J.G. Mandersloot

Lipid composition and permeability of liposomes.

Abstract 1. 1. Layered latticed liquid crystals (liposomes) in dilute electrolyte solutions have been prepared from a number of synthetic lecithins with variations in length and number of double bonds of the paraffin chains. Using the property of these structures to act as practical, ideal osmometers, their permeability behaviour towards glycerol and erythritol has been studied. The penetration of such non-electrolytes into the liposome appears to be strongly temperature dependent in a way similar to that demonstrated for glycerol permeation into erythrocytes. 2. 2. Introduction of double bonds into the paraffin chains causes a marked enhancement of the permeability. The diffusion rate of glycerol into the liposomes of (distearoyl)lecithin, (1-stearoyl-2-oleoyl)lecithin, (dioleoyl)lecithin and (dilineoyl)lecithin, increases in that order. 3. 3. Decreasing chain length also increases the permeability which is demonstrated by comparing the swelling rate of liposomes prepared from (distearoyl)lecithin, (dipalmitoyl)lecithin and (dimyristoyl)lecithin and that of those prepared from (1-stearoyl-2-myristoyl)lecithin and (1-stearoyl-2-decanoyl)lecithin. 4. 4. Comparison of the permeability of the liposomes from (1-stearoyl-2-decanoyl)lecithin and (1-stearoyl-2-myristoyl)lecithin with those obtained from (dimyristoyl)lecithin and (dipalmitoyl)lecithin, respectively, suggests that, certainly at lower temperatures, the model structures of phospholipids with asymmetric chains are much more permeable than those of lecithins with chains of equal length but with the same total number of paraffin carbon atoms. 5. 5. Liposomes of mixtures of phospholipid and cholesterol normally demonstrate a decrease in permeability which is proportional to the concentration of cholesterol.

Effects of lysophosphatidylcholines on phosphatidylcholine and phosphatidylcholine/cholesterol liposome systems as revealed by 31P-NMR, electron microscopy and permeability studies

(1) The effect of incorporation of different lysophosphatidylcholine species on the structure, barrier properties and dynamics of bilayers made of various phosphatidylcholines both the presence and absence of cholesterol have been investigated by 31P-NMR, freeze-fracture electron microscopy and K+-permeability measurements. (2) In a dispersion of lysophosphatidylcholine : cholesterol (1 : 1) the lipids are organized in extended bilayers. Upon cooling a micellar solution of 1-palmitoyllysophosphatidylcholine below the chain-melting temperature a transition to a lamellar, most likely interdigitating organization is observed. 31P-NMR shows in both situations a marked decrease in effective chemical shift anisotropy. (3) 1-Palmitoyllysophosphatidylcholine can be incorporated up to 30 mol% into liquid crystalline bilayers of dipalmitoylphosphatidylcholine and up to 35 mol% into dioleoylphosphatidylcholine bilayers. Above this concentration micellization of the bilayers occurs. In the gel state, bilayer structure is maintained up to 60 mol% of the lysocompound. (4) 1-Oleoyllysophosphatidylcholine can be incorporated to higher concentrations into liquid crystalline phosphatidylcholine bilayers than the palmitoyl analogue, which can be explained by the more cylindrical shape of the 1-oleoyllysophosphatidylcholine. (5) In marked contrast, incorporation of only 1 mol% of 1-oleoyllysophosphatidylcholine into gel state dipalmitoylphosphatidylcholine already destabilizes bilayer structure and makes the membranes completely permeable for K+. These results are discussed with respect to the mixing properties of the various lysophosphatidylcholines. (6) In general these effects are accompanied by a loss of K+-permeability barrier, which however occurs at lower lysophosphatidylcholine concentrations than needed for the start of micellization. (7) Cholesterol incorporation counteracts the bilayer destabilizing role of lysophosphatidylcholines. (8) 31P-NMR demonstrates with increasing lysophosphatidylcholine concentrations in the bilayers of phosphatidylcholines a decrease in the effective chemical shift anisotropy. As the rigid lattice spectra of lysophosphatidylcholine and phosphatidylcholine are identical, this reflects a change in the conformational and/or motional properties of the phospholipid head groups. This phenomenon might play a role in the observed permeability changes.

On the mechanism of non-electrolyte permeation through lipid bilayers and through biomembranes

Abstract The temperature effects on the permeation of polyhydroxy alcohols through the lipid bilayers of liposomes with a great variety in chemical composition were studied. Although important differences in the permeability of the various lipid bilayers were observed, Arrhenius plots demonstrated that the activation energy is independent of the degree of unsaturation or the presence of cholesterol in the paraffin barriers. The activation energies found for the penetration of a bilayer with a liquid paraffin core are 14.3 kcal for glycol, 19.4 kcal for glycerol, and 20.8 kcal for erythritol. These values are in agreement with the energies that can be expected for complete dehydration of the permeant molecules. The idea that the activation energy is determined by the number of hydrogen bonds with water is supported by the finding that a series of different diols did demonstrate practically identical activation energies. Studies on a number of biological membranes demonstrated the same activation energies for the penetration of glycerol and erythritol as found in the experiments with liposomes. These facts support the view that both the lipid bilayers and the biological membranes are penetrated by single fully dehydrated molecules.

Phosphatidylinositol as the endogenous activator of the (Na+ + K+)-ATPase in microsomes of rabbit kidney

Incubation of rabbit kidney microsomes with pig pancreatic phospholipase A2 produced residual membrane preparations with very low (Na+ + K+)-ATPase activity. The activity could be restored by recombination with lipid vesicles of negatively-charged glycerophospholipids. Vesicles of pure phosphatidylcholine and phosphatidylethanolamine were virtually inactive in this respect, but could reactivate in the presence of cholate. Incubation of the microsomes with a combination of phospholipase C (Bacillus cereus) and spingomyelinase C (Staphylococcus aureus) resulted in 90--95% release of the phospholipids. The residual membrane contained only phosphatidylinositol and still showed 50--100% of the (Na+ + K+)-ATPase activity.

Barrier properties of lecithin/lysolecithin mixtures

Light scattering, birefringence and X-ray studies showed that liposomes, with lipid molecules orientated in bilayers, are formed from egg licithin/lysolecithin mixtures up to 50 mol percent of lysolecithin; above this concentration much smaller mixed micelles are formed. Permeability studies demonstrated a dramatic increase in the permeability of the liposomes when the lyso concentration exceeds 22.5 mol percent. X-ray studies indicated a significant decrease in bilayer thickness with increasing lysolecithin concentration. It is suggested that decreased interaction energy between the lipid molecules in the bilayer is responsible for the inability of the thin bilayers to act as an effective permeability barrier.

2H-NMR, 31P-NMR and DSC characterization of a novel lipid organization in calcium-dioleoylphosphatidate membranes. Implications for the mechanism of the phosphatidate calcium transmembrane shuttle

2H-NMR, 31P-NMR and DSC investigations are presented on the structure and dynamics of the Ca2+-dioleoylphosphatidate complex which is formed upon addition of calcium to dispersions of pure dioleoylphosphatidate or of dioleoylphosphatidate in mixtures with dioleoylphosphatidylcholine (DOPC). It is concluded that the phosphate region in the polar headgroup of dioleoylphosphatidate is immobilized, while the oleate chains remain liquid and have increased disorder. In mixtures of dioleoylphosphatidate and DOPC in the presence of calcium a dioleoylphosphatidate-rich phase is segregated, in which the molecular behaviour of phosphatidate is rather similar to that of the pure Ca2+-dioleoylphosphatidate complex. A hypothetical model is proposed for the structure of this complex and this is correlated with the dioleoylphosphatidate-mediated transmembrane transport of calcium (Smaal, E.B., Mandersloot, J.G., De Kruijff, B. and De Gier, J. (1986) Biochim. Biophys. Acta 860, 99-108). Data indicate that this transmembrane shuttle is an inverted organization of phosphatidate molecules enclosing calcium ions in an anhydrous core.

Ca2+-induced changes in the barrier properties of cardiolipin/phosphatidylcholine bilayers

(1) A selective increase in permeability is induced in cardiolipin/phosphatidylcholine bilayers at Ca2+ concentrations of 1--3 mM. At higher concentrations of Ca2+ the permeability barrier is completely destroyed. (2) The selective increase in permeability is correlated with the formation of lipid particles visualized by freeze-fracture electron microscopy and an isotropic signal in 31P-NMR spectra. (3) Lowering the Ca2+ concentration shows reduction in permeability but the formation of the lipid particles is a non-reversible process. (4) At higher Ca2+ concentrations, 31P-NMR spectra and freeze-fracture results indicate the formation of the hexagonal phase, explaining the disappearance of the permeability barrier.

The fusion of abnormal plasma lipoprotein (LP-X) and the erythrocyte membrane in patients with cholestasis studied by electronmicroscopy

Adhesion followed by fusion of LP-X vesicles with the erythrocyte membrane is an important contribution to the erythrocyte enlargement in patients with intra or extra hepatic cholestasis. Adhesion of LP-X vesicles is demonstrated by thin section and freeze-etch electronmicroscopy. Fusion of LP-X with the erythrocyte membrane is deduced from biochemical data and freeze-etch electronmicroscopy in that the uptake of cholesterol and lecithin coincides with the increase in smooth areas on the fracture faces of the erythrocyte membrane.

Consequences of the interaction of calcium with dioleoylphosphatidate-containing model membranes: calcium-membrane and membrane-membrane interactions

Calcium binds to dioleoylphosphatidate/dioleoylphosphatidylcholine (DOPA/DOPC) (20:80, mol%) multilamellar vesicles in the presence of a calcium ionophore with stoichiometry of about 0.6 nmol calcium per nmol phosphatidate and an apparent dissociation constant of about 1.7 mM. Experiments on the behaviour of monomolecular films at an air/water interface show that calcium-phosphatidate binding results in a decrease in the area of the polar region of the phosphatidate molecule, probably caused by headgroup dehydration and partial charge neutralization. At calcium concentration higher than about 3 mM calcium neutralizes the negatively charged membrane surface of DOPA/DOPC (20:80, mol%) large unilamellar vesicles, and vesicle aggregation is observed. At 10 mM of calcium this results in a low level of vesicle fusion. These observed processes are not attended with calcium-induced phosphatidylcholine transbilayer movement in the membranes of DOPA/DOPC (20:80, mol%) large unilamellar vesicles. When these findings are compared with the results of a previous study on the permeability behaviour of large unilamellar vesicles of the same phospholipid composition under comparable conditions (Smaal, E.B., Mandersloot, J.G., De Kruijff, B. and De Gier, J. (1986) Biochim. Biophys. Acta 860, 99-108) the following conclusions can be drawn. At low millimolar calcium concentrations (less than 2.5 mM) calcium does not occupy all the binding sites of the membrane, no membrane-membrane interactions are observed and a selective translocation of calcium and calcium-chelating anions is appearing. The mechanism of this translocation may be explained by the formation of uncharged dehydrated complexes of calcium, phosphatidate and calcium chelator, which can pass the membrane via transient occurring non-bilayer structures. Between 3 and 10 mM of calcium an a selective permeability increase of the vesicular membrane is found, which is not a consequence of vesicle fusion but apparently of vesicle aggregation, possibly causing packing defects in the membrane.

Essential adaptation of the calcium influx assay into liposomes with entrapped arsenazo III for studies on the possible calcium translocating properties of acidic phospholipids

An adapted version of the Ca2+-influx assay of Weissmann et al. (Weissmann, G., Anderson, P., Serhan, C., Samuelson, E. and Goodman, E. (1980) Proc. Natl. Acad. Sci. USA 77, 1506-1510) is presented for studies on the possible ionophoretic properties of acidic phospholipids. This method is based on the use of the metallochromic dye arsenazo III enclosed in liposomal vesicles, to indicate the Ca2+ influx. An essential control is introduced to discriminate between Ca2+-arsenazo III complex formation inside the vesicles, as a consequence of Ca2+ influx, and outside the vesicles, as a consequence of arsenazo III leakage from the vesicles. Furthermore, some minor improvements are added, like the use of large unilamellar vesicles instead of multilamellar vesicles, and the use of dual wavelength spectrophotometry. Using this method, it was found that dioleoylphosphatidylcholine vesicles, containing 20 mol% dioleoylphosphatidylglycerol, were impermeable to Ca2+. In this system a selective Ca2+ permeability could be induced by the addition of the fungal Ca2+ ionophore A23187. In contrast, dioleoylphosphatidylcholine vesicles, containing 20 mol% dioleoylphosphatidic acid, incubated in the presence of Ca2+ were permeable to both Ca2+ and arsenazo III.