A study of the Interaction Between Cetirizine and Plasma Membrane of Eosinophils, Neutrophils, Platelets and Lymphocytes using A fluorescence Technique

The effect of cetirizine on plasma membrane fluidity and heterogeneity of human eosinophils, neutrophils, platelets and lymphocytes was investigated using a fluorescence technique. Membrane fluidity and heterogeneity were studied by measuring the steady-state fluorescence anisotropy and fluorescence decay of 1-(4- trimethylammonium-phenyl)-6-phenyl-1, 3, 5-hexatriene (TMA-DPH) incorporated in the membrane. The results demonstrate that cetirizine (1 mug/ml) induced a significant increase in the Hpid order in the exterior part of the membrane and a decrease in membrane heterogeneity in eosinophils, neutrophils and platelets. Moreover, cetirizine blocked the PAF induced changes in membrane fluidity in these cells. Cetirizine did not influence significantly the plasma membrane of lymphocytes. These data may partially explain the effect ofcetirizine on inflammatory cell activities.     

迷迭香酸对羟自由基所致小鼠肝线粒体损伤的保护作用

探索迷迭香酸对羟自由基致小鼠肝脏线粒体氧化损伤的保护作用。采用羟自由基(.OH),诱导小鼠肝线粒体损伤后,通过测定线粒体肿胀度、膜流动性、丙二醛(MDA)含量及琥珀酸脱氢酶(SDH)活性等指标以确定迷迭香酸对小鼠肝线粒体羟自由基损伤的保护作用。结果迷迭香酸剂量依赖地抑制线粒体肿胀,提高膜流动性,降低MDA的生成,增强SDH活性,差异显著。本实验证明迷迭香酸可以抑制.OH所致的线粒体损伤。     

Chenodeoxycholate induction of mitochondrial permeability transition pore is associated with increased membrane fluidity and cytochrome c release: protective role of carvedilol

Chenodeoxycholate (CDCA) is a primary bile acid mostly implicated in cholestatic liver injury. In this study, we have investigated the involvement of membrane fluidity and cytochrome c release in CDCA-induced mitochondrial permeability transition (MPT), and the preventive role of carvedilol. Treatment of calcium-loaded hepatic mitochondria with CDCA was found to cause osmotic swelling and release of cytochrome c, associated with an increase in membrane fluidity, in both protein and lipid regions. Carvedilol and cyclosporine A (CyA) reduced both cytochrome c release and alterations in membrane fluidity induced by CDCA. The hydroxylated metabolite of carvedilol, BM-910228, had no effect. Thus, modulation of membrane fluidity, plays an important role in MPT pore opening promoted by CDCA. As a result, we have delineated a pathway for the preventive role of carvedilol in mitochondrial dysfunction induced by CDCA.     

Controlling the membrane fluidity of yeasts during coupled thermal and osmotic treatments

Yeasts are often exposed to variations in osmotic pressure in their natural environments or in their substrates when used in fermentation industries. Such changes may lead to cell death or activity loss. Previous work by our team has allowed us to relate the mortality of cells exposed to a combination of thermal and osmotic treatments to leakage of cellular components through an unstable membrane when lipid phase transition occurs. In this study, yeast viability was measured after numerous osmotic and thermal treatments. In addition, the fluidity of yeast membranes was assessed according to a(w) and temperature by means of 1,6-diphenyl-1,3,5-hexatriene (DPH) anisotropy measurement. The results show that there is a negative correlation between the overall fluidity variation undergone by membranes during treatments and yeast survival. Using a diagram of membrane fluidity according to a(w) and temperature, we defined dehydration and rehydration methods that minimize fluidity fluctuations, permitting significantly increased yeast survival. Thus, such membrane fluidity diagram should be a potential tool for controlling membrane state during dehydration and rehydration and improve yeast survival. Overall fluidity measurements should now be completed by accurate structural analysis of membranes to better understand the plasma membrane changes occurring during dehydration and rehydration.     

Enterocyte mitochondrial dysfunction due to oxidative stress

The endogenous production of H2O2 in isolated rat intestinal mitochondria and oxidant induced damage to mitochondria were examined. There was an appreciable amount of H2O2 production in presence of succinate, glutamate and pyruvate, while the presence of rotenone with succinate further increased production. Superoxide generated by the X-XO system induced membrane permeability transition (MPT), calcium influx, lipid peroxidation and changes in membrane fluidity in mitochondria. A decreased mitochondrial ATPase activity and uncoupling of respiration was also observed. Spermine inhibited swelling induced by X-XO and also blocked the calcium influx and reversed the membrane fluidity changes.     

Size Effects on the Interaction of QDs with the Mitochondrial Membrane In Vitro

The mitochondrial toxicity induced by GSH-CdTe Quantum dots (QDs) of different sizes was investigated. The decreases in absorbance and transmission electron microscopy images show that QDs induce the swelling of mitochondria. Results of flow cytometry indicate that QDs cause a reduction of mitochondrial membrane potential (MMP). A remarkable increase in fluidity of protein regions of mitochondrial membrane is observed, whereas the lipid regions are not obviously affected. Cyclosporin A (CsA) effectively prevents the QD-induced mitochondrial swelling. On the basis of these results, it is proposed that QDs induce mitochondrial permeability transition (MPT). Moreover, with increasing QDs size, a pronounced MPT is observed. The difference between the membrane fluidity induced by QDs and Cadmium ion and the ineffective protective effects of EDTA suggests that the mitochondrial toxicity of QDs cannot be only attributed to the release of metal ion. The protective effects of HSA indicate that the interaction of QDs with pore-forming protein gives rise to the increase in membrane fluidity. This hypothesis is demonstrated by the interaction of QDs with model membranes and proteins using differential scanning calorimetry and isothermal titration microcalorimetry. In conclusion, as the size of QDs increases, the binding affinity of QDs with membrane protein increases, and therefore causes a pronounced mitochondrial damage.     

5-methoxytryptophol preserves hepatic microsomal membrane fluidity during oxidative stress

Lipid peroxidation is a degenerative chain reaction in biological membranes that may be initiated by exposure to free radicals. This process is associated with changes in the membrane fluidity and loss of several cell membrane-dependent functions. 5-methoxytryptophol (ML) is an indole isolated from the mammalian pineal gland. The purpose of this study was to investigate the effects of ML (0. 01mM-10mM) on membrane fluidity modulated by lipid peroxidation. Hepatic microsomes obtained from rats were incubated with or without ML (0.01-10 mM). Then lipid peroxidation was induced by FeCl(3), ADP, and NADPH. Membrane fluidity was determined using fluorescence spectroscopy. Malonaldehyde (MDA) +4-hydroxyalkenals (4-HDA) concentrations were estimated as an indicator of the degree of lipid peroxidation. With oxidative stress, membrane fluidity decreased and MDA+4-HDA levels increased. ML (0.01-3 mM) reduced membrane rigidity and the rise in MDA+4-HDA formation in a concentration-dependent manner. 10 mM ML protected against lipid peroxidation but failed to prevent the membrane rigidity. In the absence of oxidative reagents, ML (0.3-10 mM) decreased membrane fluidity whereas MDA+4-HDA levels remained unchanged. This indicates that ML may interact with membrane lipids. The results presented here suggest that ML may be another pineal indoleamine (in addition to melatonin) that resists membrane rigidity due to lipid peroxidation.     

Effect of nedocromil sodium on polymorphonuclear leukocyte plasma membrane

The effect of nedocromil sodium on the plasma membrane fluidity of polymorphonuclear leukocytes (PMNs) was investigated by measuring steady-state fluorescence anisotropy of 1-[4-trimethylammonium-phenyl]-6-phenyl- 1,3,5-hexatriene (TMA-DPH) incorporated in the membrane. Our results show that nedocromil sodium 300 muM significantly decreased membrane fluidity of PMNs. The decrease in membrane fluidity of PMNs induced by fMLP was abolished in the presence of nedocromil sodium. These data suggest that nedocromil sodium interferes with the plasma membranes of PMNs and modulates their activities.     

Increase in membrane fluidity in liposomes and plant protoplasts upon osmotic swelling.

Osmotic gradient across the membrane of nonsonicated liposomes and rose petal protoplasts are shown to induce swelling. Concomitantly, the lipid fluidity as measured by fluorescence depolarization is increased, probably due to increase in molar free volume. It is suggested that osmotic swelling can affect cell physiology via changes in membrane fluidity.     

Increase in membrane fluidity in liposomes and plant protoplasts upon osmotic swelling

Osmotic gradient across the membrane of nonsonicated liposomes and rose petal protoplasts are shown to induce swelling. Concomitantly, the lipid fluidity as measured by fluorescence depolarization is increased, probably due to increase in molar free volume. It is suggested that osmotic swelling can affect cell physiology via changes in membrane fluidity.     

Antioxidant activity of two dibenzocyclooctene lignans on the aged and ischemic brain in rats.

The effects of two dibenzocyclooctene lignans on peroxidative damage of aging and ischemic rat brain were studied. Incubation of eight-month-old rat brain mitochondria and membrane suspension with Fe(2+)-cysteine resulted in the formation of malondialdehyde (MDA) and decrease of ATPase activity. Schisanhenol (Sal) (10(-4) M) completely inhibited the peroxidative damages of brain mitochondria and membrane of rats. The swelling and disintegration of brain mitochondria, as well as the reduction of brain membrane fluidity induced by Fe(2+)-cysteine were also prevented by Sal. The results of imitative experiment of ischemia and reperfusion of brain mitochondria and membrane in vitro indicated that Sal significantly impeded production of MDA and loss of ATPase activity induced by reoxygenation following anoxia. Oral administration of Sal induced increase of cytosol glutathione-peroxidase of brain in mice under the condition of reoxygenation following anoxia. The other compound schizandrin (Sin B) also has similar activity. But its potency is weaker than that of Sal. All these results indicate that Sal and Sin B have protective action against oxidative stress.     

Influence of membrane fluidity modifiers on lysosomal osmotic sensitivity

Since lysosomes are prone to osmotic lysis, we have examined the correlation between their physical state and sensitivity to osmotic challenge, using agents which modify membrane fluidity. The latency loss of beta-hexosaminidase after an incubation in hypotonic sucrose medium was followed under different conditions of membrane fluidity, recorded by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3, 5-hexatriene. Increasing fluidity of the lysosomal membranes with benzyl alcohol (BA) and greater rigidity caused by cholesteryl hemisuccinate (CHS) increased and decreased the enzyme latency loss, respectively. The effects of BA and CHS treatments on osmotic sensitivity were reversible subsequently by reciprocal treatments of the lysosomes with CHS and BA, respectively. The results indicate that the physical state of the membrane does indeed affect lysosomal osmotic stability.     

Propidium uptake and ATP release in A549 cells share similar transport mechanisms

The lipid bilayer of eukaryotic cells’ plasma membrane is almost impermeable to small ions and large polar molecules, but its miniscule basal permeability in intact cells is poorly characterized. This report describes the intrinsic membrane permeability of A549 cells toward the charged molecules propidium (Pr²⁺) and ATP^4?. Under isotonic conditions, we detected with quantitative fluorescence microscopy, a continuous low-rate uptake of Pr (～150 × 10^?21 moles (zmol)/h/cell, [Pr]_o = 150 μM, 32°C). It was stimulated transiently but strongly by 66% hypotonic cell swelling reaching an influx amplitude of ～1500 (zmol/h)/cell. The progressive Pr uptake with increasing [Pr]_o (30, 150, and 750 μM) suggested a permeation mechanism by simple diffusion. We quantified separately ATP release with custom wide-field-of-view chemiluminescence imaging. The strong proportionality between ATP efflux and Pr²⁺ influx during hypotonic challenge, and the absence of stimulation of transmembrane transport following 300% hypertonic shock, indicated that ATP and Pr travel the same conductive pathway. The fluorescence images revealed a homogeneously distributed intracellular uptake of Pr not consistent with high-conductance channels expressed at low density on the plasma membrane. We hypothesized that the pathway consists of transiently formed water pores evenly spread across the plasma membrane. The abolition of cell swelling-induced Pr uptake with 500 μM gadolinium, a known modulator of membrane fluidity, supported the involvement of water pores whose formation depends on the membrane fluidity. Our study suggests an alternative model of a direct permeation of ATP (and other molecules) through the phospholipid bilayer, which may have important physiological implications.     

Interaction of 1,3,4-thiadiazolium mesoionic derivatives with mitochondrial membrane and scavenging activity: Involvement of their effects on mitochondrial energy-linked functions

The aim of this work was to assess the significance of the interaction of the 1,3,4-thiadiazolium derivatives MI-J, MI-4F and MI-2,4diF with mitochondrial membrane and their effects on energy-linked functions. Mitochondrial swelling in the absence of substrate was inhibited by all derivatives; however, the fluorine derivatives were most effective. MI-4F decreased swelling by ~32% even at the lowest concentration (65 nmol mg(-1) protein), reaching ~67% at the concentration of 130 nmol mg(-1) protein. Swelling of mitochondria in the presence of oxidizable substrates was also strongly decreased by all derivatives. This effect was more pronounced when using glutamate plus malate, and also fluorine derivatives, which promoted complete inhibition at all concentrations (6.5-130 nmol mg(-1) protein). Swelling occurred when succinate was the substrate in the presence of MI-J (6.5-65 nmol mg(-1) protein); however, the shrinkage rate was strongly decreased. MI-4F and MI-2,4diF also inhibited swelling, with total inhibition occurring at a concentration of 65 nmol mg(-1) protein. Lipid peroxidation induced by Fe(3+)-ADP/2-oxoglutarate in isolated mitochondria was inhibited time- and dose-dependently by the derivatives, reaching complete inhibition at the highest concentration (80 nmol mg(-1) protein). However, when lipid peroxidation was initiated by peroxyl radicals generated from AAPH, the inhibition was less intense, reaching ~50%, ~40% and ~58% with MI-J, MI-4F and MI-2,4diF (80 nmol mg(-1) protein), respectively. The mesoionic compounds also showed superoxide radical scavenging ability of ~22%, ~32% and ~40% (80 nmol mg(-1) protein), respectively. Fluorescence polarization experiments showed that the derivatives are able to enter the bilayer, decreasing its fluidity in the hydrophobic DMPC membrane region and ordering the fluid phase. Our results suggest that MI-J, MI-4F and MI-2,4diF interact significantly, albeit in different modes, with mitochondrial membrane, and that fluorine derivatives seem to alter the membrane's properties more markedly.     

Concanavalin A-induced increase in the membrane fluidity of chicken erythrocytes.

To determine the fluidity of the membrane lipid phase, chicken erythrocytes were labeled with a stearic acid derivative spin label. When chicken erythrocytes were treated with concanavalin A (Con A), ESR spectra showed a change in the peaks of the labels in membrane lipids, indicating an increase of membrane fluidity. The degree of the increase in fluidity of the membrane lipid phase depended on the valency of the lectin used. Tetravalent Con A induced an increase of membrane fluidity at a concentration as low as 30 micrograms/ml, while a monovalent derivative of Con A did not affect membrane fluidity. This increase in membrane fluidity was observed within 10 min after the addition of Con A. If bound Con A was removed with methyl alpha-D-mannoside later than 60 min after its addition, a complete return of the fluidity to the normal level could not be observed. However, no change was found in the composition of phospholipids or in the fatty acid compositions of phosphatidylcholine and phosphatidylethanolamine of chicken erythrocytes after the addition of Con A, indicating that this increase in membrane fluidity is not caused by a change of lipid composition. The clustering of membrane receptors of chicken erythrocytes for Con A was demonstrated when the two-dimensional distribution of ferritin-conjugated Con A on the membranes was assayed by transmission electron microscopy. Furthermore, it was shown that major receptors for Con A of chicken erythrocytes were transmembrane glycoproteins having apparent molecular weights of 100K, 45, and 33K.     

The fluidity of Chinese hamster ovary cell and bull sperm membranes after cholesterol addition

Cell plasma membrane fluidity is affected by membrane lipid and protein composition as well as temperature. Altering the cholesterol content of a membrane can change membrane fluidity at different temperatures and this may affect cell survival during cryopreservation. In these experiments, we examined the effect that adding cholesterol to the membranes of Chinese hamster ovary cells (CHO) and bull sperm had on cell plasma membrane fluidity and cell survival when cells were cooled to 5 degrees C or were cryopreserved. Cells were treated with 0, 1.5 or 5.0mg cholesterol-loaded cyclodextrin (CLC), stained with N-((4-(6-phenyl-1,3,5-hexatrienyl)phenyl)propyl)trimethylammonium-p-toluenesulfonate (TMAP-DPH) to evaluate membrane fluidity and with propidium iodide to evaluate cell viability, prior to analysis by flow cytometry at 23, 5 degrees C, and after cryopreservation. CHO cells exhibited a single cell population with all cells having similar membrane fluidity. Membrane fluidity did not change when temperature had been reduced and then returned to 23 degrees C (P<0.05), however, adding cholesterol to the cells induced membranes to become more rigid (P<0.05). Bull sperm samples consisted of two cell subpopulations, one having relatively higher membrane fluidity than the other, regardless of cholesterol treatment or temperature. In addition, cells possessing the highest membrane fluidity did not survive cooling or cryopreservation efficiently. CLC treatment did not significantly alter membrane fluidity after temperature changes, but did maintain higher percentages of spermatozoa surviving cooling to 5 degrees C and cryopreservation (P<0.05). In conclusion, adding cholesterol to cell resulted in detectable membrane fluidity changes in CHO cells and increased survival of bull sperm after cooling to 5 degrees C and after cryopreservation.     

Proton-induced endocytosis is dependent on cell membrane fluidity,lipid-phase order and the membrane resting potential

Recently it has been shown that decreasing the extracellular pH of cells stimulates the formation of inward membrane invaginations and vesicles, accompanied by an enhanced uptake of macromolecules. This type of endocytosis was coined as proton-induced uptake (PIU). Though the initial induction of inward membrane curvature was rationalized in terms of proton-based increase of charge asymmetry across the membrane, the dependence of the phenomenon on plasma membrane characteristics is still unknown. The present study shows that depolarization of the membrane resting potential elevates PIU by 25%, while hyperpolarization attenuates it by 25%. Comparison of uptake in suspended and adherent cells implicates that the resting-potential affects PIU through remodeling the actin-cytoskeleton. The pH at the external interface of the cell membrane rather than the pH gradient across it determines the extent of PIU. PIU increases linearly upon temperature increase in the range of 4–36 °C, in correlation with the membrane fluidity. The plasma membrane fluidity and the lipid phase order are modulated by enriching the cell's membrane with cholesterol, tergitol, dimethylsulfoxide, 6-ketocholestanol and phloretin and by cholesterol depletion. These treatments are shown to alter the extent of PIU and are better correlated with membrane fluidity than with the lipid phase order. We suggest that the lipid phase order and fluidity influence PIU by regulating the lipid order gradient across the perimeter of the lipid-condensed microdomains (rafts) and alter the characteristic tension line that separates the higher ordered lipid-domains from the lesser ordered ones.     

Exploiting the Role of Resveratrol in Rat Mitochondrial Permeability Transition

Resveratrol (RSV), a natural polyphenolic antioxidant, has been considered an anticarcinogenic agent as it triggers tumor cell apoptosis through activation of the mitochondrial pathway. In our study, the effects of RSV on mitochondria, especially on the mitochondrial permeability transition (MPT) process, were investigated by multiple methods. We found that RSV induced a collapse of membrane potential and matrix swelling related to MPT. We further demonstrated that Ca²⁺ was necessary for this RSV-induced MPT opening. In addition, RSV induced the inner membrane permeabilization to H⁺ and K⁺, the depression of respiration and changes in membrane fluidity. The results suggested that RSV-induced MPT was accompanied by mitochondrial dysfunction. But the prohibition on lipid peroxidation and different effects of low- and high-dose RSV on membrane fluidity and respiration showed that the interaction of RSV and the mitochondria could not be the result of a single simple mechanism.     

HDL derived from the different phases of conjugated diene formation reduces membrane fluidity and contributes to a decrease in free cholesterol efflux from human THP-1 macrophages

Oxidized HDL (ox-HDL) has been reported to reduce free cholesterol efflux from cells. In this study we investigate the effect of different stages of ox-HDL on macrophage membrane fluidity and its effect on free cholesterol efflux from macrophages as a cell function influenced by ox-HDL. HDL was oxidized by means of conjugated diene production using copper as a prooxidant. Fluidity of HDL and human THP-1 macrophage membranes was evaluated by changes in fluorescence anisotropy (r) by DPH probe where lower (r) values give higher fluidity. We found that ox-HDL derived from the propagation phase (PP-HDL) and the decomposition phase (DP-HDL) became less fluid ((r): 0.263+/-0.001, 0.279+/-0.002, respectively) than HDL from the lag phase (LP-HDL) and native HDL (nat-HDL) ((r): 0.206+/-0.001) (P<0.05). Macrophages incubated with PP-HDL and DP-HDL had less fluid membranes ((r): 0.231+/-0.001, 0.243+/-0.002, respectively) than those incubated with LP-HDL and nat-HDL ((r): 0.223+/-0.001) (P<0.05). Consequently, fluidity was reduced not only in ox-HDL but also in the cell membranes exposed to ox-HDL. A significant negative correlation was observed between macrophage membrane fluorescence anisotropy (r) and free cholesterol efflux from these cells (-0.876; P<0.05). Thus, lower membrane fluidity was associated with lower free cholesterol efflux from cells. In conclusion, the increase in the HDL oxidation process leads to a lost of macrophage membrane fluidity that could contribute to an explanation of the reduction of free cholesterol efflux from cells by ox-HDL.     

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.