Lipid packing and transbilayer asymmetries of mixed lipid vesicles.

Predictions, based on a previously developed theory, of the radii and asymmetric lipid distribution of mixed phosphatidylcholine/lysophosphatidylcholine and phosphatidylcholine/cholesterol vesicles of variable composition are presented. The results compare well with available experimental data, except for cis-unsaturated phosphatidylcholine/cholesterol vesicles at high concentrations of cholesterol. It is concluded that specific lipid-lipid interactions need not be invoked for saturated and trans-unsaturated phosphatidylcholine mixed with lysophosphatidylcholine or cholesterol. A discussion of the effect of packing stresses on induced flip-flop and non-spherical vesicles is also presented.     

Kinetics and mechanism of phosphatidylcholine and cholesterol exchange between chylomicrons and high-density lipoproteins.

The exchange of phosphatidylcholine and unesterified cholesterol between rat mesenteric lymph chylomicrons and human high-density lipoproteins was studied in vitro by incubation of radiolabelled chylomicrons (with [N-methyl-14C]phosphatidylcholine and [7(n)-3H]cholesterol) with unlabelled high-density lipoproteins. The kinetic analysis was based on the extent of radioisotope exchange, which was determined by the proportion of label appearing in the high-density lipoprotein elution peak after rapid fractionation on analytical agarose columns. Under our experimental conditions, no net transfer of either phosphatidylcholine or cholesterol is observed. The kinetics of exchange of both phosphatidylcholine and cholesterol are biphasic. Over the first 30 min a maximum of 25% of the phosphatidylcholine and 33% of the cholesterol in chylomicrons exchanges rapidly into the high-density-lipoprotein fraction. Thereafter both lipids continue to exchange for up to 3 h at a much lower rate. For the rapid exchange process the calculated exchange rates for phosphatidylcholine and cholesterol are proportional to the concentrations of both chylomicrons and high-density lipoproteins. The second-order rate constants are (10.5 +/- 0.5) X 10(-5) microM-1 X min-1 for phosphatidylcholine and (32.1 +/- 4.5) X 10(-5) microM-1 X min-1 for cholesterol. The kinetics of the exchange process thus suggest that a significant proportion of both phosphatidylcholine and unesterified cholesterol is rapidly exchangeable between these lipoproteins, and that this exchange is mediated by a 'bimolecular', or collisional, mechanism.     

Effects of cholesterol surface transfer on cholesterol and phosphatidylcholine synthesis in cultured rat arterial smooth muscle cells

The purpose of this study was to examine the effects of cholesterol surface transfer between lipid vesicles and rat arterial smooth muscle cells on endogenous synthesis of cholesterol and phosphatidylcholine. Lipid vesicles containing cholesterol and egg phosphatidylcholine in different proportions were used as the extracellular lipid source. The rate of cellular cholesterol and phosphatidylcholine synthesis was determined from the [14C]acetate incorporation into these lipid classes. [3H]Cholesterol in lipid vesicles, with a cholesterol/phospholipid (C/P) mole ratio of 1:1, was rapidly transferred into rat smooth muscle cells, with a half-time of about 3.6 hours in the absence of serum proteins. Incubation of cells for 5 hours with vesicles of a high C/P mole ratio (i.e. 1.5:1) at vesicle-cholesterol concentrations above 100 micrograms/ml resulted in a marked reduction of cellular cholesterol synthesis, whereas the rate of phosphatidylcholine synthesis was increased. Cells incubated with lipid vesicles of C/P 1:2 did not show any change in cellular cholesterol or phosphatidylcholine synthesis. Incubation of cells with egg phosphatidylcholine vesicles at concentrations above 300 micrograms/ml, on the other hand, stimulated endogenous synthesis of cholesterol without affecting cellular phosphatidylcholine synthesis. The main conclusion is that cholesterol surface transfer may influence cellular lipid metabolism in the absence of mediating serum lipoproteins in a model system with cultured cells and lipid vesicles.     

Possible approaches to creating phospholipid preparations--acceptors of membrane cholesterol

The capability of liposomes with plant phosphatidylcholine to extract cholesterol from erythrocyte membrane was investigated. It was shown, that plant phosphatidylcholine extracted 42% of membrane cholesterol, without causing haemolysis. This cholesterol extraction was responsible for the decrease of membrane microviscosity and for the increase of Na+, K+-ATPase activity. It is suggested that it is possible to create the extractive agent of cholesterol with plant phosphatidylcholine.     

Molecular interactions in the hydrogen belts of membranes glucose-6-phosphatase,lysophosphatidylcholine, and cholesterol

Microsomal glucose-6-phosphatase from rat liver is activated by phosphatidylcholine but inhibited by lysophosphatidylcholine. Inhibition occurs not by membrane lysis but in an intact bilayer; it is reversible; and it is overcome by addition of cholesterol but not if the cholesterol-hydroxyl group is blocked. An analog of lysophosphatidylcholine deprived of hydrogen bonding sites, 1-ether-2-deoxylysophosphatidylcholine, is a partial activator, and its effect on the enzyme in a phosphatidylcholine bilayer is not modulated by cholesterol. It appears to be one of the functions of cholesterol to buffer the lysophospholipids in membranes by complexing with them through hydrogen bonding in the hydrogen belt region. Lysophosphatidylcholine/cholesterol association is favored over phosphatidylcholine/cholesterol association.     

Is a role of phospholipase A(2) in cholesterol gallstone formation phospholipid species-dependent?

Phospholipase A(2) plays a role in cholesterol gallstone formation by hydrolyzing bile phospholipids into lysolecithin and free fatty acids. This study investigated its effects on cholesterol crystallization in model bile systems. Supersaturated model bile solutions with different cholesterol saturation indexes (1.2, 1.4, and 1.6) were prepared using cholesterol, taurocholate, and egg yolk phosphatidylcholine, soybean phosphatidylcholine, palmitoyl-oleoyl phosphatidylcholine, or palmitoyl-linoleoyl phosphatidylcholine. Then the effect of digestion of phosphatidylcholine by phospholipase A(2) on bile metastability was assessed by spectrophotometry and video-enhanced differential contrast microscopy. Addition of phospholipase A(2) caused the release of free fatty acids in a time-dependent manner. Cholesterol crystallization was enhanced by an increased crystal growth rate in model bile containing hydrophilic species such as soybean or palmitoyl-linoleoyl phosphatidylcholine, consisting predominantly of polyunsaturated fatty acids. Because phospholipase A(2) enhanced cholesterol crystallization in bile containing hydrophilic phosphatidylcholine species, but not hydrophobic phosphatidylcholine species, release of polyunsaturated fatty acids by hydrolysis may be responsible for such enhancement. Therefore, the role of phospholipase A(2) in cholesterol gallstone formation depends on the phospholipid species present in bile, so that phospholipid species selection during hepatic excretion is, in part, crucial to the cholesterol stone formation.     

Molecular interactions in the hydrogen belts of membranes. Glucose-6-phosphatase, lysophosphatidylcholine, and cholesterol

Microsomal glucose-6-phosphatase from rat liver is activated by phosphatidylcholine but inhibited by lysophosphatidylcholine. Inhibition occurs not by membrane lysis but in an intact bilayer; it is reversible; and it is overcome by addition of cholesterol but not if the cholesterol-hydroxyl group is blocked. An analog of lysophosphatidylcholine deprived of hydrogen bonding sites, 1-ether-2- deoxylysophosphatidylcholine , is a partial activator, and its effect on the enzyme in a phosphatidylcholine bilayer is not modulated by cholesterol. It appears to be one of the functions of cholesterol to buffer the lysophospholipids in membranes by complexing with them through hydrogen bonding in the hydrogen belt region. Lysophosphatidylcholine/cholesterol association is favored over phosphatidylcholine/cholesterol association.     

The effect of cholesterol on the structure of phosphatidylcholine bilayers

The effect of cholesterol on the structure of phosphatidylcholine bilayers was investigated by X-ray diffraction methods. Electron density profiles at 5 Å resolution along with chain tilt and chain packing parameters were obtained and compared for phosphatidylcholine/cholesterol bilayers and for pure phosphatidylcholine bilayers in both the gel and liquid crystalline states. The cholesterol in the bilayer was localized by noting the position of discrete elevations in the electron density profiles. Cholesterol can either increase or decrease the width of the bilayer depending on the physical state and chain length of the lipid before the introduction of cholesterol. For saturated phosphatidylcholines containing 12–16 carbons per chain, cholesterol increases the width of the bilayer as it removes the chain tilt from gel state lipids or increases the trans conformations of the chains for liquid crystalline lipids. However, cholesterol reduces the width of 18 carbon chain bilayers below the phase transition temperature as the long phospholipid chains must deform or kink to accomodate the significantly shorter cholesterol molecule. Although cholesterol has a marked effect on hydrocarbon chain organization, it was found that, within the resolution limits of the data, the phosphatidylcholine head group conformation is unchanged by the addition of cholesterol to the bilayer. The head group is oriented parallel to the plane of the bilayer for phosphatidylcholine in the gel and liquid crystalline states and this orientation is not changed by the addition of cholesterol.     

The rippled structure in bilayer membranes of phosphatidylcholine and binary mixtures of phosphatidylcholine and cholesterol

Freeze-fracture electron microscopy is used to study the rippled texture in pure dimyristoyl and dipalmitoyl phosphatidylcholine membranes and in mixtures of dimyristoyl phosphatidylcholine and cholesterol. Evidence is presented that the apparent phase transition properties of multilamellar liposomes may be dependent on the manner in which liposomes are prepared. Under certain conditions the ripple structures as visualized by freeze-fracture electron microscopy for the pure phosphatidylcholines are observed to be temperature dependent in the vicinity of the pretransition. Thus the transition can sometimes appear to be a gradual transition rather than a sharp, first-order phase transition. In mixtures of dimyristoyl phosphatidylcholine and cholesterol, the ripple repeat distance is found to increase as the cholesterol concentration is increased between 0 and 20 mol%. Above 20 mol%, no rippling is observed. A simple theory is presented for the dependence of ripple repeat spacing on cholesterol concentration in the range 0–20 mol%. This theory accounts for the otherwise inexplicable abrupt increase in the lateral diffusion coefficients of fluorescent lipids in binary mixtures of phosphatidylcholine and cholesterol when the cholesterol concentration is increased above 20 mol%.     

Molecular packing of cholesterol in phospholipid vesicles as probed by dehydroergosterol

Ergosta-5,7,9,22-tetraen-3-β-ol (dehydroergosterol) was synthesized and employed as a probe of cholesterol behavior in phospholipid bilayers. Circular dichroism (CD) spectra were obtained. The CD of dehydroergosterol in sonicated egg phosphatidylcholine vesicles was dependent on cholesterol concentration, while in unsonicated egg phosphatidylcholine liposomes and in vesicles obtained by oxctylglucoside dialysis, the CD observed was independent of cholesterol content. The CD of dehydroergosterol in sonicated sphingomyelin vesicles exhibited a different dependence on cholesterol content than seen in sonicated egg phosphatidylcholine vesicles. These data are interpreted in terms of differences between the packing of cholesterol in systems of large and small radii of curvature and in different interactions between dehydroergosterol and phosphatidylcholine and sphingomyelin.     

Interaction of cholesterol with sphingomyelin in mixed membranes containing phosphatidylcholine, studied by spin-label ESR and IR spectroscopies. A possible stabilization of gel-phase sphingolipid domains by cholesterol

The ESR spectra from different positional isomers of sphingomyelin and phosphatidylcholine spin-labeled in their acyl chain have been studied in sphingomyelin(cerebroside)-phosphatidylcholine mixed membranes that contain cholesterol. The aim was to investigate mechanisms by which cholesterol could stabilize possible domain formation in sphingolipid-glycerolipid membranes. The outer hyperfine splittings in the ESR spectra of sphingomyelin and phosphatidylcholine spin-labeled on the 5 C atom of the acyl chain were consistent with mixing of the components, but the perturbations on adding cholesterol were greater in the membranes containing sphingomyelin than in those containing phosphatidylcholine. Infrared spectra of the amide I band of egg sphingomyelin were shifted and broadened in the presence of cholesterol to a greater extent than the carbonyl band of phosphatidylcholine, which was affected very little by cholesterol. Two-component ESR spectra were observed from lipids spin-labeled on the 14 C atom of the acyl chain in cholesterol-containing membranes composed of sphingolipids, with or without glycerolipids (sphingomyelin/cerebroside and sphingomyelin/cerebroside/phosphatidylcholine mixtures). These results indicate the existence of gel-phase domains in otherwise liquid-ordered membranes that contain cholesterol. In the gel phase of egg sphingomyelin, the outer hyperfine splittings of sphingomyelin spin-labeled on the 14-C atom of the acyl chain are smaller than those for the corresponding spin-labeled phosphatidylcholine. In the presence of cholesterol, this situation is reversed; the outer splitting of 14-C spin-labeled sphingomyelin is then greater than that of 14-C spin-labeled phosphatidylcholine. This result provides some support for the suggestion that transbilayer interdigitation induced by cholesterol stabilizes the coexistence of gel-phase and "liquid-ordered" domains in membranes containing sphingolipids.     

Negatively charged phospholipids and their position in the cholesterol affinity sequence

The effects of low concentrations of cholesterol in mixtures of a negatively charged phospholipid (phosphatidylserine or phosphatidylglycerol) and another phospholipid (phosphatidylcholine, sphingomyelin or phosphatidylethanolamine) have been studied by differential scanning calorimetry. Only mixtures which showed a gel phase miscibility gap have been employed. It was demonstrated that in mixtures with phosphatidylethanolamine, cholesterol was preferentially associated with the negatively charged phospholipid, regardless whether this species represented the component with the high or with the low transition temperature in the mixture. In mixtures of a negatively charged phospholipid and phosphatidylcholine, cholesterol associated with the negatively charged phospholipid; when the phosphatidylcholine was the species with the low transition temperature, cholesterol had an affinity for the phosphatidylcholine and for the negatively charged phospholipid as well. Cholesterol, in a mixture of sphingomyelin with a high and phosphatidylserine with a low transition temperature, was preferentially associated with sphingomyelin.From these experiments it is concluded that phospholipids show a decrease in affinity for cholesterol in the following order: sphingomyelin ? phosphatidylserine, phosphatidylglycerol > phosphatidylcholine ? phosphatidylethanolamine.     

High density lipoprotein phospholipid composition is a major determinant of the bi-directional flux and net movement of cellular free cholesterol mediated by scavenger receptor BI

The role of high density lipoprotein (HDL) phospholipid in scavenger receptor BI (SR-BI)-mediated free cholesterol flux was examined by manipulating HDL(3) phosphatidylcholine and sphingomyelin content. Both phosphatidylcholine and sphingomyelin enrichment of HDL enhanced the net efflux of cholesterol from SR-BI-expressing COS-7 cells but by two different mechanisms. Phosphatidylcholine enrichment of HDL increased efflux, whereas sphingomyelin enrichment decreased influx of HDL cholesterol. Although similar trends were observed in control (vector-transfected) COS-7 cells, SR-BI overexpression amplified the effects of phosphatidylcholine and sphingomyelin enrichment of HDL 25- and 2.8-fold, respectively. By using both phosphatidylcholine-enriched and phospholipase A(2)-treated HDL to obtain HDL with a graded phosphatidylcholine content, we showed that SR-BI-mediated cholesterol efflux was highly correlated (r(2) = 0.985) with HDL phosphatidylcholine content. The effects of varying HDL phospholipid composition on SR-BI-mediated free cholesterol flux were not correlated with changes in either the K(d) or B(max) values for high affinity binding to SR-BI. We conclude that SR-BI-mediated free cholesterol flux is highly sensitive to HDL phospholipid composition. Thus, factors that regulate cellular SR-BI expression and the local modification of HDL phospholipid composition will have a large impact on reverse cholesterol transport.     

Haptenic activity of galactosyl ceramide and its topographical distribution on liposomal membranes. I. Effect of cholesterol incorporation

The relation between the immune-reaction of phosphatidylcholine liposomes containing spin-labeled galactosyl ceramide with or without cholesterol and the topographical distribution of the glycolipid in membranes was studied. In egg yolk phosphatidylcholine liposomes, both immune agglutination and antibody binding occurred, irrespectively of the presence of cholesterol, though the motion of the fatty acyl chain of spin-labeled galactosyl ceramide was restricted by cholesterol. In dipalmitoyl phosphatidylcholine liposomes, unlike in egg yolk phosphatidylcholine liposomes, the immune-reaction depended on the cholesterol content. The electron spin resonance (ESR) spectra of spin-labeled galactosyl ceramide in dipalmitoyl phosphatidylcholine liposomes indicated that cholesterol affected the topographical distribution of spin-labeled galactosyl ceramide in the liposomes. Without cholesterol, most of the spin-labeled galactosyl ceramide was clustered on the dipalmitoyl phosphatidylcholine membrane, but with increase of cholesterol, random distribution of hapten on the membrane increased. The cholesterol-dependent change in the topographical distribution of hapten on the membranes was parallel with that of immune reactivity. 'Aggregates' composed solely of galactosyl ceramide did not show any binding activity with antibody. The findings suggest that the recognition of galactosyl ceramide by antibody depended on the topographical distribution of hapten molecules. Phosphatidylcholine and/or cholesterol may play roles as 'spacers' for the proper distribution of 'active' haptens on the membranes. The optimum density of haptens properly distributed on liposomal membranes is discussed.     

Behavior of cholesterol and spin-labeled cholestane in model bile systems studied by electron spin resonance and synchrotron x-ray.

The behavior of mixed bile salt micelles consisting of sodium taurocholate, egg phosphatidylcholine, and cholesterol has been studied by ESR spin labeling and synchrotron x-ray scattering. Consistent with published phase diagrams, pure and mixed bile salt micelles have a limited capacity to incorporate and, hence, solubilize cholesterol. Excess cholesterol crystallizes out, a process that is readily detected both by ESR spin labeling using 3-doxyl-5 alpha-cholestane as a probe for cholesterol and synchrotron x-ray scattering. Both methods yield entirely consistent results. The crystallization of cholesterol from mixed bile salt micelles is indicated by the appearance of a magnetically dilute powder spectrum that is readily detected by visual inspection of the ESR spectra. Both the absence of Heissenberg spin exchange and the observation of a magnetically dilute powder spectrum provide evidence for the spin label co-crystallizing with cholesterol. In mixed bile salt micelles containing egg phosphatidylcholine, the solubility of cholesterol is increased as detected by both methods. With increasing content of phosphatidylcholine and increasing mole ratio cholesterol/phosphatidylcholine, the anisotropy of motion of the spin probe increases. The spin label 3-doxyl-5 alpha-cholestane is a useful substitute for cholesterol provided that it is used in dilute mixtures with excess cholesterol: the cholesterol/spin label mole ratio in these mixtures should be greater than 100. Despite the structural similarity between the two compounds, there are still significant differences in their physico-chemical properties.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of the bovine seminal plasma protein PDC-109 on cholesterol in the presence of phospholipids

The interaction of the major bovine seminal plasma protein PDC-109 with cholesterol was studied by employing spin-labelled analogues. It could be shown that PDC-109 does not interact directly with cholesterol molecules. However, in the presence of phospholipids we found a strong reduction of cholesterol motion by PDC-109. The fraction of immobilized cholesterol was largest for phosphorylcholine-containing lipids. This is consistent with the preferential interaction between PDC-109 and phosphatidylcholine. It is concluded that a stronger association and interaction of PDC-109 with phosphatidylcholine leads to an enhanced fraction of immobilized cholesterol analogues, but not to a phospholipid-dependent specific interaction between the protein and cholesterol. Moreover, the interaction of PDC-109 with various spin-labelled analogues of phosphatidylcholine (lysoPC, diacylPC) was investigated. In membranes of lipid vesicles the protein caused an immobilization of the phosphatidylcholine analogues mainly in the outer membrane leaflet, with no differences between diacylPC and lysoPC. The results are of relevance for understanding the physiological role of PDC-109 in the genesis of sperm cells.     

Cholesterol uptake of isolated rat hepatocytes is accelerated by several kinds of phosphatidylcholine

Uptake of cholesterol by isolated rat hepatocytes in a serum-free medium was remarkably enhanced by dispersion with several kinds of phosphatidylcholine. Of the various phosphatidylcholines tested, dilinoylphosphatidylcholine had the strongest accelerating effect, while dipalmitoylphosphatidylcholine was the weakest. The abilities to accelerate cholesterol uptake were in proportion to the content of unsaturated fatty acid in the phosphatidylcholine used. It was confirmed by electron microscopy that there is no relation between the size of the cholesterol-phosphatidylcholine complex and uptake. These data suggest that recognition of unsaturated fatty acids in phosphatidylcholine by isolated cells enhances uptake of cholesterol.     

Effect of liposomal phospholipid composition on cholesterol transfer between microsomal and liposomal vesicles.

Preincubation of rat liver microsomal vesicles at 37 degrees C in the presence of [3H]cholesterol/phospholipid liposomes results in a net transfer of cholesterol from liposomes to microsomal vesicles. This transfer follows first-order kinetics. For similar concentrations of the donor vesicles, rates of transfer are about 6-8 times lower with cholesterol/sphingomyelin liposomes compared with cholesterol/phosphatidylcholine liposomes. Also, transfer of cholesterol from cholesterol/sphingomyelin liposomes to microsomal vesicles reveals a larger activation energy than for the process from cholesterol/phosphatidylcholine liposomes. There is a significant correlation between the amount of liposomal cholesterol transferred to microsomal vesicles during preincubation and the increase found with acyl-CoA:cholesterol acyltransferase activity in these microsomes over their corresponding controls. If, however, liposomes made solely of phospholipids are substituted for the cholesterol/phospholipid liposomes in the preincubation system containing microsomal vesicles, then the acyl-CoA:cholesterol acyltransferase activity is decreased compared with the corresponding control system. Both sphingomyelin and phosphatidylcholine liposomes are equally effective in decreasing the enzyme activity. These results offer direct kinetic evidence for the positive correlation between cholesterol and sphingomyelin found in vivo in biological membranes.     

Human gallbladder mucin binds biliary lipids and promotes cholesterol crystal nucleation in model bile

The binding of phosphatidylcholine and cholesterol in model bile to human gallbladder mucin was studied by means of a rapid filtration binding assay and sucrose density gradient ultracentrifugation. Numerous low affinity binding sites for phosphatidylcholine and cholesterol were present on gallbladder mucin. Binding of phosphatidylcholine and cholesterol to mucin increased as a function of cholesterol saturation index. Proteolytic digestion of mucin disaggregated the native mucin polymer and removed hydrophobic domains on the mucin peptide core that bind l-anilino-8-naphthalenesulfonic acid. Proteolytic digestion also resulted in a 91% and 78% decrease, respectively, in the binding of phosphatidylcholine and cholesterol to mucin. The ability of trypsin-treated and native mucin to promote the nucleation of cholesterol monohydrate crystals was compared in a model bile. The incidence of cholesterol monohydrate crystal nucleation with native mucin was significantly greater at 3 days than with trypsin-treated mucin or controls (P less than 0.001). After 3, 6, and 9 days of incubation, samples containing native mucin contained significantly more crystals than controls or trypsin-digested mucin samples (P less than 0.0001 for each). These data indicate that highly purified human gallbladder mucin binds phosphatidylcholine and cholesterol in model bile. Furthermore, this study demonstrates that structural integrity of the native mucin polymer and hydrophobic domains on the peptide core are essential for the nucleation of cholesterol monohydrate crystals by mucin in model bile.     

Lateral distribution of cholesterol in membranes probed by means of a pyrene-labelled cholesterol: effects of acyl chain unsaturation

The lateral distribution of cholesterol in membranes in the fluid state was investigated by studying the variation of the molar absorption coefficient of pyrene-labelled cholesterol (Py-chol) vs. its concentration in vesicles made of phosphatidylcholine, with variable acyl chain unsaturations. Absorption measurements indicated non-ideal mixing of Py-chol in unsaturated lipids, a process mainly controlled by the cholesterol moiety of the probe. Similar abilities of cholesterol and Py-chol in perturbing the phase properties of pure saturated phosphatidylcholine were observed by DSC experiments. Immiscibility of sterols was corroborated by fluorescence polarization measurements, which indicated a weaker ordering effect of cholesterol in unsaturated membranes. The sizes and the quantities of sterol oligomers formed were calculated. A model for the lateral distribution of cholesterol in membranes is proposed and is applied to known cholesterol/phosphatidylcholine phase diagrams. Finally, the results are discussed with regard to recent models of biological membrane organization, (i.e. rafts).