Oxidative response and membrane modification of diabetic platelets challenged with PAF

Alterations in the functional activities of platelets (PLT) in type I diabetes have been widely observed. These changes play a key role in the development of cardiovascular complications in diabetes. Various functional activities of PLT are the result of the interaction of numerous stimuli with PLT plasma membrane. This study was designed to evaluate the oxidative response and membrane modifications of diabetic PLT stimulated by platelet activating factor (PAF). The oxidative response was assessed by employing luminol- and lucigenin-amplified chemiluminescence. Luminol-amplified chemiluminescence is sensitive to the release of hydrogen peroxide whereas lucigenin-amplified chemiluminescence is sensitive to the production of superoxide anion. Membrane fluidity and polarity were studied using fluorescence spectroscopy. Membrane fluidity was investigated by measuring steady-state fluorescence anisotropy of 1-[4-trimethylammonium-phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) and membrane polarity was studied by measuring the steady-state fluorescence emission and excitation spectra of 2-dimethylamino[6-lauroyl]-naphthalene (Laurdan). The diabetic group consisted of 20 type I diabetic children with good metabolic control. Our results show a significant decrease in the luminol- and lucigenin-amplified chemiluminescence of PAF stimulated PLT in the diabetic group with respect to controls. These data indicate a decrement in the release of reactive oxygen species by diabetic PLT. We observed a significant increase in steady-state fluorescence anisotropy of diabetic PLT membrane that reflects a decrease in membrane fluidity. Laurdan showed a blue shift of the fluorescence emission and excitation spectra in diabetic PLT with respect to the control group, indicating a decrease in membrane polarity. The addition of PAF to PLT induced a red shift of Laurdan spectra in both groups, indicating an increase in membrane polarity. Our study [table: see text] demonstrates an altered oxidative response to PAF stimulation of diabetic PLT, probably due to altered generation or handling of reactive oxygen species, and alterations in the physico-chemical properties of the plasma membrane which could influence various functional activities of PLT.     

Surface properties of cholesterol-containing membranes detected by Prodan fluorescence

The fluorescent membrane probe 6-propionyl-2-dimethylaminonaphthalene (Prodan) displays a high sensitivity to the polarity and packing properties of lipid membrane. Contrary to 6-lauroyl-2-dimethylaminonaphthalene (Laurdan), Prodan can also monitor the properties of the membrane surface, i.e., the polar-head pretransition. In bilayers composed of coexisting gel and liquid-crystalline phases, Prodan shows a preferential partitioning in the latter, so that the detected membrane properties mainly belong to fluid domains. In the presence of cholesterol, the packing properties of the gel phase phospholipids are modified in such a way that Prodan can penetrate and label the membrane. Although Prodan labeling of the gel phase is a function of cholesterol concentration, 3 mol percent cholesterol is sufficient for a 60% Prodan labeling with respect to the maximum labeling reached at 15 mol percent cholesterol. We present steady-state and dynamical fluorescence measurements of Prodan in bilayers in the presence of cholesterol. Our results fit the liquid-ordered/liquid-disordered phase model for cholesterol-containing membranes and show that the presence of cholesterol, in addition to modification to the phase state of the hydrophobic portion of the bilayer, strongly affects the packing and the polarity of the membrane hydrophobic-hydrophilic interface.     

Solvatochromic Modeling of Laurdan for Multiple Polarity Analysis of Dihydrosphingomyelin Bilayer

The hydration properties of the interface between lipid bilayers and bulk water are important for determining membrane characteristics. Here, the emission properties of a solvent-sensitive fluorescence probe, 6-lauroyl-2-dimethylamino naphthalene (Laurdan), were evaluated in lipid bilayer systems composed of the sphingolipids D-erythro-N-palmitoyl-sphingosylphosphorylcholine (PSM) and D-erythro-N-palmitoyl-dihydrosphingomyelin (DHPSM). The glycerophospholipids 1-palmitoyl-2-palmitoyl-sn-glycero-3-phosphocholine and 1-oleoyl-2-oleoyl-sn-glycero-3-phosphocholine were used as controls. The fluorescence properties of Laurdan in sphingolipid bilayers indicated multiple excited states according to the results obtained from the emission spectra, fluorescence anisotropy, and the center-of-mass spectra during the decay time. Deconvolution of the Laurdan emission spectra into four components based on the solvent model enabled us to identify the varieties of hydration and the configurational states derived from intermolecular hydrogen bonding in sphingolipids. Sphingolipids showed specific, interfacial hydration properties stemming from their intra- and intermolecular hydrogen bonds. Particularly, the Laurdan in DHPSM revealed more hydrated properties compared to PSM, even though DHPSM has a higher T_m than PSM. Because DHPSM forms hydrogen bonds with water molecules (in 2NH configurational functional groups), the interfacial region of the DHPSM bilayer was expected to be in a highly polar environment. The careful analysis of Laurdan emission spectra through the four-component deconvolution in this study provides important insights for understanding the multiple polarity in the lipid membrane.     

Prodan as a membrane surface fluorescence probe: partitioning between water and phospholipid phases.

Fluorescence spectral features of 6-propionyl-2-dimethylaminonaphthalene (Prodan) in phospholipid vesicles of different phase states are investigated. Like the spectra of 6-lauroyl-2-dimethylaminonaphthalene (Laurdan), the steady-state excitation and emission spectra of Prodan are sensitive to the polarity of the environment, showing a relevant shift due to the dipolar relaxation phenomenon. Because of the different lengths of their acyl residues, the partitioning of the two probes between water and the membrane bilayer differs profoundly. To account for the contribution of Prodan fluorescence arising from water, we introduce a three-wavelength generalized polarization method that makes it possible to separate the spectral properties of Prodan in the lipid phase and in water, and to determine the probe partitioning between phospholipid and water and between the gel and the liquid-crystalline phases of phospholipids. In contrast to Laurdan, Prodan preferentially partitions in the liquid-crystalline phase with respect to the gel and is sensitive to the polar head pretransition, and its partition coefficient between the membrane and water depends on the phase state, i.e., on the packing of the bilayer. Prodan is sensitive to polarity variations occurring closer to the bilayer surface than those detected by Laurdan.     

Laurdan fluorescence senses mechanical strain in the lipid bilayer membrane

The precise molecular mechanisms by which cells transduce a mechanical stimulus into an intracellular biochemical response have not yet been established. Here, we show for the first time that the fluorescence emission of an environment-sensitive membrane probe Laurdan is modulated by mechanical strain of the lipid bilayer membrane. We have measured fluorescence emission of Laurdan in phospholipid vesicles of 30, 50, and 100 nm diameter to show that osmotically induced membrane tension leads to an increase in polarity (hydration depth) of the phospholipid bilayer interior. Our data indicate that the general polarization of Laurdan emission is linearly dependent on membrane tension. We also show that higher membrane curvature leads to higher hydration levels. We anticipate that the proposed method will facilitate future studies of mechanically induced changes in physical properties of lipid bilayer environment both in vitro and in vivo.     

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.     

Phase fluctuation in phospholipid membranes revealed by Laurdan fluorescence.

The organization of lipids surrounding membrane proteins can influence their properties. We have used 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan) to study phase coexistence and phase interconversion in membrane model systems. The fluorescence properties of Laurdan provide a unique possibility to study lipid domains because of the different excitation and emission spectra of this probe in the gel and in the liquid-crystalline phase. The difference in excitation spectra allows photoselection of Laurdan molecules in one of the two phases. Using the difference in emission spectra it is then possible to observe interconversion between the two phases. We have performed experiments in dipalmitoyl-phosphatidylcholine (DPPC) vesicles at different temperatures, in particular in the region of the phase transition, where phase coexistence and interconversion between phases is likely to be maximal. We have also studied vesicles of different lipids and mixtures dilauroyl-phosphatidylcholine (DLPC), DPPC, and 50% DLPC in DPPC. Both steady-state fluorescence intensity and polarization data have been collected. To quantitate phase coexistence and interconversion we have introduced the concept of "generalized polarization." We have also performed time-resolved experiments to directly prove the interconversion process. We have found that in DLPC-DPPC mixtures, at 20 degrees C, phase interconversion occurs in approximately 30-40 ns.     

Monitoring the organization and dynamics of bovine hippocampal membranes utilizing Laurdan generalized polarization

Organization and dynamics of cellular membranes in the nervous system are crucial for the function of neuronal membrane receptors. The lipid composition of neuronal cells is unique and has been correlated with the increased complexity in the organization of the nervous system during evolution. Previous work from our laboratory has established bovine hippocampal membranes as a convenient natural source for studying neuronal receptors such as the G-protein coupled serotonin_1A receptor. In this paper, we have explored the organization and dynamics of bovine hippocampal membranes using the amphiphilic environment-sensitive fluorescent probe Laurdan. Our results show that the emission spectra of Laurdan display an additional red shifted peak as a function of increasing temperature in native as well as cholesterol-depleted membranes and liposomes made from lipid extracts of the native membrane. Interestingly, wavelength dependence of Laurdan generalized polarization (GP) in native membranes indicates the presence of an ordered gel-like phase at low temperatures, whereas characteristics of the liquid-ordered phase are observed at high temperatures. Similar experiments performed using cholesterol-depleted membranes show fluidization of the membrane with increasing cholesterol depletion. In addition, results from fluorescence polarization of DPH indicate that the hippocampal membrane is fairly ordered even at physiological temperature. The temperature dependence of Laurdan excitation GP provides a measure of the apparent thermal transition temperature and extent of cooperativity in these membranes. Analysis of time-resolved fluorescence measurements of Laurdan shows reduction in mean fluorescence lifetime with increasing temperature due to change in environmental polarity. These results constitute novel information on the dynamics of hippocampal membranes and its modulation by cholesterol depletion monitored using Laurdan fluorescence.     

Environmental polarity estimation in living cells by use of quinoxaline-based full-colored solvatochromic fluorophore PQX and its derivatives

Fluorosolvatochromic probes have recently attracted interest for live cell imaging. We recently tested our quinoxaline-based fluorosolvatochromic probes, PQX and MPQX, for in vivo imaging applications. PQX and MPQX are characterized by donor (pyrrole site)-acceptor (quinoxaline site) structure, and the peak emission wavelength of these compounds varies over the visible wavelength range depending on the polarity of the solvent used, for a variety of solvents from hexane to water. A linear relationship was obtained between peak emission wavenumber and E(T)(N) (normalized solvent polarity). When tested on cultured HEp-2 cells, the results showed that the PQX and MPQX were not harmful at the applied concentrations (10(-5) M), and site-dependent fluorescence spectra in living cells were observed with PQX treatment, which indicates that PQX penetrates into the plasma membrane, followed by delocalization throughout the cells. MPQX was also able to penetrate the cell membrane, distribute throughout the cells and emit fluorescence, but did not show site-dependent intracellular fluorescence spectra. These results suggest that PQX is a remarkable tool for investigating the local polarity environment in cells after appropriate conjugation to biomolecules.     

A study of quercetin effects on phospholipid membranes containing cholesterol using Laurdan fluorescence

Quercetin (QCT) is an important bioactive natural compound found in numerous edible plants. Since the lipid bilayer represents an essential compound of the cell membrane, QCT's direct interaction with this structure is of great interest. Therefore, we proposed to study the effects of QCT on DMPC liposomes containing cholesterol (Chol), and for this purpose Laurdan fluorescence was used. As a fluorescent probe, Laurdan is able to detect changes in membrane phase properties. When incorporated in lipid bilayers, Laurdan emits from two different excited states, a non-relaxed one when the bilayer packing is tight and a relaxed state when the bilayer packing is loose. The main tool for quantifying QCT's effects on phospholipid membranes containing Chol has been the analysis, the decomposition of Laurdan emission spectra in sums of two Gaussian functions on energy. This kind of approach has allowed good analysis of the balance between the two emitting states of Laurdan. Our results show that both Laurdan emission states are present to different extents in a wide temperature range for DMPC liposomes with Chol. QCT is decreasing the phase transition temperature in pure DMPC liposomes as proved by generalized polarization (GP) values. QCT also quenches Laurdan fluorescence, depending on the temperature and the presence of Chol in the membrane. Stern-Volmer constants were calculated for different lipid membrane compositions, and the conclusion was that the relaxed state favors the nonradiative transitions of the fluorophore.     

Laurdan Fluorescence Lifetime Discriminates Cholesterol Content from?Changes in Fluidity in Living Cell Membranes

Detection of the fluorescent properties of Laurdan has been proven to be an efficient tool to investigate membrane packing and ordered lipid phases in model membranes and living cells. Traditionally the spectral shift of Laurdan’s emission from blue in the ordered lipid phase of the membrane (more rigid) toward green in the disordered lipid phase (more fluid) is quantified by the generalized polarization function. Here, we investigate the fluorescence lifetime of Laurdan at two different emission wavelengths and find that when the dipolar relaxation of Laurdan’s emission is spectrally isolated, analysis of the fluorescence decay can distinguish changes in membrane fluidity from changes in cholesterol content. Using the phasor representation to analyze changes in Laurdan’s fluorescence lifetime we obtain two different phasor trajectories for changes in polarity versus changes in cholesterol content. This gives us the ability to resolve in vivo membranes with different properties such as water content and cholesterol content and thus perform a more comprehensive analysis of cell membrane heterogeneity. We demonstrate this analysis in NIH3T3 cells using Laurdan as a biosensor to monitor changes in the membrane water content during cell migration.     

Pyrene as a membrane depth gauge: wavelength selective fluorescence approach to monitor pyrene localizations in the membrane

We take the advantage of pyrene's unique spectral properties as a reliable polarity indicator to monitor pyrene localizations in the membrane depth by using wavelength selective fluorescence approach. We show that fine structure of pyrene fluorescence emission spectra and excimerization rate in model and native phospholipid membranes depend on the excitation wavelength. This phenomenon is not observed in neat solvents. In membranes, the dependence on the excitation wavelength reflects selective excitation of pyrene molecules located close to the membrane-water polar interface, or deep in the hydrophobic core of the membrane, verified with the aid of pyrene derivatives of fatty acids of various lengths.     

Probing the interactions of alcohols with biological membranes with the fluorescent probe Prodan.

Prodan [6-propionyl-2-(dimethylamine)naphthalene] is a hydrophobic fluorescent probe which is extremely sensitive to both the polarity and the hydrogen-bond donating capacity of the solvent. In binary mixtures of solvents, the hydrogen-bond donating effect on Prodan fluorescence saturates at relatively low concentrations of protic solvent while the polarity effect is proportional to the mixture's dielectric constant. The fluorescence emission maximum is approximately a linear function of the dielectric constant in both protic and aprotic solvents, and this allows estimation of the dielectric constant in both environments. In phospholipid bilayers and biological membranes, Prodan exhibits two distinct emission peaks: blue (430-445 nm) and green (470-505 nm). Temperature determines the relative intensity of the two peaks, but their wavelengths depend on the type of membrane and appear to reflect a specific membrane environment. In phospholipid vesicles, alcohols reduce the fluorescence intensity of the blue peak and produce a red-shift in the emission maximum of the green peak. Taking the partition coefficients of the alcohols into account, short-chain alcohols are much more effective than longer-chain alcohols in red-shifting the emission maximum of the green peak. Alcohols have similar effects on Prodan fluorescence in liver microsomal and mitochondrial membranes, synaptosomal membranes, and red blood cell plasma membranes. However, in liver organelle membranes the red-shift of the green peak is the dominant effect while in plasma membranes the quenching of the fluorescence of the blue peak is dominant. These effects are observed at low (pharmacological) ethanol concentrations and provide a unique tool for probing the interactions of ethanol with biological membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of the Fluorescent Probe Laurdan to Investigate Structural Organization of the Vesicular Stomatitis Virus (VSV) Membrane

We have used 6-dodecanoil-2-dimethylaminonaphtalene (Laurdan) to study the membrane fluidity of Vesicular Stomatitis Virus (VSV) during virus activation at acidic pH 5.8). The fluorescence properties of Laurdan provide a unique possibility to study lipid organization because of the different excitation and emission spectra of this probe in the gel and liquid crystalline phase. Acidification to pH 5.8 (the pH which triggers VSV fusion with target membranes) generates a decrease in VSV membrane fluidity that could be reversed perfectly after neutralization. We conclude that lipid reorganization of the VSV membrane in the endocytic vesicles is needed for virus activation.     

Fluorescence Studies of Red Blood Cell Membranes from Individuals with Huntington''s Disease

Fluorescence spectroscopic methods were used to investigate and compare the properties of erythrocyte membranes from individuals with Huntington's Disease (HD) and from normal individuals. Erythrocyte ghosts were labeled with four different fluorescent probes: 1,6-diphenylhexatriene (DPH); 6-lauroyl-2-(dimethylamino)-naphthalene (Laurdan); 2-(4-maleimide anilino)-naphthalene-6-sulfonic acid (MIANS) and 5-(iodoacetamidoethyl)aminoaphthalene-1-sulfonic acid (IAEDANS). DPH is sensitive to the microviscosity of the membranes. Laurdan is highly sensitive to the polarity and relaxation time of its environment. MIANS and IAEDANS both react covalently with sulfhydryl groups in membrane proteins. Erythrocyte membranes were labeled separately with each of these four probes, and we measured the centers of gravity of the fluorescence emission, the fluorescence anisotropies, and the fluorescence lifetimes. In 10 separate experiments, including a total of 24 patients and 14 control individuals, we found no significant differences between the two groups for any of the probes or spectral parameters. These results do not support the existence of a "generalized membrane defect" in individuals with HD.     

Bimodal distribution and fluorescence response of environment-sensitive probes in lipid bilayers

A remarkable heterogeneity is often observed in the spectroscopic properties of environment-sensitive fluorescence probes in phospholipid bilayers. To explain its origin, we provided a detailed investigation of the fluorescence excitation and emission spectra of 4'-dimethylamino-3-hydroxyflavone (probe F) in bilayer vesicles with the variations of fatty acid composition, polar heads, temperature, and cholesterol content. Probe F, due to excited-state intramolecular proton transfer, exhibits two bands in emission that are differently sensitive to intermolecular interactions-thereby allowing us to distinguish universal (dipole-dipole) and specific (H-bonding) interactions within the bilayer. Spectroscopic, quenching, and anisotropy data suggest the presence of two forms of probe F at different locations in the bilayer: an H-bond free form located below sn(1)-carbonyls and an H-bonded form located at the polar membrane interface. We provide a quantitative analysis of the distribution of the probe between these two locations as well as the polarity of these locations, and show that both the distribution and the polarity contribute to the probe response. Moreover, analysis of literature data on other environment-sensitive probes (Prodan, Laurdan, Nile Red, NBD lipids, etc.) in lipid bilayers allows us to suggest that the bimodal distribution in the lipid bilayer is probably a general feature of low-polar molecules with polar groups capable of H-bonding interactions.     

Therapeutic lithium alters polar head-group region of lipid bilayer and prevents lipid peroxidation in forebrain cortex of sleep-deprived rats

Lithium is regarded as a unique therapeutic agent for the management of bipolar disorder (BD). In efforts to explain the favourable effects of lithium in BD, a wide range of mechanisms was suggested. Among those, the effect of clinically relevant concentrations of lithium on the plasma membrane was extensively studied. However, the biophysical properties of brain membranes isolated from experimental animals exposed to acute, short-term and chronic lithium have not been performed to-date. In this study, we compared the biophysical parameters and level of lipid peroxidation in membranes isolated from forebrain cortex (FBC) of therapeutic lithium-treated and/or sleep-deprived rats. Lithium interaction with FBC membranes was characterized by appropriate fluorescent probes. DPH (1,6-diphenyl-1,3,5-hexatriene) and TMA-DPH (1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulphonate) were used for characterization of the hydrophobic lipid core and Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) for the membrane-water interface. Lipid peroxidation was determined by immunoblot analysis of 4-HNE-(4-hydroxynonenal)-protein adducts. The organization of polar head-group region of FBC membranes, measured by Laurdan generalized polarization, was substantially altered by sleep deprivation and augmented by lithium treatment. Hydrophobic membrane interior characterized by steady-state anisotropy of DPH and TMA-DPH fluorescence was unchanged. Chronic lithium had a protective effect against peroxidative damage of membrane lipids in FBC. In summary, lithium administration at a therapeutic level and/or sleep deprivation as an animal model of mania resulted in changes in rat FBC membrane properties.     

Examining the effects of cholesterol on model membranes at high temperatures: Laurdan and Patman see it differently

At high temperature, the presence of cholesterol in phospholipid membranes alters the influence of membrane dipoles, including water molecules, on naphthalene-based fluorescent probes such as Laurdan and Patman (solvatochromism). Although both of these probes report identical changes to their emission spectra as a function of temperature in pure phosphatidylcholine bilayers, they differ in their response to cholesterol. Computer simulations of the spectra based on a simple model of solvatochromism indicated that the presence of cholesterol reduces the probability of bilayer dipole relaxation and also blunts the tendency of heat to enhance that probability. While the overall effect of cholesterol on membrane dipoles was detected identically by the two probes, Laurdan was influenced much more by the additional effect on temperature sensitivity than was Patman. A comparison of the fluorescence data with simulations using a coarse-grained bilayer model (de Meyer et al., 2010) suggested that these probes may be differentially sensitive to two closely related properties distinguishable in the presence of cholesterol. Specifically, Patman fluorescence correlated best with the average phospholipid acyl chain order. On the other hand, Laurdan fluorescence tracked more closely with the area per lipid molecule which, although affected generally by chain order, is also impacted by additional membrane-condensing effects of cholesterol. We postulate that this difference between Laurdan and Patman may be attributed to the bulkier charged headgroup of Patman which may cause the probe to preferentially locate in juxtaposition to the diminutive headgroup of cholesterol as the membrane condenses.     

Constraints to growth of annual nettle (Urtica urens) in an elevated CO2 atmosphere: Decreased leaf area ratio and tissue N cannot be explained by ontogenetic drift or mineral N supply

Plant sterols differ from cholesterol in having an alkyl group at Δ-24, and, in the case of stigmasterol, also a Δ-22 double bond. The effects of 10 mol% of three plant sterols (campesterol, β -sitosterol, stigmasterol) and cholesterol on the molecular dynamics and phase behavior in multilamellar liposomes made from different phosphatidylcholine (PC) molecular species have been compared, utilizing the fluorescent probe Laurdan (2-dimethyl-amino-6-laurylnaphthalene). Laurdan reports the molecular mobility in the hydrophilic/hydrophobic interface of the membrane by determining the rate of dipolar relaxation of water molecules close to the glycerol backbone of PC. Our results showed that the Δ-24 alkyl group of plant sterols did not affect their ability to reduce molecular mobility in this region of the PC membranes. However, the plant sterols had a decreased capacity compared to cholesterol to inhibit formation of co-existing domains of gel and liquid-crystalline phases in membranes composed of equimolar dilauroyl-PC and dipalmitoyl-PC. The Δ-22 double bond present in stigmasterol decreased the ability of this sterol, compared to the other phytosterols, to reduce the molecular mobility at the hydrophobic/hydrophilic interface in membranes made of a saturated PC molecular species. However, in membranes made from 16:0/18:2-PC, a lipid species common in plant plasma membranes, stigmasterol was as efficient as other sterols in affecting the polarity and molecular mobility at the hydrophilic/hydrophobic interface of the membrane at 25°C, but was, in contrast to the other sterols, without effect at 0°C. Our results thus confirm as well as contradict the results of previous studies of the interactions between saturated PC and sterols, where other membrane regions were probed. The physiological relevance of the findings is discussed.