Galya Staneva

The role of sphingomyelin in regulating phase coexistence in complex lipid model membranes: Competition between ceramide and cholesterol

The structure, thermotropic phase behavior, dynamic motion and order parameters of bilayer dispersions of egg phosphatidylcholine, egg sphingomyelin, egg ceramide and cholesterol have been determined. The coexistence of gel, liquid-ordered and liquid-disordered structure has been determined by peak fitting analysis of synchrotron X-ray powder patterns. Order parameters and extent of distribution of 16-doxyl-stearic acid spin probe between ordered and disordered environments has been estimated by ESR spectral simulation methods. The presence of ceramide in proportions up to 20 mol% in phosphatidylcholine is characterized by gel-fluid phase coexistence at temperatures up to 46 degrees C depending on the amount of ceramide. Cholesterol tends to destabilize the ceramide-rich domains formed in phosphatidylcholine while sphingomyelin, by formation of stable complexes with ceramide, tends to stabilize these domains. The stability of sphingomyelin-ceramide complexes is evident from the persistence of highly ordered structure probed by ESR spectroscopy and appearance of a sharp wide-angle X-ray reflection at temperatures higher than the gel-fluid transition of ceramide alone in egg phosphatidylcholine bilayers. The competition between ceramide and cholesterol for interaction with sphingomyelin is discussed in terms of control of lipid-mediated signaling pathways by sphingomyelinase and phospholipase A2.

Membrane microdomains: Role of ceramides in the maintenance of their structure and functions

Free-standing giant unilamellar vesicles were used to visualize the complex lateral heterogeneity, induced by ceramide in the membrane bilayer at micron scale using C(12)-NBD-PC probe partitioning under the fluorescence microscope. Ceramide gel domains exist as leaf-like structures in glycerophospholipid/ceramide mixtures. Cholesterol readily increases ceramide miscibility with glycerophospholipids but cholesterol-ceramide interactions are not involved in the organization of the liquid-ordered phase as exemplified by sphingomyelin/cholesterol mixtures. Sphingomyelin stabilizes the gel phase and thus decreases ceramide miscibility in the presence of cholesterol. Gel/liquid-ordered/liquid-disordered phase coexistence was visualized in quaternary phosphatidylcholine/sphingomyelin/ceramide/cholesterol mixtures as occurrence of dark leaf-like and circular domains within a bright liquid phase. Sphingomyelin initiates specific ceramide-sphingomyelin interactions to form a highly ordered gel phase appearing at temperatures higher than pure ceramide gel phase in phosphatidylcholine/ceramide mixtures. Less sphingomyelin is engaged in formation of liquid-ordered phase leading to a shift in its formation to lower temperatures. Sphingomyelinase activity on substrate vesicles destroys micron L(o) domains but induces the formation of a gel-like phase. The activation of phospholipase A(2) by ceramide on heterogeneous membranes was visualized. Changes in the phase state of the membrane bilayer initiates such morphological processes as membrane fragmentation, budding in and budding out was demonstrated.

Modification of membrane heterogeneity by antipsychotic drugs: An X-ray diffraction comparative study

Lipid mixtures are used to mimic biological membranes as they allow characterization of lipid lateral domains defined by their specific lipid molecular organization. Therapeutic agents such as antipsychotic drugs (AP) that may interact with lipids arrangement are likely to modify membrane biological properties. The present study describes the effect of 2 typical and 5 atypical antipsychotic drugs on an aqueous co-dispersion of a lipid mixture made of egg phosphatidylcholine (PC)/brain sphingomyelin (SM)/cholesterol (1/1/1 mol/mol/mol). Lamellar liquid-ordered (Lo) and liquid-disordered (Ld) phase coexistence was identified in the control and antipsychotic-added mixtures at 37 degrees C using synchrotron small-angle X-ray scattering methods (XRD). Intensity of the Bragg peaks was used to generate electron density profiles (EDP) allowing bilayer thickness calculation. All antipsychotic except from amisulpride induced a Lo phase bilayer thickness (d(pp)) decrease. Chlorpromazine, haloperidol, amisulpride and 9-0H-risperidone induced a Ld d(pp) increase while ziprazidone, risperidone and clozapine induced a Ld d(pp) decrease, indicating that antipsychotic atypicality is not associated with a specific d(pp) modification on our lipid model mixture. Results are discussed in terms of competition of antipsychotic compounds with cholesterol and mode of reorganization of lateral domains. A pharmacological relevance of these changes is also discussed.

Comparison of the liquid-ordered bilayer phases containing cholesterol or 7-dehydrocholesterol in modeling Smith-Lemli-Opitz syndrome

The phase behavior of egg sphingomyelin (ESM) mixtures with cholesterol or 7-dehydrocholesterol (7-DHC) has been investigated by independent methods: fluorescence microscopy, X-ray diffraction, and electron spin resonance spectroscopy. In giant vesicles, cholesterol-enriched domains appeared as large and clearly delineated domains assigned to a liquid-ordered (Lo) phase. The domains containing 7-DHC were smaller and had more diffuse boundaries. Separation of a gel phase assigned by X-ray examination to pure sphingomyelin domains coexisting with sterol-enriched domains was observed at temperatures less than 38°C in binary mixtures containing 10-mol% sterol. At higher sterol concentrations, the coexistence of liquid-ordered and liquid-disordered phases was evidenced in the temperature range 20°–50°C. Calculated electron density profiles indicated the location of 7-DHC was more loosely defined than cholesterol, which is localized precisely at a particular depth along the bilayer normal. ESR spectra of spin-labeled fatty acid partitioned in the liquid-ordered component showed a similar, high degree of order for both sterols in the center of the bilayer, but it was higher in the coexisting disordered phase for 7-DHC. The differences detected in the models of the lipid membrane matrix are said to initiate the deleterious consequences of the Smith-Lemli-Opitz syndrome.

Effect of sphingosine on domain morphology in giant vesicles

Sphingosine is a bioactive molecule which is known to participate in the regulation of a number of cellular processes such as apoptosis, cell differentiation, growth, etc. Sphingosine was observed to exhibit different domain morphology depending on the surrounding lipid matrix in biomimetic systems such as giant vesicles. Our current results showed that in a glycerophospholipid matrix sphingosine segregated in gel leaf-like domains whereas cholesterol presence increased its miscibility by melting gel domains in a concentration-dependent manner. Sphingosine and cholesterol did not form merging liquid domains on the micron scale as observed for sphingomyelin and cholesterol. However, we were able to visualize that sphingosine appears as a stabilizer and amplifier of domains in liquid-ordered phase by increasing the temperature of their formation and fraction. These results imply that sphingosine acts as a modulator of the lipid domain formation and thus it could exert its biological role, not only through direct binding to proteins, but also indirectly by influencing their sorting in membranes and modulating the processes of signal transduction.

Resveratrol alters the lipid composition, metabolism and peroxide level in senescent rat hepatocytes

Investigations were performed on the influence of resveratrol on the lipid composition, metabolism, fatty acid and peroxide level in plasma membranes of hepatocytes, isolated from aged rats. Hepatocytes were chosen due to the central role of the liver in lipid metabolism and homeostasis. The obtained results showed that the level of sphingomyelin (SM) and phosphatidylserine (PS) was augmented in plasma membranes of resveratrol-treated senescent hepatocytes. The saturated/unsaturated fatty acids ratio of the two most abundant membrane phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), was decreased as a result of resveratrol treatment. The neutral sphingomyelinase was found to be responsible for the increase of SM and the decrease of ceramide in plasma membranes of resveratrol-treated senescent hepatocytes. Using labeled acetate as a precursor of lipid synthesis we demonstrated, that resveratrol treatment resulted in inhibition mainly of phospholipid synthesis, followed by fatty acids synthesis. Resveratrol induced reduction of specific membrane-associated markers of apoptosis such as localization of PS in the external plasma membrane monolayer and ceramide level. Finally, the content of lipid peroxides was investigated, because the unsaturated fatty acids, which were augmented as a result of resveratrol treatment, are an excellent target of oxidative attack. The results showed that the lipid peroxide level was significantly lower, ROS were slightly reduced and GSH was almost unchanged in resveratrol-treated hepatocytes. We suggest, that one possible biochemical mechanism, underlying the reported resveratrol-induced changes, is the partial inactivation of neutral sphingomyelinase, leading to increase of SM, the latter acting as a native membrane antioxidant. In conclusion, our studies indicate that resveratrol treatment induces beneficial alterations in the phospholipid and fatty acid composition, as well as in the ceramide and peroxide content in plasma membranes of senescent hepatocytes. Thus, the presented results imply that resveratrol could improve the functional activity of the membrane lipids in the aged liver by influencing specific membrane parameters, associated with the aging process.

Docosahexaenoic acid promotes micron scale liquid-ordered domains. A comparison study of docosahexaenoic versus oleic acid containing phosphatidylcholine in raft-like mixtures

The understanding of the functional role of the lipid diversity in biological membranes is a major challenge. Lipid models have been developed to address this issue by using lipid mixtures generating liquid-ordered (Lo)/liquid-disordered (Ld) immiscibility. The present study examined mixtures comprising Egg sphingomyelin (SM), cholesterol (chol) and phosphatidylcholine (PC) either containing docosahexaenoic (PDPC) or oleic acid (POPC). The mixtures were examined in terms of their capability to induce phase separation at the micron- and nano-scales. Fluorescence microscopy, electron spin resonance (ESR), X-ray diffraction (XRD) and calorimetry methods were used to analyze the lateral organization of the mixtures. Fluorescence microscopy of giant vesicles could show that the temperature of the micron-scale Lo/Ld miscibility is higher for PDPC than for POPC ternary mixtures. At 37°C, no micron-scale Lo/Ld phase separation could be identified in the POPC containing mixtures while it was evident for PDPC. In contrast, a phase separation was distinguished for both PC mixtures by ESR and XRD, indicative that PDPC and POPC mixtures differed in micron vs nano domain organization. Compared to POPC, the higher line tension of the Lo domains observed in PDPC mixtures is assumed to result from the higher difference in Lo/Ld order parameter rather than hydrophobic mismatch.

Making a tool of an artifact: the application of photoinduced Lo domains in giant unilamellar vesicles to the study of Lo/Ld phase spinodal decomposition and its modulation by the ganglioside GM1.

Electroformed giant unilamellar vesicles containing liquid-ordered Lo domains are important tools for the modeling of the physicochemical properties and biological functions of lipid rafts. Lo domains are usually imaged using fluorescence microscopy of differentially phase-partionioning membrane-embedded probes. Recently, it has been shown that these probes also have a photosensitizing effect that leads to lipid chemical modification during the fluorescence microscopy experiments. Moreover, the lipid reaction products are able as such to promote Lo microdomain formation, leading to potential artifacts. We show here that this photoinduced effect can also purposely be used as a new approach to study Lo microdomain formation in giant unilamellar vesicles. Photosensitized lipid modification can promote Lo microdomain appearance and growth uniformly and on a faster time scale, thereby yielding new information on such processes. For instance, in egg phosphatidylcholine/egg sphingomyelin/cholesterol 50:30:20 (mol/mol) giant unilamellar vesicles, photoinduced Lo microdomain formation appears to occur by the rarely observed spinodal decomposition process rather than by the common nucleation process usually observed for Lo domain formation in bilayers. Moreover, temperature and the presence of the ganglioside GM1 have a profound effect on the morphological outcome of the photoinduced phase separation, eventually leading to features such as bicontinuous phases, phase percolation inversions, and patterns evoking double phase separations. GM1 also has the effect of destabilizing Lo microdomains. These properties may have consequences for Lo nanodomains stability and therefore for raft dynamics in biomembranes. Our data show that photoinduced Lo microdomains can be used to obtain new data on fast raft-mimicking processes in giant unilamellar vesicles.

The interaction of antipsychotic drugs with lipids and subsequent lipid reorganization investigated using biophysical methods

The interaction of antipsychotic drugs (AP) with lipids and the subsequent lipid reorganization on model membranes was assessed using a combination of several complementary biophysical approaches (calorimetry, plasmon resonance, fluorescence microscopy, X-ray diffraction and molecular modeling). The effect of haloperidol (HAL), risperidone (RIS), and 9-OH-risperidone (9-OH-RIS) was examined on single lipid and mixtures comprising lipids of biological origin. All APs interact with lipids and induced membrane reorganization. APs showed higher affinity for sphingomyelin than for phosphatidylcholine. Cholesterol increased AP affinity for the lipid bilayer and led to the following AP ranking regarding affinity and structural changes: RIS >9-OH-RIS >HAL. Liquid-ordered domain formation and bilayer thickness were differentially altered by AP addition. Docking calculations helped understanding the observed differences between the APs and offer a representation of their conformation in the lipid bilayer. Present results indicate that AP drugs may change membrane compartmentalization which could differentially modulate the signaling cascade of the dopamine D2 receptor for which APs are ligands.

Testosterone replacement therapy improves erythrocyte membrane lipid composition in hypogonadal men.

Aim: The aim of this study was to investigate the effects of testosterone replacement therapy (TRT) on erythrocyte membrane (EM) lipid composition and physico-chemical properties in hypogonadal men. Methods: EM isolated from three patients before and after TRT with injectable testosterone undecanoate or testosterone gel were used for analysis of the phospholipid and fatty acid composition, cholesterol/phospholipid ratio, membrane fluidity, ceramide level and enzyme acivities responsible for sphingomyelin metabolism. Results: TRT induced increase of phosphatidylethanolamine (PE) in the EMs and sphingomyelin. Reduction of the relative content of the saturated palmitic and stearic fatty acids and a slight increase of different unsaturated fatty acids was observed in phosphatidylcholine (PC). TRT also induced decrease of the cholesterol/total phospholipids ratio and fluidization of the EM. Discussion: The TRT induced increase of PE content and the reduction of saturation in the PC acyl chains induced alterations in the structure of EM could result in higher flexibility of the erythrocytes. The increase of the SM-metabolizing enzyme neutral sphingomyelinase, which regulates the content of ceramide in membranes has a possible impact on the SM signaling pathway. Conclusion: We presume that the observed effect of TRT on the composition and fluidity of the EM contributes for improvement of blood rheology and may diminish the thrombosis risk. Larger studies are needed to confirm the findings of this pilot study.