Does propofol alter membrane fluidity at clinically relevant concentrations? An ESR spin label study

General anesthetics have been shown to perturb the membrane properties of excitable tissues. Due to their lipid solubility, anesthetics dissolve in every membrane, penetrate into organelles and interact with numerous cellular structures in multiple ways. Several studies indicate that anesthetics alter membrane fluidity and decrease the phase-transition temperature. However, the required concentrations to induce such effects on the properties of membrane lipids are by far higher than clinically relevant concentrations. In the present study, the fluidizing effect of the anesthetic agent propofol (2,6-diisopropyl phenol: PPF), a general anesthetic extensively used in clinical practice, has been investigated on liposome dimyristoyl-L-alpha phosphatidylcholine (DMPC) and cell (erythrocyte, Neuro-2a) membranes using electron spin resonance spectroscopy (ESR) of nitroxide labeled fatty acid probes (5-, 16-doxyl stearic acid). A clear effect of PPF at concentrations higher than the clinically relevant ones was quantified both in liposome and cell membranes, while no evident fluidity effect was measured at the clinical PPF doses. However, absorption spectroscopy of merocyanine 540 (MC540) clearly indicates a PPF fluidizing capacity in liposome membrane even at these clinical concentrations. PPF may locally influence the structure and dynamics of membrane domains, through the formation of small-scale lipid domains, which would explain the lack of ESR information at low PPF concentrations.     

Occurrence of norspermine in Euglena gracilis.

A series of fluorescent probes which locate a graded series of depths from the surface to the centre of the lipid bilayer have been used to measure the fluidity gradient in liposomes and natural membranes. In dioleoyl phosphatidylcholine liposomes and in cells which have a high content of unsaturated phospholipids, a region of high microviscosity is detected near the cis double bond/s. The significance of this phenomenon is discussed in terms of the penetration and lateral movement of membrane protein.     

Dynamic fluorescence investigations of the effect of osmotic shocks on the microenvironments of charged and uncharged dipalmitoyl-D,L-α-phosphatidylcholine liposomes

Neutral methylanthracene (MA), anionic trisodium 8-hydroxy-1,3,6-pyrenetrisulfonate, (pyranine), and cationic 3,6-diamino-10-methylacridinium chloride (acriflavine), have been used as fluorescence probes to investigate effects of osmotic shrinkage on neutral, cationic and anionic dipalmitoyl-D,L-α-phosphatidylcholine liposomes. The determined fluorescence polarizations in the liposomes and in solvents of known viscosities afforded the estimation of the microviscosities of the environments of these probes. The viscosity reported by pyranine for anionic and that by acriflavine for cationic single compartment liposomes, ～1.0 cP, indicate the aqueous environments of these probes. Increased viscosities following osmotic shrinkages have been rationalized in terms of changing the nature of the liposome entrapped water. Following the release of free water, some bound water is also released as the result of osmotic shrinkage. The determined shrinkage rates support this postulate. The viscosity of the environment of pyranine in cationic, 9.6 ± 0.3 cP, and that of acriflavine in anionic single compartment liposomes, 74 ± 5 cP, indicate electrostatic attractions of the probes to the charged liposome surface. Osmotic shrinkage results in lowering the viscosity of the environments of the probes presumably because the more concentrated sodium chloride replaces them from their sites. The high viscosities reported by MA, ～ 1000 cP, suggest the intercalation of this probe in the phospholipid bilayers. Osmotic shrinkage does not alter the environment of MA. However, in the presence of cholesterol, the viscosities reported by MA are greater than in its absence. These data contradict previous NMR, ESR and X-ray results as well as those obtained in the present work from osmotic shrinkage rates. The need for care in interpreting data obtained by the use of fluorescence probes is emphasized.     

Fluidity of natural membranes and phosphatidylserine and ganglioside dispersions. Effect of local anesthetics, cholesterol and protein.

The microviscosity of artificial lipid membranes and natural membranes was measured by the fluorescence polarization technique employing perylene as the probe. Lipid dispersions composed of brain gangliosides exhibited greater microviscosity than phosphatidylserine (268 cP vs 173 cP, at 25 degrees C). Incorporation of cholesterol (30-50%) increased the microviscosity of lipid phases by 200-500 cP. Cholesterol's effect on membrane fluidity was completely reversed by digitonin but not by amphotericin B. Incorporation of membrane proteins into lipid vesicles gave varying results. Cytochrome b5 did not alter membrane fluidity. However, myelin proteolipid produced an apparent increase in microviscosity, but this effect might be due to partitioning of perylene between lipid and protein binding sites since tha latter have a higher fluorescence anisotropy than the lipid. The local anesthetics tetracain and butacaine increased the fluidity of lipid dispersions, natural membranes and intact ascites tumor cell membranes. The effect of anesthetics appears to be due to an increased disordering of lipid structure. The fluidity of natural membranes at 25 degrees C varied as follows: polymorphonuclear leukocytes, 335 cP; bovine brain myelin, 270 cP; human erythrocyte, 180 cP; rat liver microsomes, 95 cP; rat liver mitochondria, 90 cP. In most cases the microviscosity of natural membranes reflects their cholesterol: phospholipid ratio. The natural variations in fluidity of cellular membranes probably reflect important functional requirements. Similarly, the effects of some drugs which alter membrane permeability may be the result of their effects on membrane fluidity.     

Fluidity of natural membranes and phosphatidylserine and ganglioside dispersions: Effects of local anesthetics,cholesterol and protein

The microviscosity of artificial lipid membranes and natural membranes was measured by the fluorescence polarization technique employing perylene as the probe. Lipid dispersions composed of brain gangliosides exhibited greater microviscosity than phosphatidylserine (268 cP vs 173 cP, at 25 °C). Incorporation of cholesterol (30–50%) increased the microviscosity of lipid phases by 200–500 cP. Cholesterol's effect on membrane fluidity was completely reversed by digitonin but not by amphotericin B. Incorporation of membrane proteins into lipid vesicles gave varying results. Cytochrome b₅ did not alter membrane fluidity. However, myelin proteolipid produced an apparent increase in microviscosity, but this effect might be due to partitioning of perylene between lipid and protein binding sites since the latter have a higher fluorescence anisotropy than the lipid. The local anesthetics tetracaine and butacaine increased the fluidity of lipid dispersions, natural membranes and intact ascites tumor cell membranes. The effect of the anesthetics appears to be due to an increased disordering of lipid structure. The fluidity of natural membranes at the 25 °C varied as follows:polymorphonuclear leukocytes, 335 cP; bovine brain myelin, 270 cP; human erytherocyte, 180 cP; rat liver microsomes, 95 cP; rat liver mitochondria, 90 cP. In most cases the microviscosity of natural membranes reflects their cholesterol : phospholipid ratio. The natural variations in fluidity of cellular membranes probably reflect important fuctional requirements. Similarly, the effects of some drugs which alter membrane permeability may be the result of their effects on membrane fluidity.     

Membrane fluidity changes of liposomes in response to various odorants. Complexity of membrane composition and variety of adsorption sites for odorants.

Three kinds of liposomes prepared from phosphatidylcholine (PC), azolectin, and azolectin-containing membrane proteins of the canine erythrocytes were used as models for olfactory cells. To explore properties of the adsorption sites of odorants, membrane fluidity changes in response to various odorants were measured with various fluorescence dyes which monitor the fluidity at different depths and different regions of the membranes. (a) Application of various odorants changed the membrane fluidity of azolectin liposomes. The patterns of membrane fluidity changes in response to odorants having a similar odor were similar to each other and those in response to odorants having different odors were different from each other. These results suggested that odorants having a similar odor are adsorbed on a similar site and odorants having different odors are adsorbed on different sites. (b) Such variation of the pattern was not seen in liposomes of a simple composition (PC liposome). (c) In the proteoliposomes whose composition was more complex than that of azolectin liposomes, the patterns of membrane fluidity changes varied among odorants having a similar odor. It was concluded that liposomes of complex membrane composition have the variety of adsorption sites for odorants.     

Two-step impact of Amphotericin B (AmB) on lipid membranes: ESR experiment and computer simulations

Abstract

In this study, the electron spin resonance (ESR) method was used to examine the effect of Amphotericin B (AmB) molecules on the fluidity of model membranes made of dipalmitoylphosphatidylcholine (DPPC). The changes occurring under increased AmB concentrations in the spectroscopic parameters of spin probes placed in liposomes were determined. Three probes were used, penetrating the membrane at different depths which allowed the changes in its fluidity to be found in the transverse section. A computer model of the surface layer of membrane, with AmB admixture, was developed and subjected to computer simulation. The effect of changing concentration of the admixture on the binding energy in the system of dipoles representing the surface of the membrane was examined. The ESR studies showed that the process of accumulation of AmB molecules in the membrane has two stages, marked by local maxima in the ESR spectra. The first appears for concentrations of ca. 0.25–0.5% and the second appears for ca. 2.5–3% AmB of its molar ratio to DPPC. The computer simulations permitted reconstructing the two-stage mechanism of interaction between the molecules and the membrane. They demonstrated that, at low concentrations, the AmB molecules position themselves flat on the membrane surface. After the threshold concentration is exceeded, they re-orientate to a vertical position. This process leads to the perforation of the membrane.     

The Structural State and Form of Free and Biopolymer-Encapsulated Phosphatidylcholine Liposomes in the Absence and Presence of Natural Plant Antioxidants

Electron spin resonance (ESR) and atomic force microscopy (AFM) were used to study liposomes that were prepared from soybean phosphatidylcholine (PC); they incorporated plant antioxidants (ginger, allspice, and black-pepper extracts; clove oil; etc.) that were encapsulated in biopolymers (sodium caseinate or sodium caseinate–maltodextrin covalent conjugates). Plant antioxidants were shown to cause a 15–25% decrease in the microviscosity of deep-lying regions of the liposome lipid bilayer by ESR with a 16-doxylstearic acid spin probe. A ginger extract exerted the greatest effect (24%). Sodium caseinate and its covalent conjugates with maltodextrins (dextrose equivalents (DEs) 2 and 10) increased the microviscosity by 30–35% as compared with free and antioxidant-incorporating liposomes. AFM showed that antioxidants increased the cross-sectional area and volume of liposomes and that the polymers made liposomes denser and their structure more compact.     

Effect of holotoxin A1 on microviscosity of model and biological membranes

Behavior of hydrophobic probes of 5-doxylstearol acid and pyrene in phosphatidylcholine liposomes modified by sterols isolated from Far East holothurian tissues was studied. The introduction of delta 7-sterol xylosides and delta 5-sterol sulfates into liposomes was shown to result in an increase of the membranes microviscosity. When the liposome suspension was added with holotoxin A1 a supplementary increase of microviscosity was observed, which was obviously a consequence of glycosides +non built into the membranes.     

Orientation and motion of spin-labels in rabbit small intestinal brush border vesicle membranes

The temperature dependence of the packing (order) and fluidity (microviscosity) of rabbit small, intestinal brush border vesicle membranes and of liposomes made from their extracted lipids has been investigated by using a variety of lipid spin probes. The lipids in the brush border membrane are present essentially as a bilayer. Compared to other mammalian membranes, the brush border membrane appears to be characterized by a relatively high packing order as well as microviscosity. At body temperature, the lipid molecules undergo rapid, anisotropic motion, which is essentially a fast rotation about an axis approximately perpendicular to the bilayer normal. Both the order (motional anisotropy) and the microviscosity increase with decreasing temperature and with increasing distance from the center of the bilayer. Qualitatively similar motional or fluidity gradients have been reported for other mammalian and bacterial membranes. The liposomes made from the extracted lipids have a somewhat lower packing order and a slightly higher fluidity than brush border vesicle membranes. The differences are, however, small indicating that the packing and the fluidity (microviscosity) of the membrane are primarily determined by the lipid composition. Membrane-associated proteins and cytoskeleton cannot play a dominant role in determining the order and fluidity of the lipid bilayer. Discontinuities are observed in the temperature dependence of various spectral parameters, the order parameter S, the rotational correlation time tau, and 2,2,6,6-tetramethylpiperidinyloxy partitioning. They are assigned to phase transitions and/or phase separations of the membrane lipids. These discontinuities occur at about 30, 20, and 13 degrees C for 5-doxyl-, 12-doxyl-, and 16-doxylstearic acid, respectively. The apparent transition temperature depends on the location of the spin probe along the bilayer normal, being higher the closer the probe is to the membrane surface. This indicates the possibility that chain melting is progressive and spreads with increasing temperature from the center of the membrane outward.     

Glutaryl phosphatidylcholine effects on pH and composition dependent behavior of liposomes studied by spin-labeling method

The influence of glutaryl phosphatidylcholine on the molecular organization of phosphatidylcholine liposomes was studied by spin-labeling technique. The ESR signals given by the 5-nitroxide stearic acid label showed that the presence of glutaryl lecithin (i) significantly increased the negative charge density of the polar liposome surface with increasing proton concentration depending on the bulk solution pH, and (ii) apparently decreased the packing (order) of the hydrophobic region close to the surface, essentially in the presence of saturated phospholipids. The spectral information--S (order parameter) and alpha N (isotropic nitrogen coupling constant)--resulted in the location of the probe near or in the polar zone of the membrane or in the hydrophobic region, depending on the protonation/deprotonation of the fatty acid carboxyl group of the probe. The microviscosity of the inner region of the membrane monitored by the 12- and 16-probes was not significantly altered by glutaryl lecithin. On the other hand, glutaryl lecithin has a lesser effect on liposomes containing anionic polar head groups, such as dipalmitoyl phosphatidylglycerol or phosphatidylinositol, the anionic charge of which already had the same effect on protonation of the polar surface. The temperature dependence of dipalmitoyl phosphatidylcholine liposome dynamic behavior indicates that the glutaryl lecithin effect is completely different above and below the gel-to-liquid crystalline phase transition point.     

The disordering effect of hyoscyamine drugs on phospholipid membranes

The effect of hyoscyamine drugs on the fluidity of dipalmitoylphosphatidylcholine liposomes has been studied by differential scanning calorimetry (DSC), electron spin resonance spectroscopy (ESR), fluorescence polarization and freeze-fracture electron microscopic techniques. DSC results indicate that anisodamine, anisodine, atropine and scopolamine all increase the fluidity of dipalmitoylphosphatidylcholine liposomes but with different degrees of efficiency. The increasing of fluidity of dipalmitoylphosphatidylcholine liposomes by hyoscyamine drugs is in a dose-dependent way. Increase of the fluidity of phosphatidylcholine liposomes by anisodamine was also shown by the other three methods. The possible mechanism of hyoscyamine-membrane interaction is discussed.     

The disordering effect of hyoscyamine drugs on phospholipid membranes

The effect of hyoscyamine drugs on the fluidity of dipalmitoylphosphatidylcholine liposomes has been studied by differential scanning calorimetry (DSC), electron spin resonance spectroscopy (ESR), fluorescence polarization and freeze-fracture electron microscopic techniques. DSC results indicate that anisodamine, anisodine, atropine and scopolamine all increase the fluidity of dipalmitoylphosphatidylcholine liposomes but with different degrees of efficiency. The increasing of fluidity of dipalmitoylphosphatidylcholine liposomes by hyoscyamine drugs is in a dose-dependent way. Increase of the fluidity of phosphatidylcholine liposomes by anisodamine was also shown by the other three methods. The possible mechanism of hyoscyamine-membrane interaction is discussed.     

Fluidity of liposome membranes doped with metalloporphyrins: an ESR study

Changes in membrane fluidity of porphyrin-doped liposomes have been investigated to assess the kinetics of the fluidization process. Metal complexes of tert-butylphenyl meso-substituted porphyrin, containing ions of Mg, Mn, Fe, Co, Ni and Cu, were used as dopants. Liposomes were obtained by sonication of hen egg yolk lecithin (EYL). Electron paramagnetic resonance (ESR) was applied using two spin probes, TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) and 16-DOXYL-stearic acid [2-ethyl-2-(15-methoxy-15-oxopentadecyl)-4,4-dimethyl-3-oxazolidinyloxyl], localized at different sites within the membrane to determine the spectroscopic parameters: partition (F) and rotation correlation time (tau), related to the membrane's fluidity. It was found, that porphyrins considerably fluidize the membranes, and the dynamics of this process depends on the kind of the compound used and the membrane's area surveyed by the probes. The Cu complex proved to be the most effective one within the surface layer, whereas the Mn complex most strongly fluidized the deeper parts of the lipid double-layer. Variations in fluidity observed after the porphyrins had been introduced into the liposome were found to stabilize inside the double-layer and within the surface layer after ca. 25 and 50 h, most probably due to hydration of the hydrophilic part of the membrane.     

Change in the dynamics of a spin probe in complement-dependent lysis of a liposome

The mobility of phospholipid chains in membranes of liposomes consisting of egg lecitin, cholesterol, dicetylphosphate, sensitized by the lipopolysaccharide antigen F. tularensis by the action of a homologous antiserum and a rabbit complement preparation was studied using 5- and 16-doxylstearate spin probes. It was shown that, during the immune lysis of liposome membranes, changes in the dynamics of spin probes occur, which correlate with the formation of transmembrane channels and exit of the fluorescent marker from the interior of liposomes. It was found that the ratio of the intensities I1/I2 of two low-field extrema in the ESR spectrum is most sensitive to changes in the liposome membrane that are induced by immune components.     

Effect of phosphatidylinositol replacement by diacylglycerol on various physical properties of artificial membranes with respect to the role of phosphatidylinositol response

In an attempt to gain insight into the physiological role of phosphatidylinositol turnover enhanced by extracellular stimuli, the physical properties of artificial membranes (egg yolk phosphatidylcholine/bovine brain phosphatidylserine) containing phosphatidylinositol or diacylglycerol were studied by ESR using spin probes and freeze-fracture electron microscopy. Diacylglycerol lost both the ability to form lipid bilayer structures and its susceptibility to calcium ions. Yeast phosphatidylinositol included in dipalmitoylphosphatidylcholine liposomes lowered the phase transition temperature of dipalmitoylphosphatidylcholine and expanded the temperature range of phase transition. However, diacylglycerol at the same concentration did not undergo the effects caused by phosphatidylinositol but the phase transition temperature was slightly raised. Phase separation of phosphatidylserine induced by calcium ions was enhanced when the phosphatidylinositol was replaced by diacylglycerol in phosphatidylcholine/phosphatidylserine/phosphatidylinositol (3:5:2, by molar ratio) mixtures. The mobility of phosphatidylcholine spin probe was decreased in phosphatidylcholine/phosphatidylserine/diacylglycerol (3:5:2, by molar ratio) liposomes compared with phosphatidylcholine/phosphatidylserine/phosphatidylinositol (3:5:2, by molar ratio) liposomes. An additional component from protonated stearic acid spin probes was observed in phosphatidylcholine/phosphatidylinositol (8:2, by molar ratio) liposomes at 40 degrees C, whereas the component was not seen in phosphatidylcholine/diacylglycerol (8:2, by molar ratio) liposomes. This may indicate the alteration of surface charge induced by the replacement of phosphatidylinositol by diacylglycerol. Indeed, in the presence of 1 mM Ca2+, the additional component was removed by an electrostatic interaction between Ca2+ and phosphatidylinositol molecules in phosphatidylcholine/phosphatidylinositol liposomes at 40 degrees C. These results support the hypothesis that the enhanced turnover of phosphatidylinositol may play a triggering role for various cellular responses to exogenous stimuli by altering membrane physical states.     

Interaction of cytoplasmic l-glycerol-3-phosphate dehydrogenase with liposomes

NAD-linked l-glycerol-3-phosphate dehydrogenase binds to phosphatidylcholine liposomes as shown by the changes in the properties of both the enzyme and the membrane. The surface potential and the fluidity of the liposome membrane (monitored at the 5th C atom depth) change due to the presence of the enzyme, whereas the enzyme is activated by the liposomes. These findings suggest the occurrence of peripheral protein-lipid interactions.     

Interaction of cytoplasmic L-glycerol-3-phosphate dehydrogenase with liposomes

NAD-linked L-glycerol-3-phosphate dehydrogenase binds to phosphatidylcholine liposomes as shown by the changes in the properties of both the enzyme and the membrane. The surface potential and the fluidity of the liposome membrane (monitored at the 5th C atom depth) change due to the presence of the enzyme, whereas the enzyme is activated by the liposomes. These findings suggest the occurrence of peripheral protein-lipid interactions.     

Effect of phosphatidylinositol replacement by diacylglycerol on various physical properties of artificial membranes with respect to the role of phosphatidylinositol response

In an attempt to gain insight into the physiological role of phosphatidylinositol turnover enhanced by extracellular stimuli, the physical properties of artificial membranes (egg yolk phosphatidylcholine/bovine brain phosphatidylserine) containing phosphatidylinositol or diacylglycerol were studied by ESR using spin probes and freeze-fracture electron microscopy. Diacylglycerol lost both the ability to form lipid bilayer structures and its susceptibility to calcium ions. Yeast phosphatidylinositol included in dipalmitoylphosphatidylcholine liposomes lowered the phase transition temperature of dipalmitoylphosphatidylcholine and expanded the temperature range of phase transition. However, diacylglycerol at the same concentration did not undergo the effects caused by phosphatidylinositol but the phase transition temperature was slightly raised. Phase separation of phosphatidylserine induced by calcium ions was enhanced when the phosphatidylinositol was replaced by diacylglycerol in phosphatidylcholine/ phosphatidylserine/phosphatidylinositol (3:5:2, by molar ratio) mixtures. The mobility of phosphatidylcholine spin probe was decreased in phosphatidylcholine/ phosphatidylserine/diacylglycerol (3:5:2, by molar ratio) liposomes compared with phosphatidylcholine/phosphatidylserine/phosphatidylinositol (3:5:2, by molar ratio) liposomes. An additional component from protonated stearic acid spin probes was observed in phosphatidylcholine/phosphatidylinositol (8:2, by molar ratio) liposomes at 40°C, whereas the component was not seen in phosphatidylcholine/diacylglycerol (8:2, by molar ratio) liposomes. This may indicate the alteration of surface charge induced by the replacement of phosphatidylinositol by diacylglycerol. Indeed, in the presence of 1 mM Ca²⁺, the additional component was removed by an electrostatic interaction between Ca²⁺ and phosphatidylinositol molecules in phosphatidylcholine/phosphatidylinositol liposomes at 40°C. These results support the hypothesis that the enhanced turnover of phosphatidylinositol may play a triggering role for various cellular responses to exogenous stimuli by altering membrane physical states.     

Metalloporphyrin intercalation in liposome membranes: ESR study

Liposomes characterized by membranes featuring diverse fluidity (liquid-crystalline and/or gel phase), prepared from egg yolk lecithin (EYL) and dipalmitoylphosphatidylcholine (DPPC), were doped with selected metalloporphyrins and the time-related structural and dynamic changes within the lipid double layer were investigated. Porphyrin complexes of Mg(II), Mn(III), Fe(III), Co(II), Ni(II), Cu(II), Zn(II), and the metal-free base were embedded into the particular liposome systems and tested for 350 h at 24°C using the electron spin resonance (ESR) spin probe technique. 5-DOXYL, 12-DOXYL, and 16-DOXYL stearic acid methyl ester spin labels were applied to explore the interior of the lipid bilayer. Only the 16-DOXYL spin probe detected evident structural changes inside the lipid system due to porphyrin intercalation. Fluidity of the lipid system and the type of the porphyrin complex introduced significantly affected the intermolecular interactions, which in certain cases may result in self-assembly of metalloporphyrin molecules within the liposome membrane, reflected in the presence of new lines in the relevant ESR spectra. The most pronounced time-related effects were demonstrated by the EYL liposomes (liquid-crystalline phase) when doped with Mg and Co porphyrins, whereas practically no spectral changes were revealed for the metal-free base and both the Ni and Zn dopants. ESR spectra of the porphyrin-doped gel phase of DPPC liposomes did not show any extra lines; however, they indicated the formation of a more rigid lipid medium. Electronic configuration of the porphyrin’s metal center appeared crucial to the degree of molecular reorganization within the phospholipid bilayer system.