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.

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).

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. A similar twofold acceleration of doxorubicin flip-flop rate across membranes was observed with neutral mild detergents, including Tween 20, Nonidet P-40 and Triton X-100, and certain Pgp modulators, such as verapamil and progesterone. Concentrations of these agents, similar to those required for membrane fluidization, inhibited Pgp ATPase activity in a mode similar to that observed with the anesthetics. The mode of inhibition, i.e. lack of evidence for classical enzyme inhibition and the correlation of Pgp ATPase inhibition with membrane fluidization over a wide range of concentrations and structures of drugs favors the direct inhibition of Pgp ATPase activity by membrane fluidization. The unusual sensitivity of Pgp to membrane fluidization, as opposed to acceleration of ATPase activity of ion transporters, could fit the proposed function of Pgp as a 'flippase', which is in close contact with the membrane core.

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. All the dyes tested were substrates of reconstituted P-glycoprotein and cellular P-glycoprotein and were excluded to a variable degree from MDR cells. The transmembrane movement rate proved the major factor determining the efficacy of the P-glycoprotein-mediated exclusion of rhodamine dyes from MDR cells. Thus, rhodamine B, the poorest cellular P-glycoprotein substrate, exhibited a high affinity toward reconstituted P-glycoprotein, but was the fastest membrane-traversing dye. In contrast, tetramethylrosamine, the best cellular MDR probe, exhibited high affinity toward reconstituted P-glycoprotein and slow transmembrane movement rate. Therefore, an anticancer drug with a fast transmembrane movement rate is expected to overcome the MDR phenomenon. Furthermore, the widely used MDR marker, rhodamine 123, was a poor cellular MDR substrate compared with other rhodamine dyes, especially tetramethylrosamine, which was a superior cellular MDR substrate for functional dye-exclusion studies.

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.

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. Transbilayer movement rates of drugs and modulators were estimated from their equilibration rate throughout artificial multilamellar vesicles. The equilibration rate of five selected modulators was faster than the equilibration rate of five representative MDR-type drugs tested, which was comparable with the rate of rhodamine 123 equilibration. Moreover, the carrier-type peptide ionophore, valinomycin, which is freely mobile in the membrane, inhibited Pgp-mediated efflux of rhodamine 123 from MDR cells. In contrast, the channel-forming ionophore gramicidin D, a Pgp substrate that flip-flops slowly across the membrane, did not modulate cellular Pgp activity. Pgp, with a turnover number of about 900 min-1 can keep pace with the influx of an MDR-drug like rhodamine 123 exhibiting a transbilayer movement with a lifetime of minutes. On the other hand, Pgp would fail to protect MDR cells against cytotoxic drugs that are freely mobile through biological membranes and that re-enter cells faster than their Pgp-mediated active efflux rate. The relatively fast transbilayer movement exhibited by MDR modulators suggest that in contrast to MDR-type drugs, MDR modulators traverse the plasma membrane faster than the maximal expulsion rate of Pgp.

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 micromol of Pi/min/mg of protein, suggesting the presence of an endogenous Pgp substrate. This basal ATPase activity was stimulated (up to 5.2 micromol of Pi/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.

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. J., Regev, R., and Assaraf, Y. G. (1994) J. Biol. Chem. 269, 26058-26065). P-glycoprotein mediated the ATP-dependent uptake of 86Rb+-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 86Rb+ 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.

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. I. (1993) J. Biol. Chem. 268, 4556-4566), differential screening of a cDNA library from pyrimethamine-resistant (PyrR100) cells was used. This library was screened with total cDNA from wild-type and PyrR100 cells. Surprisingly, several differentially overexpressed cDNA clones were isolated from PyrR100 cells, many of which mapped to the mitochondrial genome. Several lines of evidence establish mitochondria as a new target for the cytotoxic activity of pyrimethamine. (a) At > or = 10 microM, pyrimethamine inhibited mitochondrial respiration in viable wild-type cells. (b) Electron microscopy revealed degenerated mitochondrial membrane cristae in PyrR100 cells. (c) Some mitochondrially encoded transcripts were prominently elevated, whereas the normally stable 12 S/16 S rRNA was decreased in PyrR100 cells. (d) Metabolic pulse-chase labeling suggested an increased turnover rate of mitochondrially synthesized proteins in PyrR100 cells. (e) The specific activity of the key respiratory enzymatic complex cytochrome c oxidase was reduced by 6-fold in PyrR100 cells. (f) Consequently, the rate of respiration in intact PyrR100 cells was reduced by 3-fold. We conclude that pyrimethamine and possibly lipophilic analogues of methotrexate possess a folinic acid nonrescuable toxicity involving disruption of mitochondrial inner membrane structure and respiratory function, thereby establishing a new organellar target for the cytotoxic effect elicited by lipid-soluble antifolates.

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. These chemosensitizers yielded a marked increase in the accumulation of taxol in parental hamster and human carcinoma cells that are devoid of P-gp. These chemosensitizers and non-ionic detergents brought about a pronounced increase in the accumulation of structurally and mechanistically diverse lipophilic chromophores in parental and MDR hamster cells. Furthermore, non-toxic concentrations of these non-ionic detergents yielded a marked potentiation of taxol cytotoxicity in parental cells. These findings were consistent with a chemosensitizer-mediated, P-gp-independent increase in membrane permeability. Thus, several aspects of chemosensitizers' interaction with lipid bilayers and biomembranes were studied. In this respect, like various mild detergents, chemosensitizers induced a dose-dependent leakage of carboxyfluorescein encapsulated in liposomes. Like specialized membrane fluidizers, various chemosensitizers induced a dose-dependent membrane fluidization (and sometimes rigidification) in both liposomes and various wild-type and MDR animal and human cells, as revealed by diphenylhexatriene fluorescence polarization. Furthermore, a favorable correlation was observed between the ability of chemosensitizers to permeabilize lipid bilayers and their capacity to potentiate anticancer-drug cytotoxicity. Thus, we propose that chemosensitizer-mediated changes in the physical properties of biomembranes, including altered fluidity and increased permeability, may be important factors in achieving potentiation of anticancer-drug cytotoxicity in wild-type and MDR mammalian cells. This study offers a basis for the chemosensitizer-mediated potentiation of drug toxicity to healthy tissues, thus emphasizing the importance of a prior evaluation of the potential untoward toxicity when simultaneously using MDR chemosensitizers and cytotoxic agents in the clinic.

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. Therefore, P-glycoprotein was extracted from canalicular membrane vesicles and reconstituted into proteoliposomes that are relatively impermeable to cations. P-glycoprotein activity in the proteoliposomes was dependent on ATP hydrolysis since it was not observed with non-hydrolyzable analogs of ATP. Maximal ATP-dependent 86Rb+ uptake occurred at 50 nM gramicidin D and at 500 nM valinomycin thus possibly reflecting higher affinity of P-glycoprotein for gramicidin D. Nigericin, which does not participate in the multidrug resistance phenomenon, did not support an ATP-dependent uptake of 86Rb+. ATP hydrolysis increased the amount of 86RB+ transported into the proteoliposomes. Furthermore, preincubation of the proteoliposomes in the presence of gramicidin D and 86Rb+, allowing for maximal ATP-independent 86Rb+ uptake to occur, did not interfere with subsequent ATP-dependent uptake, indicating the latter to constitute an active transport mechanism. The ATP-dependent component of 86Rb+ uptake occurred neither with liposomes nor with proteoliposomes reconstituted with proteins extracted from sinusoidal vesicles that lack P-glycoprotein. The ATP-dependent uptake was blocked by the known inhibitors of the ATPase activity associated with P-glycoprotein, oligomycin and vanadate, as well as by its established substrates, daunorubicin, doxorubicin, vinblastine, and the tripeptide N-acetyl-leucyl-leucyl-norleucinal. Thus, the reconstituted P-glycoprotein catalyzes the ATP-dependent 86Rb+ uptake that appears to occur by an energy-dependent translocation of the 86Rb(+)-ionophore complex. In this case, the actual substrate of P-glycoprotein is the ionophore-cation complex, which is both hydrophobic and positively charged as are most of the substrates of P-glycoprotein. This is the first demonstration of transport of a naturally occurring polypeptide by proteoliposomes reconstituted with physiologically expressed P-glycoprotein.

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. It is concluded that the reconstituted transhydrogenase can utilize both a membrane potential and a proton gradient, separately or combined, where the relative contributions of these components depend on the activity of the ATPase. In the reconstituted vesicles, the maximally active transhydrogenase is apparently driven by an electrochemical proton gradient where the membrane potential and the proton gradient contribute one-third and two-thirds, respectively. The rate-dependent relative generation of a membrane potential and pH gradient presumably reflects the proton pump characteristics of the ATPase and/or buffering/permeability characteristics of the vesicles rather than the properties of the transhydrogenase per se. These results are discussed in relation to current models for transhydrogenase-linked proton translocation.

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. Drug concentrations at least one order of magnitude lower were needed to induce leakage from vesicles containing only phosphatidylcholine. Under these conditions, contents leakage occurred with no measurable aggregation or membrane intermixing. On the other hand, much higher concentrations of both drugs were required to induce leakage from vesicles containing predominantly cardiolipin. Release of contents occurred upon aggregation of the vesicles and collapse of the vesicular organization, as well as formation of paracrystalline structure when dodecylamine was employed or amorphous material when gramicidin A was used. In contradistinction to other model systems, phosphatidylcholine was needed for fusion induced by the cationic amphipaths, and its presence reduced the threshold concentration of the drugs needed to induce leakage of the contents. The similar effects of the two drugs on membranes imply that, at least in these model membranes, the relevant feature of both drugs is only their amphiphatic nature.

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]. In both of these respects, the transhydrogenase-ATPase vesicles were severalfold more efficient than beef heart submitochondrial particles. By measuring the ATP-driven transhydrogenase and the oligomycin-sensitive ATPase activities simultaneously and under the same conditions at low ATP concentrations, i.e. below 15 microM, the ATP-driven transhydrogenase/oligomycin-sensitive ATPase activity ratio was found to be about 3. This value is consistent with the stoichiometries of three protons translocated per ATP hydrolyzed and one proton translocated per NADPH formed and with a mechanism where the two enzymes interact through a delocalized proton-motive force.

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. Addition of valinomycin increased the pH shift twice with a concomitant 50% inhibition of the light-driven transhydrogenase, whereas nigericin inhibited the pH shift completely and the light-driven transhydrogenase partially. Taken together, these results suggest that transhydrogenase and bacteriorhodopsin interact through a delocalized proton-motive force. Possible partial reactions of transhydrogenase were investigated with transhydrogenase-bacteriorhodopsin vesicles energized by light. Reduction of oxidized 3-acetylpyridine adenine dinucleotide by NADH, previously claimed to represent partial reactions, was found to require NADPH. Similarly, reduction of thio-NADP+ by NADPH required NADH. It is concluded that these reactions do not represent partial reactions.

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. Reduction in the efficiency of energy transfer was temperature and time dependent, and was also dependent upon the ratio between the reconstituted Sendai virus envelopes (donor) and recipient cells (acceptor). No reduction in the efficiency of energy transfer was observed when non-fusogenic, reconstituted viral envelopes were incubated with cell membranes.

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. It is suggested that the high efficiency of melittin is due to its amphipathic nature. Its hydrophobic moiety is probably anchored in one liposome while the positively charged hydrophilic moiety attracts another liposome.

Interaction of acidic liposomes with red blood cells. Induction of endocytosis and shedding of particles.

Calcium ions induced tight binding of massive amounts of liposomes containing cardiolipin, phosphatidyl-ethanolamine and phosphatidylcholine to erythrocytes. Initially, liposome-liposome fusion occurred and only subsequently the resulting large structures adhered to cells. Large clumps composed of liposomes and cells were formed. Upon prolonged incubation, the clumps were dissipated spontaneously and excess liposomes were released. A constant amount of phospholipid (15-25 nmol/10(8) cells) were incorporated into the cell membranes. Upon disaggregation, the cells shed erythrocyte particles. The latter were isolated and shown to contain lipids from both cellular and liposomal origin. The particles lacked spectrins and contained variable amounts of band 3 content. Liposomes induced endocytosis in reticulocytes but not in mature erythrocytes. In most cases, the liposomes themselves were not engulfed by the cells and remained attached to their surface. The relevance of this phenomenon to delivery to liposome contents into cells is discussed.

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).