Increase In Membrane Viscosity Research Articles

Effects of grafted polymers on the lipid membrane fluidity

Since the membrane fluidity controls the cellular functions, it is important to identify the factors that determine the cell membrane viscosity. Cell membranes are composed of not only lipids and proteins but also polysaccharide chain-anchored molecules, such as glycolipids. To reveal the effects of grafted polymers on the membrane fluidity, in this study, we measured the membrane viscosity of polymer-grafted giant unilamellar vesicles (GUVs), which were prepared by introducing the poly (ethylene glycol) (PEG)-anchored lipids to the ternary GUVs composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol. The membrane viscosity was obtained from the velocity field on the GUV generated by applying a point force, based on the hydrodynamic model proposed by Henle and Levine. The velocity field was visualized by a motion of the circular liquid ordered (Lo) domains formed by a phase separation. With increasing PEG density, the membrane viscosity of PEG-grafted GUVs increased gradually in the mushroom region and significantly in the brush region. We propose a hydrodynamic model that includes the excluded volume effect of PEG chains to explain the increase in membrane viscosity in the mushroom region. This work provides a basic understanding of how grafted polymers affect the membrane fluidity.

Mapping cisplatin-induced viscosity alterations in cancer cells using molecular rotor and fluorescence lifetime imaging microscopy.

.Significance: Despite the importance of the cell membrane in regulation of drug activity, the influence of drug treatments on its physical properties is still poorly understood. The combination of fluorescence lifetime imaging microscopy (FLIM) with specific viscosity-sensitive fluorescent molecular rotors allows the quantification of membrane viscosity with high spatiotemporal resolution, down to the individual cell organelles.Aim: The aim of our work was to analyze microviscosity of the plasma membrane of living cancer cells during chemotherapy with cisplatin using FLIM and correlate the observed changes with lipid composition and cell’s response to treatment.Approach: FLIM together with viscosity-sensitive boron dipyrromethene-based fluorescent molecular rotor was used to map the fluidity of the cell’s membrane. Chemical analysis of membrane lipid composition was performed with time-of-flight secondary ion mass spectrometry (ToF-SIMS).Results: We detected a significant steady increase in membrane viscosity in viable cancer cells, both in cell monolayers and tumor spheroids, upon prolonged treatment with cisplatin, as well as in cisplatin-adapted cell line. ToF-SIMS revealed correlative changes in lipid profile of cisplatin-treated cells.Conclusions: These results suggest an involvement of membrane viscosity in the cell adaptation to the drug and in the acquisition of drug resistance.

Effect of intra-membrane C60 fullerenes on the modulus of elasticity and the mechanical resistance of gel and fluid lipid bilayers.

Penetration and partition of C60 to the lipid bilayer core are both relevant to C60 toxicity, and useful to realise C60 biomedical potential. A key aspect is the effect of C60 on bilayer mechanical properties. Here, we present an experimental study on the mechanical effect of the incorporation of C60 into the hydrophobic core of fluid and gel phase zwitterionic phosphatidylcholine (PC) lipid bilayers. We demonstrate its incorporation inside the hydrophobic lipid core and the effect on the packing of the lipids and the vesicle size using a combination of infrared (IR) spectroscopy, atomic force microscopy (AFM) and laser light scattering. Using AFM we measured the Young's modulus of elasticity (E) of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in the absence (presence) of intra-membranous C60 at 24.5 °C. E of fluid phase supported bilayers is not altered by C60, but E increases with incorporation of C60 in gel phase bilayers. The increase is higher for longer hydrocarbon chains: 1.6 times for DPPC and 2 times for DSPC. However the mechanical resistance of gel phase bilayers of curved bilayered structures decreases with the incorporation of C60. Our combined results indicate that C60 causes a decrease in gel phase lipid mobility, i.e. an increase in membrane viscosity.

Influence of membrane viscosity on capsule dynamics in shear flow

AbstractMost previous numerical studies on capsule dynamics in shear flow have ignored the role of membrane viscosity. Here we present a numerical method for large deformation of capsules using a Kelvin–Voigt viscoelastic model for the membrane. After introducing the model and the related numerical implementation, we present a comprehensive analysis of the influence of the membrane viscosity on buckling, deformation and dynamics. We observe that the membrane viscosity leads to buckling in the range of shear rate in which no buckling is observed for capsules with purely elastic membrane. For moderate to large shear rates, the wrinkles on the capsule surface appear in the same range of the membrane viscosity that was reported earlier for artificial capsules and red blood cells based on experimental measurements. In order to obtain stable shapes, it is necessary to introduce the bending stiffness. It is observed that the range of the bending stiffness required is also in the same range as that reported for the red blood cells, but considerably higher than that estimated for artificial capsules. Using the stable shapes obtained in the presence of bending stiffness, we analyse the influence of membrane viscosity on deformation, inclination and tank-treading frequency of initially spherical capsules. Membrane viscosity is observed to reduce the capsule deformation, and introduce a damped oscillation in time-dependent deformation and inclination. The time-averaged inclination angle shows a non-monotonic trend with an initial decrease reaching a minimum and a subsequent increase with increasing membrane viscosity. A similar non-monotonic trend is also observed in the tank-treading frequency. We then consider the influence of the membrane viscosity on the unsteady dynamics of an initially oblate capsule. The dynamics is observed to change from a swinging motion to a tumbling motion with increasing membrane viscosity. Further, a transient dynamics is also observed in which a capsule starts with one type of dynamics, but settles with a different dynamics over a long time.

A shift to 50°C provokes death in distinct ways for glucose- and oleate-grown cells of Yarrowia lipolytica

Based on the observation that shocks provoked by heat or amphiphilic compounds present some similarities, this work aims at studying whether cells grown on oleate (amphiphilic pre-stress) acquire a tolerance to heat shock. In rich media, changing glucose for oleate significantly enhanced the cell resistance to the shock, however, cells grown on a minimal oleate medium lost their ability to grow on agar with the same kinetic than glucose-grown cells (more than 7-log decrease in 18min compared with 3-log for oleate-grown cells). Despite this difference in kinetics, the sequence of events was similar for oleate-grown cells maintained at 50°C with a (1) loss of ability to form colonies at 27°C, (2) loss of membrane integrity and (3) lysis (observed only for some minimal-oleate-grown cells). Glucose-grown cells underwent different changes. Their membranes, which were less fluid, lost their integrity as well and cells were rapidly inactivated. But, surprisingly, their nuclear DNA was not stained by propidium iodide and other cationic fluorescent DNA-specific probes but became stainable by hydrophobic ones. Moreover, they underwent a dramatic increase in membrane viscosity. The evolution of lipid bodies during the heat shock depended also on the growth medium. In glucose-grown cells, they seemed to coalesce with the nuclear membrane whereas for oleate-grown cells, they coalesced together forming big droplets which could be released in the medium. In some rare cases of oleate-grown cells, lipid bodies were fragmented and occupied all the cell volume. These results show that heat triggers programmed cell death with uncommon hallmarks for glucose-grown cells and necrosis for methyl-oleate-grown cells.

Mutual effects of MinD–membrane interaction: I. Changes in the membrane properties induced by MinD binding

In Escherichia coli and other bacteria, MinD, along with MinE and MinC, rapidly oscillates from one pole of the cell to the other controlling the correct placement of the division septum. MinD binds to the membrane through its amphipathic C-terminal α-helix. This binding, promoted by ATP-induced dimerization, may be further enhanced by a consequent attraction of acidic phospholipids and formation of a stable proteolipid domain. In the context of this hypothesis we studied changes in dynamics of a model membrane caused by MinD binding using membrane-embedded fluorescent probes as reporters. A remarkable increase in membrane viscosity and order upon MinD binding to acidic phospholipids was evident from the pyrene and DPH fluorescence changes. This viscosity increase is cooperative with regards to the concentration of MinD-ATP, but not of the ADP form, indicative of dimerization. Moreover, similar changes in the membrane dynamics were demonstrated in the native inverted cytoplasmic membranes of E. coli, with a different depth effect. The mobility of pyrene-labeled phosphatidylglycerol indicated formation of acidic phospholipid-enriched domains in a mixed acidic-zwitterionic membrane at specific MinD/phospholipid ratios. A comparison between MinD from E. coli and Neisseria gonorrhea is also presented.

Dynamics of fluid vesicles in shear flow: Effect of membrane viscosity and thermal fluctuations

The dynamical behavior of vesicles is investigated in simple shear flow. A simulation technique is presented that combines a three-dimensional particle-based mesoscopic model (multiparticle collision dynamics) for the solvent with a dynamically triangulated surface model for the membrane. In this model, thermal fluctuations of the solvent and of the membrane are consistently taken into account. The membrane viscosity can be varied by changing the bond-flip rate of the dynamically triangulated surface. Vesicles are found to transit from steady tank-treading to unsteady tumbling motion with increasing membrane viscosity. At small reduced volumes, the shear induces a transformation from a discocyte to a prolate shape at low membrane viscosity. On the other hand, at high membrane viscosity, the shear induces a transformation from prolate to discocyte, or tumbling motion accompanied by shape oscillations between these two states. Thermal fluctuations induce intermittent tumbling and smooth out the transitions. This effect can be understood from a simplified stochastic model.

Fluid Vesicles with Viscous Membranes in Shear Flow

The effect of membrane viscosity on the dynamics of vesicles in shear flow is studied. We present a new simulation technique, which combines three-dimensional multiparticle collision dynamics for the solvent with a dynamically triangulated membrane model. Vesicles are found to transit from steady tank treading to unsteady tumbling motion with increasing membrane viscosity. Depending on the reduced volume and membrane viscosity, shear can induce both discocyte-to-prolate and prolate-to-discocyte transformations. This behavior can be understood from a simplified model.

Modulation of anthracycline activity in canine mammary tumour cells in vitro by medroxyprogesterone acetate

Failure of chemotherapy with anthracyclines as a result of drug resistance and toxicity is a major problem in the clinical management of neoplasia. The aim of the present study was to evaluate the activity of medroxyprogesterone acetate (MPA) as a chemosensitiser on anthracycline cytotoxicity. The study investigated whether such an effect could be related to an increase in lipid peroxidation, nitric oxide production, membrane fluidity and intracellular anthracycline concentration. The results showed that anthracyclines decreased nitric oxide production but increased membrane viscosity (polarisation constant) and lipid hydroperoxide formation in canine mammary tumour cells. Moreover, it was found that both drug-induced cytotoxicity and membrane viscosity increased in the presence of MPA. Conversely, lipid hydroperoxides decreased in MPA-supplemented cells. Medroxyprogesterone acetate did not show any effect on nitric oxide production. The two anthracyclines used (doxorubicin and idarubicin) showed differential intranuclear accumulation in canine mammary tumour cells, and MPA significantly modified intracellular concentration of anthracyclines.

Analysis of red blood cell motion through cylindrical micropores: effects of cell properties

Filtration through micropores is frequently used to assess red blood cell deformability, but the dependence of pore transit time on cell properties is not well understood. A theoretical model is used to simulate red cell motion through cylindrical micropores with diameters of 3.6, 5, and 6.3 microns, and 11-microns length, at driving pressures of 100-1000 dyn/cm2. Cells are assumed to have axial symmetry and to conserve surface area during deformation. Effects of membrane shear viscosity and elasticity are included, but bending resistance is neglected. A time-dependent lubrication equation describing the motion of the suspending fluid is solved, together with the equations for membrane equilibrium, using a finite difference method. Predicted transit times are consistent with previous experimental observations. Time taken for cells to enter pores represents more than one-half of the transit time. Predicted transit time increases with increasing membrane viscosity and with increasing cell volume. It is relatively insensitive to changes in internal viscosity and to changes in membrane elasticity except in the narrowest pores at low driving pressures. Elevating suspending medium viscosity does not increase sensitivity of transit time to membrane properties. Thus filterability of red cells is sensitively dependent on their resistance to transient deformations, which may be a key determinant of resistance to blood flow in the microcirculation.

Elevated protein tyrosine phosphatase activity and increased membrane viscosity are associated with impaired activation of the insulin receptor kinase in old rats

Insulin resistance is very common in the elderly, and may be associated with glucose intolerance or frank diabetes. In previous studies we demonstrated that insulin resistance in old Wistar rats is associated with decreased autophosphorylation and activation of the hepatic insulin receptor kinase (IRK) in vivo. We now show that this defect can be reproduced in vitro, where the extent of insulin-induced activation of IRK in liver membranes of old rats was decreased by approximately 50% compared with young controls. The defect could be largely abolished after solubilization of the membranes with Triton X-100. We also show that: (a) the viscosity of membranes from the old rats was significantly (P < 0.001, n = 4) higher (by 15%) compared with young controls; (b) incubation of plasma membranes from old animals with lecithin liposomes, which lowered their cholesterol levels, partially abolished the defect in IRK activation; and (c) Triton extracts of liver membranes prepared from old rats did not interfere with the activation of IRK derived from young controls. Additionally, non-membrane components did contribute to the development of this defect. We observed a significant (approximately 30%) (P < 0.001, n = 18) elevation of cytosolic protein tyrosine phosphatase (PTP) activity directed against the beta subunit of the insulin receptor in livers of old rats. No such elevation of PTP activity could be demonstrated with synthetic substrates. Our findings are consistent with a model in which increased membrane viscosity as well as enhancement of a cytosolic PTP activity both markedly inhibit the activation in vivo of the hepatic IRK in old animals.

The effect of endotoxin on the neonatal erythrocyte

Intestinal ischemia is considered a major factor in the development of necrotizing enterocolitis (NEC). Despite this, the majority of affected infants lack documentation of clinical events associated with obvious gut hypoperfusion. Recent evidence in adults suggests that endotoxin may impair flow in the microcirculation through alterations in erythrocyte deformability. As the gut serves as a semipermeable reservoir of endotoxin in the stressed neonate, such localized activity may result in intestinal ischemia at the microcirculatory level through alterations in the red cell membrane. This study evaluates the role of endotoxin on neonatal erythrocyte membrane viscosity. Paired anticoagulated whole blood specimens were obtained from the umbilical cord of 10 neonates at delivery. Samples were incubated with either 2 μg/mL of E coli endotoxin (LPS) or an equal volume of saline (control). Following incubation, erythrocytes were isolated, washed, and incorporated with the fluorescent membrane probe TMA-DPH. Membrane viscosity was assessed by spectroscopic analysis of the fluorescent emissions induced by excitation of the probe at 365 nm. Results were calculated as anisotropy and analyzed for differences by ANOVA. Endotoxin resulted in a significant increase in red cell membrane viscosity as compared to control (LPS 291.2 ± 5.1 sv Control 271.7 ± 3.3, P < .01). As the effects of endotoxin are known to be primarily the result of white blood cell (WBC) activation, this study was repeated in an additional 10 neonates in whom WBCs were removed prior to endotoxin/saline incubation. In the absence of WBCs, endotoxin failed to induce any significant alteration in membrane viscosity (LPS 288.8 ± 4.4 v Control 292.2 ± 4.7, P = NS). Endotoxin results in increased membrane viscosity in the neonatal erythrocyte. These changes are modulated by the WBC. Such membrane alterations may impair flow in the intestinal microcirculation and serve as a potential inciting event in the development of NEC.

Estimation of the oxygen gradient across phospholipid bilayers of mitochondria from reperfused rabbit hearts after ischemia.

Mitochondria isolated from myocardium exposed to 30 minute ischemia followed by 30 minute reperfusion showed an increase in membrane viscosity and a decrease in wobbling angle of phospholipids, compared with those from the normally perfused myocardium in anesthetized open-chest rabbits. The values for the membrane viscosity were used to estimate the oxygen gradient across the lipid bilayers of mitochondrial membranes with a model of cylindrical diffusion. The effective diffusion coefficient for oxygen, DO2, was approximated to be 6.5 and 6.3 x 10(-5) cm2/sec in the control and ischemic-reperfused area, respectively, by comparing reported DO2 values with values for membrane viscosity. For the surface area of the inner mitochondrial membrane including cristae and for the oxygen consumption rate of the myocardium, reported values for rats and cats, respectively, were employed. Using these values, oxygen gradients across the lipid bilayers of mitochondrial lipid membranes were estimated to be only 0.055 and 0.057 nM in the control and 30 minute ischemic-reperfused myocardium, respectively. If the mitochondrial membranes are hydrated because of the ischemia-reperfusion, the absorption coefficient of the membrane to oxygen will decrease and the oxygen gradient will be increased. In the present study, however, the fluorescence life time of DPH, the hydrophobic fluorophore, showed no shortening despite the ischemia-reperfusion. Hence, no indication of membrane hydration was obtained.

A study of the stability of W/O/W multiple emulsions

The stability of W/O/W emulsions has been studied in batch agitators. The surfactants suitable for W/O/W emulsions were screened and the factors affecting the emulsion stability were also studied. Results showed that polyamine E644 was an excellent emulsifier for W/O emulsions since the emulsion stabilized by it has not only good stability but also shows smaller swelling. The stability of the emulsion increases with increase in membrane viscosity and concentration of surfactant, but decreases with increase in concentration of the internal reagent in the internal phase and the carriers in the membrane. Raising the agitation speed and the time for preparing the W/O emulsion is beneficial to membrane stability due to formation of smaller internal droplets, but raising the speed for dispersing the W/O emulsion in the external phase results in an increase in membrane breakage.

132 CALCIUM REGULATED MEMBRANE-ATTACHED HEMOGLOBIN AND MECHANICAL PROPERTIES OF DENSITY FRACTIONATED ERYTHROCYTES HEMBRANES: IMPLICATIONS FOR HEMOLYTIC DISEASES AND RBC AGEING

The present study was designed to evaluate the influence of calcium (Ca)1, regulated membrane attached hemoglobin (Hbm) on the viscoelastic properties of density fractionated human RBC membranes. (Ca)1 of RBC was elevated using ionophore A23187 (10μM); mechanical properties of RBC membranes were determined via micropipette technique. Salient results included: 1) a 300% increase in membrane viscosity of aged cells following ionophore treatment corresponding to elevated Hbm levels; 2) no change in membrane viscosity and Hbm, levels of young cells after ionophore exposure; 3) no dependence between elastic shear modulus and intracellular calcium concentration. 4) Comparison to osmotically shrunk cells reveals an additional calcium concentration dependent association of hemoglobin to the membrane. This implies nembrane mechanical properties are independent from the intracellular viscosity. Our results appear relevant to hemolytic diseases characterized by elevated (Ca)1, (e.g. sickle cell anemia) and to age associated changes in deformability. Supported by DFG Fr 752/1-1 u. 1-2 by NIH grants HL 15162 and HL 15722 and Dy AHA-GLAA award 5371G.

Biophysical correlates of lysophosphatidylcholine- and ethanol-mediated shape transformation and hemolysis of human erythrocytes. Membrane viscoelasticity and NMR measurement

The effects of monopalmitoylphosphatidylcholine (MPPC or lysophosphatidylcholine) and a series of short-chain primary alcohols (ethanol, 1-butanol and 1-hexanol) on cell shape, hemolysis, viscoelastic properties and membrane lipid packing of human red blood cells (RBCs) were studied. For MPPC, the effective membrane concentration to induce the formation of stage 3 echinocytes ( 8 · 10 6 molecules per cell) was one order of magnitude lower than that needed to induce 50% hemolysis ( 7 · 10 7 molecules per cell). In contrast, short-chain alcohols induced both shape changes and hemolysis within close concentration range ( 2.5 · 10 8 to 3.5 · 10 8 molecules per cell). Viscoelastic properties of the RBCs were studied by micropipette aspiration and correlated with shape change. Ethanol-treated RBCs showed a decrease in membrane elastic modulus and an increase in membrane viscosity in the recovery phase at the early stage of shape change. MPPC-treated cells showed the same type of viscoelastic changes, but these were not observed until the formation of stage 2 echinocytes. High-resolution solid-state 13C nuclear magnetic resonance technique was applied to study membrane lipid packing in the ghost membrane by following the chemical shift of hydrocarbon chains. Both MPPC and ethanol caused the 13C-NMR chemical shift to move upfield, indicating that membrane lipids were expanded due to the intercalation of these exogenous molecules. Using data obtained from model compounds, we convert values of chemical shift into a lipid packing parameter, i.e., number of gauche bonds for fatty acyl hydrocarbon chains. Approximately 10 8 interacting molecules per cell are required to induce a detectable change of lipid packing by both MPPC and ethanol. The results indicate that homolysis occurs at a smaller surface area for MPPC- than ethanol-treated RBCs. Our findings suggest that progressive changes in the molecular packing in the membrane lead eventually to hemolysis, but the mode responsible for shape transformation varies with these amphipaths.

Substrate stimulation of 7 alpha-hydroxylase, an enzyme located in the cholesterol-poor endoplasmic reticulum.

We examined the role of cholesterol in altering the activity of the microsomal cytochrome P-450 enzyme, cholesterol-NADPH:oxygen oxidoreductase (cholesterol 7 alpha-hydroxylase). Liposomes were used to deliver cholesterol to hepatic microsomes. Formation of 7 alpha-hydroxycholesterol was quantitated by isotope dilution/gas chromatography-mass spectrometry. As the liposomal cholesterol/phospholipid molar ratio increased, 7 alpha-hydroxylase activity increased, whereas the activity of another microsomal cytochrome P-450 enzyme, ethylmorphine N-demethylase, decreased. To determine if the degree of stimulation was affected by the endogenous activity (without liposomes), microsomes, from rats fed chow alone or chow containing cholestyramine, taurocholate, or cholesterol were challenged with cholesterol-enriched liposomes. The degree of stimulation was dependent upon the endogenous activity: cholestyramine-fed much greater than cholesterol = chow control greater than taurocholate-fed. To determine if cholesterol stimulates 7 alpha-hydroxylase by increasing membrane viscosity, microsomes were incubated with liposomes having the same cholesterol/phospholipid molar ratio as microsomes, but different viscosities. Dipalmitoylphosphatidylcholine (high viscosity) liposomes increased microsomal viscosity and decreased 7 alpha-hydroxylase activity. In contrast, dioleoylphosphatidylcholine (low viscosity) liposomes decreased microsomal viscosity and increased enzyme activity. Since greater viscosity inhibits 7 alpha-hydroxylase, cholesterol cannot stimulate the enzyme by increasing membrane viscosity. The data suggest that cholesterol stimulates production of 7 alpha-hydroxycholesterol by providing substrate.

Effect of membrane conductance on proton/electron stoichiometry of cytochrome c oxidase activity in plant mitochondria

Proton translocation linked to cytochrome c oxidase activity in antimycin-inhibited potato mitochondria has been investigated using the classical oxygen pulse technique. At 25°C, proton translocation took place with an apparent H + e − ratio of 1.10 ± 0.06 with ascorbate plus N, N, N′, N′-tetramethylphenylenediamine (TMPD) and 1.19 ± 0.05 with ascorbate plus ferrocyanide. The decrease in temperature from 25°C to 5°C resulted in an increase in membrane viscosity associated with a decrease in the rate constant of the proton back-flow. Under these conditions, the extent of proton ejection was strongly enhanced and the H + e − ratio of ascorbate/TMPD was increased up to 1.73 ± 0.06 and reached 3.65 ± 0.20 for the span succinate-O 2. In the presence of increasing concentrations of the uncoupler CCCP, at 5°C the H + e − ratio observed or extrapolated at zero time was lowered as the rate of proton back-flow was enhanced. The same relationship was obtained between the H + e − ratio and the rate of proton back-flow after enhancement of proton conductance by either temperature or CCCP. It results that in both cases the calculated H + e − ratio extrapolated to zero time was underestimated. Extrapolation of H +/e − ratio in potato mitochondria under conditions of zero proton back-flow provides values which approach 2 for the cytochrome c oxidase complex and 4 for the span succinate-O 2.

Enhanced membrane phospholipid metabolism in human pancreatic adenocarcinoma cell lines detected by low-resolution 1H nuclear magnetic resonance spectroscopy.

The low-resolution proton nuclear magnetic resonance (NMR) spectra of human pancreatic adenocarcinomas maintained in nude mice and in culture exhibit characteristic features. First, the high values of longitudinal (T1) and transverse (T2) relaxation times were attributed to disturbances in the exchange of ions and water molecules in cancerous cells. Second, a new peak situated at about 1.8 +/- 0.2 ppm from the peak of tissular water was observed. It was higher in spectra recorded from the proliferative peripheral zone of the tumor than from the central necrotic zone and was not observed in healthy control pancreatic tissue. Histological examination of the xenografts by transmission electron microscopy indicated intense phospholipid metabolism with marked development of the plasma membrane and the presence of numerous secretory granules, lysosomes, and multivesicular bodies in the cytoplasm. The new 1H NMR low-resolution peak was thought to reflect an increase in membrane viscosity stemming from alterations in the structure and metabolism of membrane phospholipids. Whatever its origin, the 1.8-ppm peak is a particular feature of cancerous pancreatic cells, which should be readily detectable by NMR in vivo.

Lipid peroxidation and phospholipase A 2 activity in liposomes composed of unsaturated phospholipids: a structural basis for enzyme activation

The effect of lipid peroxidation on membrane structure and phospholipase A 2 activity was studied using liposomes composed of bovine liver phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The phospholipids were mixed at set ratios and sonicated to yield small unilamellar vesicles. The liposome preparations were subjected to lipid peroxidation as induced by cumene hydroperoxide and hematin. Under these conditions, a sharp increase in lipid peroxidation was noted over a 30 min incubation period and was accompanied by loss of polyunsaturated fatty acids (PUFA). Liposomes enriched in PE were most extensively peroxidized with a preferred oxidation of this phospholipid. The extent of PC oxidation was also greater in liposomes containing the largest proportions of PE. Analysis of liposome anisotropy, via steady-state fluorescence polarization of diphenylhexatriene indicated that progressive increases in either PE content or the level of lipid peroxidation increased the apparent microviscosity of the vesicles. Moreover, lipid peroxidation increased anisotropy more effectively than variations in the ratios of PE vs. PC. Thus, peroxidation of 5–10% of the phospholipids produced the same anisotropy increase as a 20% increase in the ratio of PE vs. PC. Analysis of vesicle turbidity suggested that fusion was also more readily achieved through lipid peroxidation. When liposomes were incubated with 0.4 U/ml of snake venom phospholipase A 2, a direct correlation was found between the degree of lipid peroxidation and the extent of phospholipid hydrolysis. The more unsaturated phospholipid, PE, was most extensively hydrolyzed following peroxidation. Increasing the proportion of PE also resulted in more extensive phospholipid hydrolysis. These findings indicate that lipid peroxidation produces a general increase in membrane viscosity which is associated with vesicle instability and enhanced phospholipase A 2 attack. A structural basis for membrane phospholipase A 2 activation as a consequence of lipid peroxidation is discussed in light of these findings.