Cholesterol inhibition of the temporary increase of membrane fluidity of lymphocytes induced by mitogenic lectins

The mitogenic response of human peripheral lymphocytes to lectins can be decreased by brief treatment of the cells with lecithin-cholesterol liposomes. This fact indicates that the temporary increase of membrane fluidity, which occurs within 30 min after addition of mitogenic lectins, is an important early event for the subsequent activation of lymphocytes. This temporary increase of membrane fluidity is accompanied by neither a decrease in cellular cholesterol level nor by particular acceleration of the incorporation of polyunsaturated fatty acids into phospholipids. These facts suggest that this change in membrane fluidity is not due to the alteration of membrane lipid composition, but can be regarded as a result of temporary perturbation of membrane lipid bilayers induced by binding of the lectins to their membrane receptors.     

Spectroscopic characterization of vesicle formation on heated human erythrocytes and the influence of the antiviral agent amantadine

EPR investigations on the vesiculation process of heated human erythrocytes were performed, using different fatty acid spin labels. Spectrin denaturation and vesiculation do not influence the fluidity of the lipid phase of the remaining membrane of human erythrocytes: Vesicles released differ in chemical composition as well as in the lipid fluidity of their membrane from the intact human erythrocyte membrane. A reduced cholesterol-to-phospholipid ratio and a depletion of spectrin was found. By changing the ionic concentration of the suspension medium an effect on membrane spectra and on vesicle release was established. The adamantane derivative amantadine causes fluidization of the human erythrocyte membrane and inhibits vesicle release. Based on these results, a model for the mechanism by which adamantane-like molecules could interact with membranes is proposed.     

Age-Related Differences in Synaptosomal Peroxidative Damage and Membrane Properties

Young, adult, and old rats were used to study the effect of age on the integrity and functioning of brain synaptosomes. An evaluation was made of the differences in lipid composition, membrane fluidity, Na+, K(+)-ATPase activity, and susceptibility to in vitro lipid peroxidation. There was an age-related increase in synaptosomal free fatty acids, with no modification in acyl chain composition, and a decrease in membrane phospholipids which increased the cholesterol/phospholipid mole ratio. With altered lipid composition, there was a corresponding age-dependent decrease in membrane fluidity, a reduction of Na+, K(+)-ATPase activity, and an overall greater susceptibility to in vitro lipid peroxidation. Furthermore, lipid peroxidation promoted strong modifications of the membrane fluidity, lipid composition, and Na+,K(+)-ATPase activity just as aging did, thus indicating a possible contribution of oxidative damage to ageing processes. The cases studied revealed that the greater responsiveness of old membranes to in vitro lipid peroxidation resulted in the highest degree of membrane alteration, indicating that all pathological states known to promote a peroxidative injury can have even more dramatic consequences when they take place in old brain.     

伴刀豆球蛋白A和层粘连蛋白与膜受体结合下膜分子运动及微丝组装变化的比较

研究伴刀豆球蛋白A和层粘连蛋白分别与小鼠腹腔巨噬细胞膜受体结合下引起细胞膜分子运动的变化和对微丝组装的影响.结果表明,伴刀豆球蛋白A和层粘连蛋白作用下均导致膜表面蛋白分子的侧向扩散速率减慢,膜脂流动性降低,加快膜内微丝组装并使微丝含量增加.两配体作用下引起细胞上述反应有相似性.     

伴刀豆球蛋白A和层粘连蛋白与膜受体结合下膜分子运动及微丝组装变化的比较

研究伴刀豆球蛋白A和层粘连蛋白分别与小鼠腹腔巨噬细胞膜受体结合下引起细胞膜分子运动的变化和对微丝组装的影响.结果表明,伴刀豆球蛋白A和层粘连蛋白作用下均导致膜表面蛋白分子的侧向扩散速率减慢,膜脂流动性降低,加快膜内微丝组装并使微丝含量增加.两配体作用下引起细胞上述反应有相似性.     

Effect of streptozotocin-induced diabetes on rat liver Na+/K+-ATPase.

Na+/K+-ATPase during diabetes may be regulated by synthesis of its alpha and beta subunits and by changes in membrane fluidity and lipid composition. As these mechanisms were unknown in liver, we studied in rats the effect of streptozotocin-induced diabetes on liver Na+/K+-ATPase. We then evaluated whether fish oil treatment prevented the diabetes-induced changes. Diabetes mellitus induced an increased Na+/K+-ATPase activity and an enhanced expression of the beta1 subunit; there was no change in the amount of the alpha1 and beta3 isoenzymes. Biphasic ouabain inhibition curves were obtained for diabetic groups indicating the presence of low and high affinity sites. No alpha2 and alpha3 isoenzymes could be detected. Diabetes mellitus led to a decrease in membrane fluidity and a change in membrane lipid composition. The diabetes-induced changes are not prevented by fish oil treatment. The results suggest that the increase of Na+/K+-ATPase activity can be associated with the enhanced expression of the beta1 subunit in the diabetic state, but cannot be attributed to changes in membrane fluidity as typically this enzyme will increase in response to an enhancement of membrane fluidity. The presence of a high-affinity site for ouabain (IC50 = 10-7 M) could be explained by the presence of (alphabeta)2 diprotomeric structure of Na+/K+-ATPase or an as yet unknown alpha subunit isoform that may exist in diabetes mellitus. These stimulations might be related, in part, to the modification of fatty acid content during diabetes.     

Fluorescence photobleaching recovery measurements of surface lateral mobilities on normal and SV40-transformed mouse fibroblasts

Lateral mobilities of fluorescent cell surface probes have been measured on normal (3T3) and transformed (SV3T3) cultured mouse fibroblasts. There is little discernible difference in the mobilities of a lipid analogue (diI), a fluorescent ganglioside derivative (GM1), and tetramethylrhodamine-labeled succinylated concanavalin A. The two cell lines showed expected differences in their abilities to grow in agar, to grow without serum, and to be agglutinated by lectins, indicating that changes of these properties in transformed cells are probably not mediated through increased overall membrane fluidity but are associated with distinct alterations in the mobilities of cell surface receptors. Both fluorescent dextran derivatives and antimouse cell surface antibodies were distinctly less mobile on SV3T3 cells, and the mobile fraction of Con A receptors was lower on SV3T3 cells.     

Structural changes in BHK cell plasma membrane caused by the binding of vesicular stomatitis virus

Spin label electron spin resonance techniques using a nitroxide derivative of stearic acid were used to detect changes in plasma membrane structure caused by the binding of vesicular stomatitis virus (VSV) to cell plasma membranes of intact BHK-21 cells. The results indicate that binding of VSV to cell surface receptors causes an increase in the observed rigidity of the plasma membrane lipid bilayer. This change in membrane structure, which appears to be caused by the cross-linking of receptors in the plane of the plasma membrane, could be prevented by treating the cells with colchicine before addition of virus and could be reversed by treating the cells with colchicine after addition of virus. Cells treated with a monovalent, water-soluble derivative of VSV G-protein (Gs) did not show an increase in plasma membrane bilayer rigidity. However, addition of anti-VSV G-protein immunoglobulin G to cells pretreated with G8 caused an increase in plasma membrane bilayer rigidity. This increased rigidity could also be reversed by the addition of colchicine. Fluorescence microscopy was used to determine the distribution of fluorescein-labeled VSV particles on the cell surface after addition of virus. Approximately 30 min after addition of virus, discrete areas on the cell surface showed fluorescent staining, which coalesced to apical regions of the cell after approximately 40 min.     

Erythrocyte membrane fluidity and changes in plasma lipid composition: a possible relationship in childhood obesity

We have studied plasma lipid patterns and erythrocyte membrane fluidity in 60 obese children and 20 normal children. Plasma levels of total cholesterol and associated low-density lipoproteins were significantly increased in 20 obese patients with respect to controls. A significant decrease in membrane fluidity, measured as an increase in the fluorescence polarization value of the probe 1,6-diphenyl-1,3,5-hexatriene, associated with an increase in the cholesterol/protein ratio has been shown in obese patients. The study of the correlation between erythrocyte membrane fluidity and plasma cholesterol has indicated that significant changes in fluidity and membrane lipid composition also occur in erythrocytes of obese patients with normal plasma lipid levels. These findings confirm that the erythrocyte membrane responds very early to modifications of plasma lipoproteins and suggest that in childhood obesity a modified transfer of cholesterol from plasma to erythrocyte membrane may take place.     

Alterations in Membrane Lipid Dynamics of Leukemic Cells Undergoing Growth Arrest and Differentiation: Dependency on the Inducing Agent

The effect of various differentiation inducers on membrane cell dynamics was studied using HL-60 and K562 leukemic cell lines. Membrane lipid dynamics was measured by the steady-state fluorescence polarization (P) method utilizing either 1,6-diphenyl-1,3,5-hexatriene (DPH) or the trimethyl ammonium derivative of DPH (TMA-DPH), which ascertains anchorage of the label to the membrane–water–lipid interface. Decrease in membrane microfluidity was observed in HL-60 cells undergoing differentiation into macrophages by 1,25-dihydroxyvitamin D₃and by K562 cells induced to differentiate by DMSO. Sodium butyrate caused an increase in membrane fluidity in K562 cells undergoing differentiation into erythroid-like cells while in HL-60 cells a dual effect was observed. At 0.4 mM concentration, in which the cells were induced to differentiate along the monocyte pathway, a decrease in membrane fluidity was observed, while at 1 mM concentration an increase in membrane fluidity occurred. Interferon-γ (IFN-γ) induced an increase in membrane fluidity in both cell lines. Using HL-60 cells fluorescently labeled by TMA-DPH, similar results indicating fluidization of the membrane following IFN-γ treatment were obtained. Advanced fluorescence lifetime measurements, evaluated either by phase modulation spectrofluorometry or by single photon correlation fluorometry confirmed that the decrease in fluorescence polarization by IFN-γ resulted from membrane fluidization and not from elongation of the probe's excited state lifetime. It is suggested that the inducer mode of action, and not the differentiation route, determine the outcome of changes in membrane microviscosity.     

Alterations of human erythrocyte membrane fluidity by oxygen-derived free radicals and calcium

Two possible reasons for the structural alterations of cell membranes caused by free radicals are lipid peroxidation and an increase in the intracellular calcium ion concentration. To characterize the alterations in membrane molecular dynamics caused by oxygen-derived free radicals and calcium, human erythrocytes were spin-labeled with 5-doxyl stearic acid, and alterations in membrane fluidity were quantified by electron spin resonance oxidase (0.07 U/mL) decreased membrane fluidity, and the addition of superoxide dismutase and catalase inhibited the effect on membrane fluidity of the hypoxanthine-xanthine oxidase system. Hydrogen peroxide (0.1 and 1 nM) also decreased membrane fluidity and caused alterations to erythrocyte morphology. In addition, a decrease in membrane fluidity was observed in erythrocytes incubated with 2.8 mM CaCl₂. On the other hand, incubation of erythrocytes with calcium-free solution decreased the changes in membrane fluidity caused by hydrogen peroxide.

These results suggest that changes in membrane fluidity are directly due to lipid peroxidation and are indirectly the result of increased intracellular calcium concentration. We support the hypothesis that alterations of the biophysical properties of membranes caused by free radicals play an important role in cell injury, and that the accumulation of calcium amplifies the damge to membranes weakened by free radicals.     

Induction of acetylcholinesterase release from erythrocytes in the presence of liposomes

When human erythrocytes are incubated with liposomes, the release of acetylcholinesterase (AChE) occurs following an induction period [Cook et al. (1980) Biochemistry 19, 4601-4607]. However, the mechanism of the induction has not been elucidated. We examined the relationships among the lipid transfer from liposomes to erythrocytes, the morphological change of erythrocytes, the fluidity of the erythrocyte membrane and the start of AChE release. The AChE release into the liposomes and into shed-vesicle fractions started simultaneously after an induction period. The morphological index (MI) of erythrocytes was approximately 2.8 at the beginning of the release, regardless of the induction period. AChE was not released from the erythrocytes of index 2.8 even in the presence of liposomes if the MI remained at 2.8. Therefore, for the release, erythrocytes needed a further increase of the MI from 2.8. As the rate of lipid transfer increased, the induction period became shorter. No significant lipid release from erythrocytes was detected during the induction period. The initiation of the AChE release was not simply affected by the change in the membrane fluidity of erythrocytes upon interaction with liposomes. These results first demonstrate that AChE release into the shed-vesicle and liposome fractions is triggered by a further increase of the MI from 2.8, which is induced by lipid transfer from liposomes to erythrocytes.     

Sphingomyelin phase transition in the sheep erythrocyte membrane

The dependence of membrane dynamics on the mole ratio of lecithin to sphingomyelin (L/S) was examined by the fluorescence depolarization of the fluidity probe DPH in membranes isolated from sheep and human erythrocytes. In these membranes L/S is the main variable of lipid composition (0.02 and 1.7, respectively). The sheep erythrocyte membrane, which is rich in sphingomyelin, displays a higher lipid microviscosity than the human erythrocyte membrane in addition to a broad gel/liquid-crystal phase transition in the range of 26–35°C. Single-walled lipid vesicles of high sphingomyelin content, when studied by the same technique, exhibited dynamic characteristics similar to those found in the sheep erythrocyte membrane. Both the apparent microviscosity and the transition temperature decreased with increasing the L/S. Membrane proteins of human and sheep erythrocytes were fluorescently labeled with the sulfhydryl reagent N-dansylaziridine and the emission spectrum was recorded as a function of temperature. In the human erythrocyte membranes a gradual increase in the ratio of emission maxima at 520 and 490 nm was observed between 6 and 40°C. At this temperature range the ratio of the above emission maxima in sheep erythrocyte membranes displayed a break between 20 and 28°C, which partially overlapped the phase transition observed for the lipid core. The effect of the lipid phase transition on membrane proteins for the lipid core. The effect of the lipid phase transition on membrane proteins was further assessed by comparing the activity of the membrane bound phospholipase A2 in the intact and detergent-solubilized sheep erythrocyte membranes. Below 31°C the lipids suppress the enzyme activity by about 90%, whereas above this temperature this suppression is progressively abolished.     

Species-dependent differences in the effect of ionic strength on potassium transport of erythrocytes: the role of lipid composition.

The Rb+(K+) efflux of erythrocytes from six mammalian species was investigated in solutions of physiological and low ionic strength. A species dependent increase of the Rb+(K+) efflux in low ionic strength solution could be observed. The rate constant of Rb+(K+) efflux of erythrocytes in physiological ionic strength solution correlates with the content of arachidonic acid of the membrane phospholipids. The same relation was observed in solution of low ionic strength with the exception of human erythrocytes. In addition, an age-dependent correlation of the rate constant of Rb+(K+) efflux from calf erythrocytes in low ionic strength solution with the content of arachidonic acid of the membrane phospholipids was found. The Rb+(K+) efflux of human erythrocytes, which is enhanced in low ionic strength solution, decreases with the decreasing temperature. The temperature-dependent ESR order parameter of a fatty acid spin label for human and cow erythrocytes in solution of physiological and low ionic strength media suggested that the effect of low ionic strength on Rb+(K+) efflux is not solely based on a change of membrane fluidity. The results are interpreted as being due to a specific influence of membrane phospholipids on the Rb+(K+) efflux.     

Red blood cell triiodothyronine uptake as membrane parameter of depression

We tested the hypothesis considering the role of hypothalamic-pituitary-thyroid axis (HPT), L-triiodothyronine (L-T3) uptake into erythrocytes, and the role of membrane lipids in the development and treatment of affective disorders. Changes in kinetic parameters (V(max), maximal velocity and K(M), apparent Michaelis constant) of L-T3 uptake into red blood cells (RBCs) and changes in membrane fluidity in a group of 24 patients with major depression were measured before treatment and after 1 month of treatment with citalopram. Parameters V(max) and K(M), as well as membrane microviscosity, were significantly increased in depressed patients both before and after treatment in comparison with healthy subjects. We concluded that the function of the membrane transporter for L-T3 in RBC is changed in depression. This change is probably connected with alteration of membrane fluidity and/or transporter-lipid interactions. We did not find any normalization of the measured parameters after 1 month of treatment. The results show the importance of composition and physical properties of the lipid bilayer for transmembrane transport of L-T3 and support the hypothesis that the HPT axis is in depression.     

Rat brain membrane-bound delta opioid receptor: loss and reactivation of binding on dialysis and aging at low temperature.

A change in the environment of rat brain membranes by dialysis from phosphate buffered saline (PBS) to 10 mM potassium phosphate (pH 7.2) led to a 35% loss in delta opioid receptor binding, while alteration of membrane structure on freezing at -20 degrees C for 55 days led to 85% loss of receptor binding. The dialysate, 200 mM KCI and NaCl restored receptor binding lost on dialysis. This K+ and Na+ restabilization of the receptor can be through cation-pi bonding, interactions that are suited to the lipid bilayer. In membranes stored at -20 degrees C, the loss of binding is attributed to increased membrane fluidity by phospholipase A2 action on membrane phospholipids, resulting in an increase of free fatty acids. K+ but not Na+ restabilization of these membrane receptors may be due to the ability of K+ to decrease membrane fluidity.     

Effects of Proteus mirabilis lipopolysaccharides with different O-polysaccharide structures on the plasma membrane of human erythrocytes

The effects of O33 and O49 P. mirabilis lipopolysaccharides (LPSs) on human erythrocyte membrane properties were examined. Physical parameters of the plasma membrane, such as membrane lipid fluidity, physical state of membrane proteins, and osmotic fragility, were determined. The fluidity of the lipids was estimated using three spin-labeled stearic acids of doxyl derivatives: 5-doxylstearic acid, 12-doxylstearic acid, and 16-doxylstearic acid. All the applied labels locate to different depths of the lipid layer and provide information on the ordering of phospholipid fatty acyl chain mobility. LPSs O49 increased the membrane lipid fluidity in the polar region of the lipid bilayer as indicated by spin-labeled 5-doxylstearic acid. An increase in fluidity was also observed in the deeper region using 12-doxylstearic acid only for O33 LPSs. The highest concentration of O33 LPSs (1 mg/ml) increased the motion of membrane proteins detected by the spin-label residue of iodoacetamide. These results showed different actions of O33 and O49 LPSs on the plasma membrane due to the different chemical structures of O-polysaccharides. P. mirabilis O33 and O49 LPSs did not induce changes in the membrane cytoskeleton, osmotic fragility and lipid peroxidation of erythrocytes. On the other hand a rise in the content of carbonyl compounds was observed for the highest concentrations of O33 LPS. This result indicated protein oxidation in the erythrocyte membrane. Lipid A, the hydrophobic part of LPS, did not change the membrane lipid fluidity and osmotic fragility of erythrocytes. Smooth and rough forms of P. mirabilis LPSs were tested for their abilities for complement-mediated immunohemolysis of erythrocytes. Only one out of seven LPSs used was a potent agent of complement-mediated hemolysis. It was rough, Ra-type of P. mirabilis R110 LPS. The O-polysaccharide-dependent scheme of reaction is presented.     

In situ assessment of erythrocyte membrane properties during cold storage

Membrane fluidity and overall protein secondary structure of human erythrocytes were studied in situ using Fourier transform infrared spectroscopy (FTIR). Erythrocyte membranes were found to have weakly cooperative phase transitions at 14 degrees C and at 34 degrees C, which were tentatively assigned to the melting of the inner membrane leaflet and the sphingolipid rich outer leaflet, respectively. Cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) resulted in a large increase in the cooperativity of these transitions, and led to the appearance of another phospholipid transition at 25 degrees C. Multiple, sharp membrane phase transitions were observed after 5 days cold storage (4 degrees C ), which indicated phase separation of the membrane lipids. Using fluorescence microscopy, it was determined that the lipid probe 1,1'-dioctadecyl-3,3,3',3-tetramethyl-indocarbocyanine perchlorate (dil-C18) remained homogeneously distributed in the erythrocyte membrane during cold storage, suggesting that lipid domains were below the resolution limit of the microscope. Using thin layer chromatography, changes in the membrane lipid composition were detected during cold storage. By contrast, assessment of the amide-II band with FTIR showed that the overall protein secondary structure of haemoglobin was stable during cold storage.     

A semisynthetic 5-n-alkylresorcinol derivative and its effect upon biomembrane properties

MSAR (1-sulfate-3-myristoyl-5-pentadecylbenzene) is a semisynthetic derivative of 5-n-pentadecylresorcinol (C15:0). MSAR exhibits hemolytic activity against sheep erythrocytes with a EH50 value of (35 +/- 1.7) microM. At low concentrations MSAR also exhibits the ability to protect cells against their hypoosmotic lysis. This protective effect is significant as, at 0.1 microM of MSAR, the extent of osmotically induced cell lysis is reduced by approx. 20%. It was demonstrated that the 9-anthroyloxystearic acid signal was most intensively quenched by MSAR molecules, suggesting a relatively deep location of these molecules within the lipid bilayer. MSAR causes an increase of the fluorescence of the membrane potential sensitive probe. This indicates an alteration of the surface charge and a decrease of the local pH value at the membrane surface. At low bilayer content (1-4 mol%) this compound causes a significant increase of the phospholipid bilayer fluidity (both under and above the main phase transition temperature) of dipalmitoylphosphatidylcholine (DPPC) liposomes. At this low content MSAR slightly decreases the main phase transition temperature (T(c)) value. The effects induced in the phospholipid bilayer by higher contents of MSAR molecules (5-10 mol%) make it impossible to determine the T(c) value and to evaluate changes of the membrane fluidity by using pyrene-labeled lipid. MSAR also causes a decrease of the activity of membrane-bound enzymes - red blood cell acetylcholinesterase (AChE) and phospholipase A2 (PLA2). MSAR decreases the AChE activity by 40% at 100 microM. The presence of MSAR in the liposomal membrane induces a complete abolishment of the lag time of the PLA2 activity, indicating that these molecules induce the formation of packing defects in the bilayer which may result from imperfect mixing of phospholipids.