Effect of cholesterol on the valinomycin-mediated uptake of rubidium into erythrocytes and phospholipid vesicles

Human erythrocytes have been treated with lipid vesicles in order to alter the cholesterol content of the cell membrane. Erythrocytes have been produced with cholesterol concentrations between 33 and 66 mol% of total lipid. The rate of valinomycin-mediated uptake of rubidium into the red cells at 37°C was lowered by increasing the cholesterol concentration of the cell membrane. Cholesterol increased the permeability to valinomycin at 20°C of small (less than 50 nm), unilamellar egg phosphatidylcholine vesicles formed by sonication. Cholesterol decreased the permeability to valinomycin at 20°C of large (up to 200 nm) unilamellar egg phosphatidylcholine vesicles formed by freezethaw plus brief sonication. It is concluded that cholesterol increases the permeability of small membrane vesicles to hydrophobic penetrating substances while above the transition temperature but has the opposite effect on large membrane vesicles and on the membranes of even larger cells.     

Cholesterol transfer from small and large unilamellar vesicles

The rates of transfer of [14C]cholesterol from small and large unilamellar cholesterol/egg yolk phosphatidylcholine vesicles to a common vesicle acceptor were compared at 37 degrees C. The rate of exchange of cholesterol between vesicles of identical cholesterol concentrations (20 mol%) did not differ from the rate of transfer from donor vesicles containing 20 mol% cholesterol to egg yolk PC vesicles. Further, the rate of transfer of [14C]cholesterol from vesicles containing 15 mol% dicetyl phosphate (to confer a negative charge) was not different from the rate of transfer from neutral vesicles. However, the half-time for transfer of [14C]cholesterol from large unilamellar donor vesicles was about 5-times greater (10.2 h, 80 nm diameter) than from small unilamellar vesicles (2.3 h, 23 nm diameter). These data suggest that increased curvature in small unilamellar vesicles reduces cholesterol-nearest neighbor interactions to allow a more rapid transfer of cholesterol into the aqueous phase.     

The effects of charge and size on the interaction of unilamellar liposomes with macrophages

The effect of the positive surface charge of unilamellar liposomes on the kinetics of their interaction with rat peritoneal macrophages was investigated using three sizes of liposomes: small unilamellar vesicles (approx. 25 nm diameter), prepared by sonication, and large unilamellar vesicles (100 nm and 160 nm diameter), prepared by the Lipoprep dialysis method. Charge was varied by changing the proportion of stearylamine added to the liposomal lipids (egg phosphatidylcholine and cholesterol, molar ratio 10:2.5). Increasing the stearylamine content of large unilamellar vesicles over a range of 0-25 mol% enhanced the initial rate of vesicle-cell interaction from 0.1 to 1.4 microgram lipid/min per 10(6) cells, and the maximal association from 5 to 110 micrograms lipid/10(6) cells. Cell viability was greater than 90% for cells incubated with large liposomes containing up to 15 mol% stearylamine but decreased to less than 50% at stearylamine proportions greater than 20 mol%. Similar results were obtained with small unilamellar vesicles except that the initial rate of interaction and the maximal association were less sensitive to stearylamine content. The initial rate of interaction, with increasing stearylamine up to 25 mol%, ranged from 0.5 to 0.7 microgram lipid/min per 10(6) cells, and the maximal association ranged from 20 to 70 micrograms lipid/10(6) cells. A comparison of the number and entrapped aqueous volume of small and large vesicles containing 15 mol% stearylamine revealed that although the number of large vesicles associated was 100-fold less than the number of small vesicles, the total entrapped aqueous volume introduced into the cells by large vesicles was 10-fold greater. When cytochalasin B, a known inhibitor of phagocytosis, was present in the medium, the cellular association of C8-LUV was reduced approx. 25% but association of SUV increased approx. 10-30%. Modification of small unilamellar vesicles with an amino mannosyl derivative of cholesterol did not increase their cellular interaction over that of the corresponding stearylamine liposomes, indicating that cell binding induced by this glycolipid may be due to the positive charge of the amine group on the sugar moiety. The results demonstrate that the degree of liposome-cell interaction with macrophages can be improved by increasing the degree of positive surface charge using stearylamine. Additionally, the delivery of aqueous drugs to cells can be further improved using large unilamellar vesicles because of their greater internal volume. This sensitivity of macrophages to vesicle charge and size can be used either to increase or reduce liposome uptake significantly by this cell type     

Spontaneous cholesterol movement between lipid vesicles and monkey small intestinal brush border membrane

[14C]Cholesterol movement between egg phosphatidylcholine-cholesterol lipid vesicles and vesicles prepared from monkey small intestinal brush border membrane (BBMV) was studied in physiological buffer at 37 degrees C. The rate of cholesterol transfer from sonicated unilamellar vesicles (ULV) to BBMV follows apparently first-order kinetics. Intermembrane cholesterol movement was strikingly similar in both the directions. However, from BBMV to ULV, the transfer rate was three times faster than that of ULV to brush border membrane (BBM). Similarity in the rate constant was observed when cholesterol transfer was studied using either large multilamellar lipid vesicles or ULV as the donor and BBMV as the acceptor membrane. Rate constant was also the same when the acceptor membrane used was either intact BBMV or ULV prepared from BBM lipids. The rate of transfer of label was not affected even when the acceptor vesicle concentration was increased over fivefold, indicating the first-order nature of the reaction. Transfer of cholesterol from ULV to BBMV was accelerated by the presence of acetone, dimethyl sulfoxide (DMSO), deoxycholate, and papain. Partially purified nonspecific lipid-exchange protein increased the rate of cholesterol transfer by about threefold. Reduction in BBM cholesterol and phospholipid content was noted by DMSO, acetone, and deoxycholate, while papain caused a small depletion of membrane protein. Cholesterol transfer is temperature dependent with an activation energy of 31 kJ X mol-1, which is almost identical in the presence or absence of nonspecific lipid-exchange protein. The molecular mechanism of intermembrane cholesterol movement is discussed in view of the kinetic data obtained.     

Uptake of antineoplastic agents into large unilamellar vesicles in response to a membrane potential

Many drugs exhibit lipophilic and cationic (basic) characteristics. Previous studies have shown that lipophilic cations can be accumulated into model membrane 'liposomal' (vesicular) systems in response to establishing a membrane potential (inside negative) across the vesicle membrane. We demonstrate here that the anticancer drugs, adriamycin and vinblastine, can be rapidly accumulated into egg phosphatidylcholine large unilamellar vesicles in response to a valinomycin-dependent K+ diffusion potential (delta psi) to achieve high effective interior concentrations. Further, trapping efficiencies approaching 100% can be easily achieved. The influence of lipid composition and the requirement for valinomycin have been examined for adriamycin. Equimolar cholesterol levels inhibit the uptake process at 20 degrees C. However, incubation at higher temperature results in enhanced uptake. Similarly, the presence of egg phosphatidylserine or incubation at elevated temperatures results in significant adriamycin uptake in the absence of valinomycin. It is shown that the adriamycin retention time in the vesicles is enhanced by an order of magnitude or more when actively trapped by the presence of a membrane potential in comparison to passive trapping procedures. It is suggested that such active trapping procedures may be of use for loading liposomal systems for drug delivery applications, and may provide avenues for controlled release of encapsulated material.     

The effect of lipid composition on the relaxivity of Gd-DTPA entrapped in lipid vesicles of defined size

The effects of lipid composition on the relaxivity of gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA) entrapped in lipid vesicles has been examined for vesicles of different sizes composed of egg phosphatidylcholine and cholesterol in various molar ratios, as well as the stability of those same vesicles in human serum at 37 degrees C. It is found that the incorporation of cholesterol decreases the apparent relaxivity of the entrapped Gd-DTPA, concomitant with an increase in vesicle stability in serum. Cholesterol has little effect on relaxivity when incorporated at ratios up to 20 mole percent, but has an increasing effect at higher mole percentages. These results correlate with the known effects of cholesterol on the osmotic water permeability coefficients of various model membrane systems and suggest that it is the water flux across the vesicle bilayer that is limiting to the T1 relaxivity of the entrapped Gd-DTPA. The incorporation of up to 20 mole percent cholesterol has little effect on the stability of the vesicles in serum, whereas vesicles containing more than 20 mole percent cholesterol show greater increases in stability. It was also found that the stability of vesicles depends upon the size of the vesicles; smaller vesicles are less stable in human serum at 37 degrees C than larger vesicles.     

Interaction of calcium and cholesterol sulphate induces membrane destabilization and fusion: implications for the acrosome reaction

Cholesterol sulphate is a potent stabilizer of membrane bilayer structure in both dielaidoylphosphatidylethanolamine and egg phosphatidylethanolamine model membranes, however, the addition of calcium abolishes this bilayer stabilization. Calcium also induces fusion and leakage of egg phosphatidylethanolamine large unilamellar vesicles containing cholesterol sulphate, but has no effect on fusion or leakage of egg phosphatidylcholine large unilamellar vesicles containing cholesterol sulphate. With egg phosphatidylethanoiamine liposomes, the initial rate, and extent of fusion, at constant calcium concentration, vary inversely with the mol percentage of cholesterol sulphate present in the vesicle membrane. The interaction of calcium and cholesterol sulphate, which causes membrane destabilization and fusion in phosphatidylethanolamine containing model systems, may play a role in the acrosome reaction in human sperm.     

Binding of antigen-bearing fluorescent liposomes to the murine myeloma tumor MOPC 315.

Small unilamellar lipid vesicles bearing the DNP-hapten on their surfaces and containing the water-soluble fluorescent dye carboxyfluorescein were formed by sonication. These vesicles were incubated with cells from the murine myeloma tumor MOPC 315, which secrete and also bear on the cell surface an immunoglobulin with affinity for the nitrophenyl hapten. At 0 degrees C the cells bound an average of several thousand vesicles at saturation. This binding was specific for the nitrophenyl hapten on the vesicle since it was abolished by an excess of soluble nitrophenyl derivative, by omission of the hapten from the vesicle, or by substitution for MOPC 315 of a tumor lacking receptors for the nitrophenyl hapten. Specific binding of vesicles was greater when cells were incubated at 37 degrees C. The study suggests that ligand-bearing vesicles can be a useful marker for cell surface immunoglobulin. However, in spite of the ability to "target" vesicles to cell surface determinants, binding did not result in increased delivery of vesicle contents to the cytoplasm.     

Cholesterol Stimulation of Penetration of Unilamellar Liposomes by Hydrophobic Compounds.

The incorporation of cholesterol into unilamellar liposomes greatly increased the transmembranous movement of hydrophobic ionophores such as nigericin. In reconstituted liposomes containing rhodopsin as the only protein, the presence of cholesterol lowers by 10-fold or more the amount of negericin required to eliminate the light-driven proton gradient. These effects are seen both above and below the transition temperature of the phospholipid used for reconstitution. Cholesterol similarly increases the ability of A-23187, 1799, or NH4SCN to collapse the proton gradient of bacteriorhodopsin vesicles. Cholesterol also lowers the concentration of nigericin or valinomycin required for a rapid translocation of Rb+ into protein-free liposomes. It also lowers the concentration of A-23187 required for the release of Ca45 trapped in protein-free liposomes. In contrast to these observations and in confirmation of previous findings, we observed that cholesterol decreased the permeability of liposomes for glucose. Thus the effects of cholesterol on the permeability of the membrane vary with the chemical nature of the permeating compounds. We have also confirmed that in multilamellar liposomes cholesterol decreases the permeability of Rb+ in the presence of valinomycin. It therefore appears that the effect of cholesterol changes with the size and structural features of the model membranes.     

Cholesterol movement between human skin fibroblasts and phosphatidylcholine vesicles

Cholesterol readily exchanges between human skin fibroblasts and unilamellar phospholipid vesicles. Only a fraction of the exchangeable cholesterol and only 10–15% of the total cellular free cholesterol is available for net movement or depletion to cholesterol-free phosphatidylcholine vesicles. [¹⁴C]Cholesterol introduced into the fibroblast plasma membrane by exchange from lipid vesicles does not readily equilibrate with fibroblast cholesterol labelled endogenously from [³H]mevalonic acid. While endogenously-synthesized [³H]cholesterol readily becomes incorporated into a pool of esterified cholesterol, little, if any, of the [¹⁴C]cholesterol introduced into the fibroblast plasma membrane by exchange from lipid vesicles becomes available for esterification. We interpret these findings as suggesting that: (1) net cholesterol movement from fibroblasts to an acceptor membrane is limited to a small percentage of the plasma membrane cholesterol, and (2) separate pools of cholesterol exist in human skin fibroblasts, one associated with the plasma membrane and the second associated with intracellular membranes, and equilibration of cholesterol between the two pools is a very limited process.     

The inhibition of erythrocyte glyceraldehyde-3-phosphate dehydrogenase. In situ PMR studies

In order to investigate the role of the plasma membrane in determining the kinetics of removal of cholesterol from cells, the efflux of [3H]cholesterol from intact cells and plasma membrane vesicles has been compared. The release of cholesterol from cultures of Fu5AH rat hepatoma and WIRL-3C rat liver cells to complexes of egg phosphatidylcholine (1 mg/ml) and human high-density apolipoprotein is first order with respect to concentration of cholesterol in the cells, with half-times (t 1/2) for at least one-third of the cell cholesterol of 3.2 +/- 0.6 and 14.3 +/- 1.5 h, respectively. Plasma membrane vesicles (0.5-5.0 micron diameter) were produced from both cell lines by incubating the cells with 50 mM formaldehyde and 2 mM dithiothreitol for 90 min. The efflux of cholesterol from the isolated vesicles follows the same kinetics as the intact, parent cells: the t 1/2 values for plasma membrane vesicles of Fu5AH and WIRL cells are 3.9 +/- 0.5 and 11.2 +/- 0.7 h, respectively. These t 1/2 values reflect the rate-limiting step in the cholesterol efflux process, which is the desorption of cholesterol molecules from the plasma membrane into the extracellular aqueous phase. The fact that intact cells and isolated plasma membranes release cholesterol at the same rates indicates that variations in the plasma membrane structure account for differences in the kinetics of cholesterol release from different cell types. In order to investigate the role of plasma membrane lipids, the kinetics of cholesterol desorption from small unilamellar vesicles prepared from the total lipid isolated from plasma membrane vesicles of Fu5AH and WIRL cells were measured. Half-times of cholesterol release from plasma membrane lipid vesicles of Fu5AH and WIRL cells were the same, with values of 3.1 +/- 0.1 and 2.9 +/- 0.2 h, respectively. Since bilayers formed from isolated plasma membrane lipids do not reproduce the kinetics of cholesterol efflux observed with the intact plasma membranes, it is likely that the local domain structure, as influenced by membrane proteins, is responsible for the differences in t 1/2 values for cholesterol efflux from these cell lines.     

Relationships between membrane lipid composition and biological properties of rat myocytes. Effects of aging and manipulation of lipid composition

Cultures of newborn rat heart myocytes undergo major age-related alterations as demonstrated by comparing 5-6-day-old cells ("young cells") and 14-15-day-old cells ("old cells"). This includes: changes from spherical to elongated shape; sphingomyelin and cholesterol level/cell increase by 100% and 50%, respectively, while the phosphatidylcholine is reduced by 15-20% with almost no change in content of total phospholipids. There is a 50% increase in total protein content/cell while DNA content remain constant. The specific activity of seven marker enzymes representing most subcellular organelles is increased. Beating rate is reduced from 160 +/- 20 to 20 +/- 20 beats min-1. All the above age-dependent alterations are affected by modification of cellular polar lipid composition. Small unilamellar vesicles of egg phosphatidylcholine added to the growth medium of old cells serve as donor of egg phosphatidylcholine to the cells and as acceptor of cellular sphingomyelin and cholesterol. Sphingomyelin-phospholipid exchange can be separated from cholesterol depletion either by using vesicles of egg phosphatidylcholine/cholesterol mixtures which serve only in the phospholipid exchange process, or by small unilamellar vesicles of sphingomyelin which act only as efficient cholesterol acceptors. Such experiments indicated that the major response of old cells is to alteration in the phosphatidylcholine to sphingomyelin mole ratio, while changes in the cholesterol level induce smaller effects. Thus, reversal of phosphatidylcholine to sphingomyelin mole ratio to the values shown by young cells reverse cellular functions and features which were altered by cell aging to levels found in young cells. This includes: increase in the beating rate back to 160 +/- 20, reduction in the total protein level and in the specific activity per DNA content of seven marker enzymes and reappearance of spherical cell shape. These results suggest that membrane lipid composition has major influence on cellular properties which as described in the accompanying paper (Yechiel, E., Barenholz, Y., and Henis, Y. I. (1985) J. Biol. Chem. 260, 9132-9136), may be mediated through the organization and dynamics of the cell membranes.     

Movement of cholesterol between vesicles prepared with different phospholipids or sizes

The rates of exchange of [4-14C]cholesterol between lipid vesicles prepared with different phospholipids and with different sizes have been measured. The first-order rate constants were higher using vesicles prepared from phosphatidylcholines with highly branched or polyunsaturated fatty acyl chains than with saturated diacyl or di-O-alkyl chains. The rate measurements indicate that the affinity of cholesterol for phospholipid does not vary significantly on change of the type of linkage (ether or ester) in phosphatidylcholine (PC) or of the positions of the fatty acyl chains in 1,2-diacyl-PC bearing one saturated and one unsaturated chain; furthermore, egg phosphatidylglycerol and egg phosphatidylethanolamine appear to have comparable affinities for cholesterol. However, the molecular packing in the bilayer and nearest-neighbor interactions involving cholesterol appear tightened more by N-palmitoylsphingomyelin than by dipalmitoyl-PC; on incorporation of 44 mol % of these phospholipids (which have the same fatty acyl chain composition) into either small or large unilamellar vesicles prepared with egg phosphatidylglycerol, the exchange rates were strikingly slower when the donor species contained sphingomyelin compared with PC. The rate of cholesterol exchange was 100% faster with small unilamellar vesicles than with large unilamellar vesicles as donors, suggesting that the looser packing in the highly curved small vesicles facilitates cholesterol desorption. The cholesterol exchange rate did not vary with the size of the acceptor vesicles, which indicates that desorption is the rate-limiting step in the exchange process in the presence of excess acceptors.     

Investigation of polyene macrolide antibiotic-induced permeability changes in vesicles by 31P nuclear magnetic resonance

The permeability of egg yolk lecithin (EYL) vesicles to Pr3+ has been measured by 31P nuclear magnetic resonance (nmr) spectroscopy. Measurable Pr3+ leakage into the internal aqueous compartment of EYL vesicles at ambient (21 degrees C) temperature required the presence of small (7--10 mol%) amounts of dicetyl phosphate (DCP). The permeability of DCP-containing vesicles is decreased by incorporation of sterol (cholesterol greater than ergosterol approximately 5.6-dihydroergosterol greater than zymosterol) into the lipid bilayer. Addition of the polyene macrolide antibiotic, nystatin, to DCP-containing EYL vesicles with and without sterol resulted in increased Pr3+ permeability at the three temperatures studied (21--37.5 degrees C). Permeability changes observed upon addition of nystatin to sterol-impregnated, DCP-containing vesicles varied with sterol structure: ergosterol approximately 5,6-dihydroergosterol greater than cholesterol approximately zymosterol. These results are compared with other polyene macrolide induced permeability changes on model and natural membrane systems. Permeability changes induced by nystatin in sterol-free EYL vesicles were generally greater than for comparable sterol-containing vesicles. This is attributed to a nonspecific interaction of the antibiotic with the latter vesicles.     

Comparison of the enthalpy state of vesicles of different size by their interaction with alpha-lactalbumin

The characteristics of small unilamellar, large unilamellar and large multilamellar vesicles of dimyristoylphosphatidylcholine and their interaction with alpha-lactalbumin are compared at pH 4. (1) By differential scanning calorimetry and from steady-state fluorescence anisotropy data of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene it is shown that the transition characteristics of the phospholipids in the large unilamellar vesicles resemble more those of the multilamellar vesicles than of the small unilamellar vesicles. (2) The size and composition of the lipid-protein complex formed with alpha-lactalbumin around the transition temperature of the lipid are independent of the vesicle type used. Fluorescence anisotropy data indicate that in this complex the motions of the lipid molecules are strongly restricted in the presence of alpha-lactalbumin. (3) The previous data and a comparison of the enthalpy changes, delta H, of the interaction of the three vesicle types with alpha-lactalbumin allow us to derive that the enthalpy state of the small unilamellar vesicles just below 24 degrees C is about 24 kJ/mol lipid higher than the enthalpy state of both large vesicle types at the same temperature. The abrupt transition from endothermic to exothermic delta H values around 24 degrees C for large vesicles approximates the transition enthalpy of the pure phospholipid.     

Interaction of short-chain lecithin with long-chain phospholipids: characterization of vesicles that form spontaneously

Stable unilamellar vesicles formed spontaneously upon mixing aqueous suspensions of long-chain phospholipid (synthetic, saturated, and naturally occurring phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin) with small amounts of short-chain lecithin (fatty acid chain lengths of 6-8 carbons) have been characterized by using NMR spectroscopy, negative staining electron microscopy, differential scanning calorimetry, and Fourier transform infrared (FTIR) spectroscopy. This method of vesicle preparation can produce bilayer vesicles spanning the size range 100 to greater than 1000 A. The combination of short-chain lecithin and long-chain lecithin in its gel state at room temperature produces relatively small unilamellar vesicles, while using long-chain lecithin in its liquid-crystalline state produces large unilamellar vesicles. The length of the short-chain lecithin does not affect the size distribution of the vesicles as much as the ratio of short-chain to long-chain components. In general, additional short-chain decreases the average vesicle size. Incorporation of cholesterol can affect vesicle size, with the solubility limit of cholesterol in short-chain lecithin micelles governing any size change. If the amount of cholesterol is below the solubility limit of micellar short-chain lecithin, then the addition of cholesterol to the vesicle bilayer has no effect on the vesicle size; if more cholesterol is added, particle growth is observed. Vesicles formed with a saturated long-chain lecithin and short-chain species exhibit similar phase transition behavior and enthalpy values to small unilamellar vesicles of the pure long-chain lecithin prepared by sonication. As the size of the short-chain/long-chain vesicles decreases, the phase transition temperature decreases to temperatures observed for sonicated unilamellar vesicles. FTIR spectroscopy confirms that the incorporation of the short-chain lipid in the vesicle bilayer does not drastically alter the gauche bond conformation of the long-chain lipids (i.e., their transness in the gel state and the presence of multiple gauche bonds in the liquid-crystalline state).     

Cryoelectron microscopy of a nucleating model bile in vitreous ice: formation of primordial vesicles

Because gallstones form so frequently in human bile, pathophysiologically relevant supersaturated model biles are commonly employed to study cholesterol crystal formation. We used cryo-transmission electron microscopy, complemented by polarizing light microscopy, to investigate early stages of cholesterol nucleation in model bile. In the system studied, the proposed microscopic sequence involves the evolution of small unilamellar to multilamellar vesicles to lamellar liquid crystals and finally to cholesterol crystals. Small aliquots of a concentrated (total lipid concentration = 29.2 g/dl) model bile containing 8.5% cholesterol, 22.9% egg yolk lecithin, and 68.6% taurocholate (all mole %) were vitrified at 2 min to 20 days after fourfold dilution to induce supersaturation. Mixed micelles together with a category of vesicles denoted primordial, small unilamellar vesicles of two distinct morphologies (sphere/ellipsoid and cylinder/arachoid), large unilamellar vesicles, multilamellar vesicles, and cholesterol monohydrate crystals were imaged. No evidence of aggregation/fusion of small unilamellar vesicles to form multilamellar vesicles was detected. Low numbers of multilamellar vesicles were present, some of which were sufficiently large to be identified as liquid crystals by polarizing light microscopy. Dimensions, surface areas, and volumes of spherical/ellipsoidal and cylindrical/arachoidal vesicles were quantified. Early stages in the separation of vesicles from micelles, referred to as primordial vesicles, were imaged 23-31 min after dilution. Observed structures such as enlarged micelles in primordial vesicle interiors, segments of bilayer, and faceted edges at primordial vesicle peripheries are probably early stages of small unilamellar vesicle assembly. A decrease in the mean surface area of spherical/ellipsoidal vesicles was correlated with the increased production of cholesterol crystals at 10-20 days after supersaturation by dilution, supporting the role of small unilamellar vesicles as key players in cholesterol nucleation and as cholesterol donors to crystals. This is the first visualization of an intermediate structure that has been temporally linked to the development of small unilamellar vesicles in the separation of vesicles from micelles in a model bile and suggests a time-resolved system for further investigation.     

Cholesterol stimulation of penetration of unilamellar liposomes by hydrophobic compounds

Summary The incorporation of cholesterol into unilamellar liposomes greatly increased the transmembranous movement of hydrophobic ionophores such as nigericin. In reconstituted liposomes containing rhodopsin as the only protein, the presence of cholesterol lowers by 10-fold or more the amount of nigericin required to eliminate the light-driven proton gradient. These effects are seen both above and below the transition temperature of the phospholipid used for reconstitution.Cholesterol similarly increases the ability of A-23187, 1799, or NH₄SCN to collapse the proton gradient of bacteriorhodopsin vesicles. Cholesterol also lowers the concentration of nigericin or valinomycin required for a rapid translocation of Rb⁺ into protein-free liposomes. It also lowers the concentration of A-23187 required for the release of Ca⁴⁵ trapped in protein-free liposomes. In contrast to these observations and in confirmation of previous findings, we observed that cholesterol decreased the permeability of liposomes for glucose. Thus the effects of cholesterol on the permeability of the membrane vary with the chemical nature of the permeating compounds. We have also confirmed that in multilamellar liposomes cholesterol decreases the permeability of Rb⁺ in the presence of valinomycin. It therefore appears that the effect of cholesterol changes with the size and structural features of the model membranes.     

Factors contributing to the distribution of cholesterol among phospholipid vesicles

The distribution of cholesterol between vesicles of different lipid composition at equilibrium has been determined. Small, sonicated unilamellar vesicles and large unilamellar vesicles were incubated at a defined temperature, and aliquots were then obtained at selected times for analysis. Inclusion of a small amount of phosphatidylserine or phosphatidylinositol in the membrane does not appreciably affect the distribution of cholesterol at equilibrium by these measurements. A membrane in the gel state is a poor acceptor of cholesterol. The length of the hydrocarbon chain on the phospholipid may also play a role. Bovine brain sphingomyelin dramatically slows the kinetics of cholesterol transfer, and the equilibrium distribution of cholesterol among vesicles containing sphingomyelin is therefore not observable in these experiments. Data obtained with vesicles containing phosphatidylethanolamine indicate a preference of cholesterol for vesicles composed of phosphatidylcholine compared to vesicles consisting primarily of phosphatidylethanolamine, at equilibrium. Experiments with a chaotropic agent indicate that the nature of the surface of the phosphatidylethanolamine bilayer, and its hydration, are important factors in the distribution of cholesterol among membranes in which phosphatidylethanolamine is present. These data suggest that membrane lipid content may play a role in the distribution of cholesterol among the membranes of a cell.     

Protein-induced fusion of phospholipid vesicles of heterogeneous sizes.

We have investigated the fusion of phospholipid vesicles induced by lysozyme and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Vesicles were composed of dimyristoylphosphatidylcholine/dioleoylphosphatidylethanolamine/ cholesterol (DMPC:DOPE:Chol, 2:1:1). Small unilamellar vesicles (SUV, diameter ca. 30 nm) obtained by extensive sonication or large unilamellar vesicles (LUV, diameters ranged from 100 to 400 nm) obtained by extrusion methods were used. Fusion of LUV induced by lysozyme and GAPDH was drastically decreased when the diameter of the vesicles increased over a value of 100 nm. Lysozyme effect was stopped at the aggregation step while GAPDH effect was stopped at the fusion (lipid mixing) step. Fusion of heterogeneous vesicle populations (SUV with LUV) was observed only with GAPDH and this happened only when the lipids were in the liquid-crystalline state.