Changes in the physical properties of the lipid phase of neutrophils during their activation

A change of the state of lipid phase of the neutrophil membrane was studied under their activation. Stimulation of neutrophils with hexachlorocyclohexane results in an increase of microviscosity of the membrane lipid layer, while stimulation with concanavalin A brings about an increase of microviscosity only in the near protein region, but in the total lipid microviscosity is reduced.     

Increase in lipid microviscosity of unilamellar vesicles upon the creation of transmembrane potential

Summary Diffusion potential of potassium ions was formed in unilamellar vesicles of phosphatidyl choline. The vesicles, which included potassium sulfate buffered with potassium phosphate, were diluted into an analogous salt solution made of sodium sulfate and sodium phosphate. The diffusion potential was created by the addition of the potassium-ionophore, valinomycin. The change in lipid microviscosity, ensuing the formation of membrane potential, was measured by the conventional method of fluorescence depolarization with 1,6-diphenyl-1,3,5-hexatriene as a probe. Lipid microviscosity was found to increase with membrane potential in a nonlinear manner, irrespective of the potential direction. Two tentative interpretations are proposed for this observation. The first assumes that the membrane potential imposes an energy barrier on the lipid flow which can be treated in terms of Boltzmann-distribution. The other interpretation assumes a decrease in lipid-free volume due to the pressure induced by the electrical potential. Since increase in lipid viscosity can reduce lateral and rotational motions, as well as increase exposure of functional membrane proteins, physiological effects induced by transmembrane potential could be associated with such dynamic changes.     

Effects of native and oxidized apolipoprotein A-I on lipid bilayer microviscosity of erythrocyte plasma membrane

Using a pyrene as a fluorescent probe, we investigated the influence of native and oxidized apolipoprotein A-I (apo A-I) and their complexes with tetrahydrocortisol (THC) on the microviscosity of the erythrocyte plasma membrane. The addition of THC to isolated membranes led to a 17% increase in the membrane microviscosity. In contrast, native apo A-I reduced the microviscosity (i.e., increased the fluidity) of the membranes by 15%. A more pronounced increase (by 25%) in the membrane fluidity was found in the presence of the complex of apo A-I with THC. Unlike native apo A-I, oxidized apo A-I and its complex with THC did not change the membrane viscosity. In view of the fact that apo A-I plays an important role in the binding of membrane cholesterol we suggest that the observed increase in the membrane fluidity under the influence of the native apo A-I is associated with the cholesterol efflux from plasma membrane. Oxidative modification of apo A-I likely disturbs the mechanisms of the cholesterol efflux and prevents the decrease in the membrane microviscosity.     

Stage-specific changes in membrane microviscosity in <Emphasis Type="Italic">Misgurnus fossilis</Emphasis> embryos

Natural and probe fluorescence as well as membrane microviscosity was studied in eggs and embryos of Misgurnus fossilis by fluorescence microscopy. The lateral mobility of the probe (pyrene) increased in loach embryos from early to late blastula, which indicates a decrease in plasma membrane microviscosity. At the later stage of mid-gastrula, the microviscosity remained largely invariant. Considering that the embryo exposure to different temperatures changes the quantum yield of fluorescence and the degree of pyrene excimerization, one can gain information about both the temperature-induced structural changes and changes in membrane microviscosity in the embryos. Natural and probe fluorescence of embryonic membranes is proposed as at tool to study morphogenetic mechanisms.     

Changes in the microviscosity of lipid bilayer membranes of various malignant cells and tumor transplantability

It is shown that cholesterol incorporation into the membranes of Zajdel hepatoma cells, lymphoblast leukemia cells L1210 and into those of ovary tumour causes an increase in the membrane phospholipid bilayer microviscosity measured by pyrene as fluorescent probe. The increase in the membrane lipid microviscosity resulted in a decrease in the activity of Na,K-ATPase and 5-nucleotidase of the tumour cells. After the injection of tumour cells with an increase of cholesterol/phospholipid ratio we observed an increase of the life-span of experimental animals as compared to the control groups.     

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The effect of hypoglycemic drugs chlorpropamide and phenformin has been tested on isolated liver plasma membranes as to their microviscosity parameters. Results reported suggest that both drugs are able to increase in vitro plasma membrane microviscosity in a dose-dependent way.     

Interaction of tumor and normal blood cells with ethylene oxide and propylene oxide block copolymers

Ethylene oxide and propylene oxide block copolymers (pluronics) are widely known as agents that promote drug penetration across biological barriers. We have studied the interaction of normal and malignant blood cells with pluronics L61 and P85 that have different hydrophobicity. SP2/0 myeloma cells accumulated pluronics while normal cells adsorb most of the polymer on the surface. Interaction of pluronics with cells resulted in drastic changes of membrane microviscosity. Tumor cell membrane microviscosity decreased after pluronics adsorption, in contrast to normal cells, whose membrane microviscosity was enhanced. We suppose that sensitivity of tumor cell membrane microviscosity to the pluronics action correlates with its permeability for molecular substances.     

Hypoglycemic drugs increase liver plasma membrane microviscosity in vitro.

The effect of hypoglycemic drugs chlorpropamide and phenformin has been tested on isolated liver plasma membranes as to their microviscosity parameters. Results reported suggest that both drugs are able to increase in vitro plasma membrane microviscosity in a dose-dependent way.     

Quantification of lipid bilayer effective microviscosity and fluidity effect induced by propofol

Electron spin resonance (ESR) spectroscopy with nitroxide spin probes was used as a method to probe the liposome microenvironments. The effective microviscosities have been determined from the calibration of the ESR spectra of the probes in solvent mixtures of known viscosities. In the first time, by measuring ESR order parameter (S) and correlation time (tau(c)) of stearic spin probes, we have been able to quantify the value of effective microviscosity at different depths inside the liposome membrane. At room temperature, local microviscosities measured in dimyristoyl-l-alpha phosphatidylcholine (DMPC) liposome membrane at the different depths of 7.8, 16.95, and 27.7 A were 222.53, 64.09, and 62.56 cP, respectively. In the gel state (10 degrees C), those microviscosity values increased to 472.56, 370.61, and 243.37 cP. In a second time, we have applied this technique to determine the modifications in membrane microviscosity induced by 2,6-diisopropyl phenol (propofol; PPF), an anaesthetic agent extensively used in clinical practice. Propofol is characterized by a unique phenolic structure, absent in the other conventional anaesthetics. Indeed, given its lipophilic property, propofol is presumed to penetrate into and interact with membrane lipids and hence to induce changes in membrane fluidity. Incorporation of propofol into dimyristoyl-l-alpha phosphatidylcholine liposomes above the phase-transition temperature (23.9 degrees C) did not change microviscosity. At 10 degrees C, an increase of propofol concentration from 0 to 1.0 x 10(-2) M for a constant lipid concentration mainly induced a decrease in microviscosity. This fluidity effect of propofol has been qualitatively confirmed using merocyanine 540 (MC540) as lipid packing probe. Above 10(-2) M propofol, no further decrease in microviscosity was observed, and the microviscosity at the studied depths (7.8, 16.95, and 27.7 A) amounted 260.21, 123.87, and 102.27 cP, respectively. The concentration 10(-2) M was identified as the saturation limit of propofol in dimyristoyl-l-alpha phosphatidylcholine liposomes.     

Acceleration of membrane senescence in cut carnation flowers by treatment with ethylene

The lipid microviscosity of microsomal membranes from senescing cut carnation (Dianthus caryophyllus L. cv. White Sim) flowers rises with advancing senescence. The increase in membrane microviscosity is initiated within 3 to 4 days of cutting the flowers and coincides temporally with petal-inrolling denoting the climacteric-like rise in ethylene production. Treatment of young cut flowers with aminoethoxyvinylglycine prevented the appearance of petal-inrolling and delayed the rise in membrane microviscosity until day 9 after cutting. When freshly cut flowers or aminoethoxyvinylglycine-treated flowers were exposed to exogenous ethylene (1 microliter per liter), the microviscosity of microsomal membranes rose sharply within 24 hours, and inrolling of petals was clearly evident. Thus, treatment with ethylene accelerates membrane rigidification. Silver thiosulphate, a potent anti-ethylene agent, delayed the rise in microsomal membrane microviscosity even when the flowers were exposed to exogenous ethylene. Membrane rigidification in both naturally senescing and ethylene-treated flowers was accompanied by an increased sterol:phospholipid ratio reflecting the selective loss of membrane phospholipid that accompanies senescence. The results collectively indicate that the climacteric-like surge in ethylene production during senescence of carnation flowers facilitates physical changes in membrane lipids that presumably lead to loss of membrane function.     

The in vitro influence of the external electrostatic field on the physical parameters of erythrocyte membranes

The in vitro influence of external electrostatic fields with 200 kV/m tension on the biophysical parameters of the erythrocyte membranes and their ghosts of white outbred rats was studied. The investigation on the parameters of erythrocyte membranes and their ghosts, particularly, their microviscosity, the amount and degree of membrane proteins submersion in lipids, polarity in depth of the membrane bilayer and its viscosity was carried out by the spectrofluorimeteric method using pyrene as a hydrophobic fluorescent probe. The analyses of literature data, findings of the current study and their comparison with the results of our previous works allow of concluding that the in vitro influence of external electrostatic fields with 200 kV/m tension on the erythrocyte membranes and their ghosts occurs at different sites of membrane. It is shown that the preliminary exposure of erythrocytes in external electrostatic fields leads to the changes of the parameters both of a membrane surface layer and the intra-membrane domains. So, the decrease in the strength of peripheral proteins binding to the erythrocyte membranes and the increase in the microviscosity of the lipid bilayer are observed. The influence of the field on the ghosts of intact erythrocytes results in alterations of the studied parameters only of the membrane surface.     

Effect of Escherichia coli lipopolysaccharide on the microviscosity of liver plasma membranes and hepatocyte suspensions and monolayers

The fluorescence probe 1,6-diphenylhexa-1,3,5-triene (DPH) was used for monitoring structural perturbations induced by lipopolysaccharide (LPS) of Escherichia coli (0111:B4) in plasma membranes of rat liver. Changes in microviscosity were observed in plasma membrane preparations from control rats after treatment with LPS and in plasma membrane preparations from liver perfused with LPS. In both systems fluorescence polarization was measured from which microviscosity was calculated. This parameter increases with LPS treatment. From temperature dependence studies was inferred that LPS interaction with plasma membrane preparations induces an increase of both the polarization term (r0/r-1)-1 and flow activation energy (delta E). Addition of LPS to hepatocyte suspensions also induces an increase on microviscosity and a delay in the fall of microviscosity induced by a temperature rise in hepatocyte monolayers grown on microcover slides. These data suggest that LPS interaction can be attributed to its binding to membrane hydrophobic regions in a non-specific manner.     

Phospholipase A activity determines the rate of respiration of the mitochondria in hibernating animals

The mechanisms for regulating the rate of respiration and oxidative phosphorylation in liver mitochondria from hibernating ground squirrels were studied. The microviscosity of the mitochondrial membrane in hibernating squirrels was found to be higher than that in active animals. Probably, a high microviscosity of the membrane causes a decreases in the rate of the transport of oxidation substrates into the mitochondrial matrix, which in turn may be one of the main reasons for the inhibition of mitochondrial respiration in hibernating squirrels. The activation of phospholipase A2 in a hypotonic medium results in the acceleration of the respiration and phosphorylation in the mitochondria from hibernating squirrels and is accompanied by the increase of the transport of substrates across the mitochondrial membrane. The inhibition of phospholipase A2 decreases Ca2+--induced acceleration of the transport of substrates and prevents the activation of the respiration and phosphorylation in a hypotonic medium.     

Changes in the membrane lipids and lymphocyte function in staphylococcal infection and the development of delayed hypersensitivity

Experimental staphylococcal infection was reproduced in rats by the intraperitoneal injection of S. aureus strain 75. The degree of the development of delayed hypersensitivity (DH) to staphylococci, microviscosity, the levels of free radical oxidation and antioxidation resistance were evaluated in the dynamics of the infectious process by the methods of chemiluminometry and fluorescent probing with pyrene. The functional activity of lymphocytes was determined by the inclusion of 3H-thymidine into DNA as the consequence of stimulation with phytohemagglutinin. The development of DH was found to depend on the microviscosity and antioxidation resistance of membrane lipids. The increase of microviscosity and the simultaneous decrease of the induction time of chemifluorescent rapid flash inhibit the development of DH, leading to the aggravation of the infectious process. The increase of fluidity and the accumulation of antioxidants facilitate the development of DH and lead to a milder course of the infectious process.     

Na,K-ATPase: radiation inactivation studies

Na,K-ATPase from duck salt gland and ox brain in the membrane-bound or solubilized form was studied by the radiation inactivation technique using ATP, CTP, GTP or p-NPP as substrates. The values of radiation inactivation size (RIS) were compared with the target size (TS) for the alpha-subunit of the enzyme obtained by an independent method as well as with analytical centrifugation data obtained for C12E8-solubilized enzyme. It was concluded that during ATP (CTP) hydrolysis the enzyme operates as an oligomeric structure; the complex formation requires the presence of K+ and adenosine triphosphate binding to the sites with a low affinity for the nucleotide. Specially designed experiments revealed that the degree of enzyme oligomerization increases with an increase in the microviscosity of the membrane lipid environment.     

Contact-mediated changes in the fluidity of membrane lipids in normal and malignant transformed mammalian fibroblasts

The degree of microviscosity, gh, (fluidity/rigidity behavior) of membrane lipids of normal and transformed mammalian fibroblasts obtained from mice, hamsters and rats was quantitatively monitored by fluorescence polarization, P, analysis of the fluorescent probe 1,6-diphenyl 1,3,5-hexatriene (DPH) when embedded in lipid regions of cellular membranes of intact viable cells. Analysis of membrane microviscosity of six different cell populations and of individual cells in each cell population have indicated that the membrane microviscosity of all cell types, both normal and transformed fibroblasts, changes as a function of the cell density in the growing cultures. The membrane microviscosity was found to be low (high lipid fluidity) in sparse conditions but high (high lipid rigidity) in dense conditions. The induced changes in membrane microviscosity are practically reversible for all cell types and a complete reversion can be obtained within a few hours after changing the cell density conditions from sparse to dense and vice versa.Comparative studies with normal and transformed fibroblasts have shown that transformed fibroblasts have a more rigid lipid layer in their cellular membranes than normal or untransformed fibroblasts. The difference in membrane microviscosity between transformed and normal fibroblasts is higher in confluent conditions as compared with subconfluent cultures. These differences in the degree of fluidity of membrane lipids that are controlled by possible differences in the cellto-cell contact in normal and transformed fibroblasts may play a major role in determining the growth behavior of normal and malignant cells that are growing as a solid tissue and may have a direct effect on the control mechanisms that determine the presence or absence of the “density dependent inhibition” of growth.     

Influence of membrane fluidity of 5'-nucleotidase activity in isolated hepatocyte plasma membrane.

The influence of membrane microviscosity on 5'-nucleotidase activity has been investigated on liver plasma membrane preparations from rats during aging and following diet restriction. In addition the microviscosity of membranes from old rats was changed in vitro by the Active Lipids. During aging the membrane microviscosity increased progressively and in parallel the activity of 5'-nucleotidase decreased. Diet restriction was able to slow down the modification of both parameters. The experiment performed with the Active Lipids further supports that membrane microviscosity modulated the enzyme activity.     

Lipid peroxidation causes an increase of lipid order and a decrease of 5'-nucleotidase activity in the liver plasma membrane.

The effect of peroxidation on 5'-nucleotidase activity as well as on membrane microviscosity has been investigated in liver plasma membranes from Wistar rats. The peroxidation was performed with 100 microM H2O2 and 200 microM FeSO4 and/or with 5 mM t-butylhydroperoxide. Treatment of the membranes with these oxidizing agents resulted in an elevation of the transition temperatures of the polarization of the lipid fluorescent probes 1,6 diphenyl-1,3,5 hexatriene (DPH), 3-p-(6-phenyl) 1,3,5 hexatriene phenylpropionic acid (PA-DPH) as well as of the fluorescent thiol reagent N-(1-pyrene) maleimide (1-PM). The peroxidation resulted in a decrease of the activity of 5'nucleotidase. Our data support that the increase of membrane microviscosity of the lipid domain regulates the activity of 5'-nucleotidase.     

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.     

Enveloped viruses as model membrane systems: microviscosity of vesicular stomatitis virus and host cell membranes.

The fluorescence probe 1,6-diphenyl-1,3,5-hexatriene was used to study and compare the dynamic properties of the hydrophobic region of vesicular stomatitis virus grown on L-929 cells, plasma membrane of L-929 cells prepared by two different methods, liposomes prepared from virus lipids and plasma membrane lipids, and intact L-929 cells. The rate of penetration of the probe into the hydrophobic region of the lipid bilayer was found to be much faster in the lipid vesicle bilayer as compared with the intact membrane, but in all cases the fluorescence anisotropy was constant with time. The L-cell plasma membranes, the vesicles prepared from the lipids derived from the plasma membranes, and intact cells are found to have much lower microviscosity values than the virus or virus lipid vesicles throughout a wide range of temperatures. The microviscosity of plasma membrane and plasma membrane lipid vesicles was found to depend on the procedure for plasma membrane preparation as the membranes prepared by different methods had different microviscosities. The intact virus and liposomes prepared from the virus lipids were found to have very similar microviscosity values. Plasma membrane and liposomes prepared from plasma membrane lipids also had similar microviscosity values. Factors affecting microviscosity in natural membranes and artificially mixed lipid membranes are discussed.