massive binding to the membranes and the slow flip-flop across the membrane. The long residence-time of the drug in the inner leaflet of the plasma membrane allows P-glycoprotein a better opportunity to remove it from the cell.

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 aim of the present study was to determine whether these agents modulate MDR by interfering with the active efflux of drugs or by allowing for accelerated passive permeation across the plasma membrane. Whereas Pluronic P85, Tween‐20, Triton X‐100 and Cremophor EL modulated MDR by inhibition of Pgp‐mediated efflux, with no appreciable effect on transbilayer movement of drugs, the anesthetics chloroform, benzyl alcohol, diethyl ether and propofol modulated MDR by accelerating transbilayer movement of drugs, with no concomitant inhibition of Pgp‐mediated efflux. At higher concentrations than those required for modulation, the anesthetics accelerated the passive permeation to such an extent that it was not possible to estimate Pgp activity. The capacity of the surface‐active agents to accelerate passive drug transbilayer movement was not correlated with their fluidizing characteristics, measured as fluorescence anisotropy of 1‐(4‐trimethylammonium)‐6‐phenyl‐1,3,5‐hexatriene. This compound is located among the headgroups of the phospholipids and does not reflect the fluidity in the lipid core of the membranes where the limiting step of drug permeation, namely drug flip‐flop, occurs.

Chlorophylls as probes for membrane fusion. Polymyxin B-induced fusion of liposomes

Abstract Chlorophylls a and b exhibited efficient energy transfer when incorporated into liposomes, at concentrations greater than 2% of total lipids. The efficiency of energy transfer decreased when lower pigment concentrations were used. This phenomenon was utilized to construct an assay for membrane fusion. Liposomes containing photosynthetic pigments at 2% of total lipids were induced to fuse in the presence of excess non-pigmented liposomes. The fusion with the non-pigmented liposomes caused dilution of the pigments resulting in lower efficiency of energy transfer. The assay was applied successfully to cation-induced fusion of acidic liposomes. The extent of fusion depended both on cations and on the phosphatidylethanolamine content. The assay was used to demonstrate that the antibiotic polymyxin B was capable of inducing fusion. The efficiency of polymyxin B-induced fusion was extremely high, and on charge ratio basis it exceeded even that of polylysine. Acidic liposomes containing chlorophylls were incubated with Ehrlich ascites tumour cells in the presence of either Ca2+ or polylysine. This led to tight binding of liposomes to cells. However, the efficiency of energy transfer between the chlorophylls, associated with the cells, did not change. This result excluded fusion or net transfer of lipids as major modes of liposome-cell interaction.

Calcium-induced fusion of proteoliposomes and protein-free liposomes Effect of their phosphatidylethanolamine content on the structure of fused vesicles

The acidic phospholipid cardiolipin was shown to be very efficient in promoting calcium-induced fusion of proteoliposomes. The degree of fusion was dependent on the phosphatidylethanolamine content of the vesicles. Addition of CaCl2 to proteoliposomes containing phosphatidylcholine and cardiolipin but without phosphatidylethanolamine did not induce fusion. Fusion of cytochrome oxidase vesicles, containing less than 50 mol% phosphatidylethanolamine resulted in monolamellar vesicles with a diameter of about 200 nm. The vesicles could be induced to fuse further by establishing an osmotic pressure across their membranes. When proteoliposomes containing more than 50 mol% phosphatidylethanolamine were fused, large vesicles with a diameter exceeding 1 micrometer were formed. They appeared in the electron microscope as a mixture of multilamellar and monolamellar vesicles. Fusion of corresponding liposomes resulted in formation of even larger structures appearing as dense multilamellar bodies and paracrystalline honeycomb-like lattices.

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). Cells from the same patients at later phases exhibited a further increase in TMR resistance. Doxorubicin was excluded from all cell samples obtained from CML patients at presentation. The resistance to TMR and doxorubicin was energy‐dependent, and was not modulated by inhibitors of MDR1 and multidrug‐resistance protein‐1 (MRP1). Transcription of mRNAs suspected as relevant to multidrug resistance was assessed using comparative reverse transcription polymerase chain reaction. All cells from the CML patients transcribed high levels of MRP3, MRP4 and MRP5 compared with healthy donors. Low levels of MDR1, MRP1, MRP2, MRP6, lung resistance‐related protein and anthracycline resistance‐associated protein were equally transcribed in cells from healthy donors and CML patients. These results indicated that neither MDR1 nor MRP1 mediate the resistance in these cells. Our results shed light on a resistance mechanism operative in CML patients, which, together with the resistance to apoptosis, is responsible for the lack of response of CML patients to induction‐type protocols used to treat acute myeloid leukaemia patients.

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. The initial phase comprised a rapid drop in fluorescence that was neither affected by cooling the cells on ice, nor by activity of mitochondria or ABC transporters. This phase reflects the association of dye with the outer leaflet of the plasma membrane. The subsequent phase of TMA‐DPH fluorescence quenching occurred in drug‐sensitive cell lines with a half‐life in the range 20–40 s. The second phase of fluorescence quenching was abolished by incubation of the cells on ice and was transiently inhibited in cells with active mitochondria. Thus, the second phase of fluorescence quenching reflects the accumulation of dye in the cytoplasmic leaflet of the plasma membrane, presumably as a result of flip‐flop of dye across the plasma membrane and slow diffusion from the inner leaflet into the cells. Whereas activity of P‐glycoprotein prevented the second phase of fluorescence quenching, the activity of multidrug resistance‐associated protein 1 had no effect on this phase. Thus, P‐glycoprotein appears to pump rhodamines from the cytoplasmic leaflet either to the outer leaflet or to the outer medium.