Effects of 2-hydroxyoleic acid on the structural properties of biological and model plasma membranes

Genetic hypertension is associated with alterations in lipid metabolism, membrane lipid composition and membrane-protein function. 2-Hydroxyoleic acid (2OHOA) is a new antihypertensive molecule that regulates the structure of model membranes and their interaction with certain peripheral signalling proteins in vitro. While the effect of 2OHOA on elevated blood pressure is thought to arise through its influence on signalling proteins, its effects on membrane lipid composition remain to be assessed. 2OHOA administration altered the lipid membrane composition of hypertensive and normotensive rat plasma membranes, and increased the fluidity of reconstituted liver membranes from hypertensive rats. In spontaneously hypertensive rats (SHR), treatment with 2OHOA increased the cholesterol and sphingomyelin content while decreasing that of phosphatidylserine-phosphatidylinositol lipids. In addition, monounsaturated fatty acid levels increased as well as the propensity of reconstituted membranes to form HII-phases. These data suggest that 2OHOA regulates lipid metabolism that is altered in hypertensive animals, and that it affects the structural properties of liver plasma membranes in SHR. These changes in the structural properties of the plasma membrane may modulate the activity of signalling proteins that associate with the cell membrane such as the Galphaq/11 protein and hence, signal transduction.     

The effect of hydroxylated fatty acid-containing phospholipids in the remodeling of lipid membranes

The synthetic fatty acid 2-hydroxyoleic acid (2OHOA) is an antitumor drug that regulates membrane lipid composition and structure. An important effect of this drug is the restoration of sphingomyelin (SM) levels in cancer cell membranes, where the SM concentration is lower than in non-tumor cells. It is well known that free fatty acid concentration in cell membranes is lower than 5%, and that fatty acid excess is rapidly incorporated into phospholipids. In a recent work, we have considered the effect of free 2OHOA in model membranes in liquid ordered (Lo) and liquid disordered (Ld) phases, by using all-atom molecular dynamics. This study concerns membranes that are modified upon incorporation of 2OHOA into different phospholipids. 2OHOA-containing phospholipids have a permanent effect on lipid membranes, making a Ld membrane surface more compact and less hydrated, whereas the opposite effect is observed in Lo domains. Moreover, the hydroxyl group of fatty acid chains increases the propensity of Ld model membranes to form hexagonal or other non-lamellar structures. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

Alterations in lipid composition and fluidity of liver plasma membranes in copper-deficient rats

In view of the importance of membrane fluidity on cell functions, the influence of phospholipid acyl groups on membrane fluidity, and the changes in lipid metabolism induced by copper (Cu) deficiency, this study was designed to examine the influence of dietary Cu on the lipid composition and fluidity of liver plasma membranes. Male Sprague-Dawley rats were divided into two dietary treatments, namely Cu deficient and Cu adequate. After 8 weeks of treatment, liver plasma membranes were isolated by sucrose density gradient centrifugation. The lipid fluidity of plasma membranes, as assessed by the intramolecular eximer fluorescence of 1,3-di(1-pyrenyl) propane, was significantly depressed by Cu deficiency. In addition, Cu deficiency significantly reduced the content of arachidonic and palmitoleic acids but increased the docosatetraenoic and docosahexaenoic acids of membrane phospholipids. This alteration in unsaturated phospholipid fatty acid composition, especially the large reduction in arachidonic acid, may have contributed to the depressed membrane fluidity. Furthermore, Cu deficiency also markedly altered the fatty acid composition of the triacylglycerols associated with the plasma membranes. Thus, the lipid composition and fluidity of liver plasma membranes are responsive to the animal's Cu status.     

Effect of a 2-hydroxylated fatty acid on Cholesterol-rich membrane domains

Abstract

2-Hydroxyoleic acid (2OHOA) is a synthetic fatty acid with antihypertensive properties that is able to alter structural membranes properties. The main purpose of this study was to analyze the effect of 2OHOA on the membrane architecture in cholesterol (Cho)-rich domains. For this purpose, model membranes mimicking the composition of lipid rafts and PC- or PE-Cho-rich domains were examined in the absence and presence of 2OHOA by synchrotron X-ray diffraction, atomic force microscopy (AFM) and microcalorimetry (DSC) techniques. Our results demonstrate that 2OHOA phase separates from lipid raft domains and affects the lateral organization of lipids in the membrane. In model raft membranes, 2OHOA interacted with the sphingomyelin (SM) gel phase increasing the thickness of the water layer, which should lead to increased bilayer fluidity. The hydrogen binding competition between 2OHOA and Cho could favour the enrichment of 2OHOA in SM domains separated from the SM-Cho domains, resulting in an enhanced phase separation into SM-2OHOA-rich liquid-disordered (non-raft) and SM-Cho-rich liquid-ordered (raft) domains. The segregation into 2OHOA-rich/Cho-poor and 2OHOA-poor/Cho-rich domains was also observed in PC bilayers.     

Lipid composition and fluidity in the jejunal brush-border membrane of spontaneously hypertensive rats. Effects on activities of membrane-bound proteins

The lipid composition and fluidity of jejunal brush-border membrane vesicles (BBMV) have been studied in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats. The activities of both Na⁺-dependent D-glucose cotransport and Na⁺-H⁺ antiport have also been determined. A significant increase in the level of free cholesterol was observed in jejunal BBMV from SHR compared to WKY rats. Since phospholipid values did not change in either group of animals, a significant enhancement in the free cholesterol/phospholipid ratio was observed in SHR. A decrease in the levels of phosphatidylethanolamine together with an increase in the values of phosphatidylserine was observed in hypertensive rats. Although the content of phosphatidylcholine (PC) and sphingomyelin (SM) was not singificantly altered in SHR, the ratio PC/SM significantly increased in these animals when compared to WKY rats. The major fatty acids present in bursh-border membranes prepared from SHR and WKY rats were palmitic (160), stearic (180), oleic (181, n-9) and linoleic (182, n-6), and the fatty acid composition was not modified by the hypertension. A decreased fluorescence polarization, i.e., increased membrane fluidity, was observed in SHR, which was not correlated to the increased ratio of cholesterol/phospholipid found in the brush-border membrane isolated from these animals. These structural changes found in SHR were associated to an enhancement in both Na⁺-dependent D-glucose transport and Na⁺-H⁺ antiport activity in the jejunal BBMV of SHR.Abbreviations BBMV brush-border membrane vesicles - DPH 1,6-diphenyl-1,3,5-hexatriene - FC free cholesterol - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - SM sphingomyelin - SHR spontaneously hypertensive rat - p steady-state fluoroscence polarization - r_s steady-state fluorescence anisotropy - WKY Wistar Kyoto     

The hypotensive drug 2-hydroxyoleic acid modifies the structural properties of model membranes

We studied the interactions of the hypotensive drug, 2-hydroxyoleic acid (2OHOA), with model membranes using the techniques of DSC, ³¹P NMR and X-ray diffraction. We demonstrate that 2OHOA alters the thermotropic behaviour of 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE), thereby promoting the formation of hexagonal phases (H_II), despite stabilizing the lamellar phase (L_α). The lattice parameters of lamellar and non-lamellar structures were not altered by the presence of 2OHOA. The molecular bases underlying the alterations in membrane structure provoked by 2OHOA were analysed by comparing the effects produced by 2OHOA with the closely related fatty acids (FAs), oleic acid (OA) and elaidic acid (EA). The capacity of C-18 FAs to induce H_II-phase formation followed the order OA>2OHOA>EA. Furthermore, while 2OHOA stabilized the L_α phase, OA destabilized it. The net negative charge of 2OHOA at physiological pH (～7.4) influenced its effect on membrane structure. By analysing the molecular architecture of 2OHOA in DEPE monolayers, interactions between the carboxylate groups of 2OHOA and the amine groups of DEPE were observed, as well as between the 2-hydroxyl group of the FA and the carbonyl oxygen of the phospholipid acyl chain. These structural characteristics provoked an increase in the P-to-N and P-to-P distances of neighbouring phospholipid headgroups in the presence of 2OHOA, with respect to those observed with OA and EA. The higher headgroup area at the lipid–water interface in presence of 2OHOA could account for the differential effect of this drug on the phase behaviour of DEPE membranes.     

Effect of liver plasma membrane fluidity on endogenous phospholipase A2 activity

Investigations have been carried out on the influence of the phospholipid composition and the physicochemical properties of rat liver plasma membranes on the endogenous activity of membrane-bound phospholipase A2. The membrane phospholipid composition was modified by the incorporation of different phospholipids in the lipid bilayer by the aid of lipid transfer proteins. The results indicate that the endogenous activity of phospholipase A2 in liver plasma membranes depends upon membrane fluidity and not upon the presence of a specific phospholipid in the enzyme's microenvironment.     

Plasma membrane stress induces relocalization of Slm proteins and activation of TORC2 to promote sphingolipid synthesis

The plasma membrane delimits the cell, and its integrity is essential for cell survival. Lipids and proteins form domains of distinct composition within the plasma membrane. How changes in plasma membrane composition are perceived, and how the abundance of lipids in the plasma membrane is regulated to balance changing needs remains largely unknown. Here, we show that the Slm1/2 paralogues and the target of rapamycin kinase complex 2 (TORC2) play a central role in this regulation. Membrane stress, induced by either inhibition of sphingolipid metabolism or by mechanically stretching the plasma membrane, redistributes Slm proteins between distinct plasma membrane domains. This increases Slm protein association with and activation of TORC2, which is restricted to the domain known as the membrane compartment containing TORC2 (MCT; ref.?). As TORC2 regulates sphingolipid metabolism, our discoveries reveal a homeostasis mechanism in which TORC2 responds to plasma membrane stress to mediate compensatory changes in cellular lipid synthesis and hence modulates the composition of the plasma membrane. The components of this pathway and their involvement in signalling after membrane stretch are evolutionarily conserved.     

Normalization of sphingomyelin levels by 2-hydroxyoleic acid induces autophagic cell death of SF767 cancer cells

The very high mortality rate of gliomas reflects the unmet therapeutic need associated with this type of brain tumor. We have discovered that the plasma membrane fulfills a critical role in the propagation of tumorigenic signals, whereby changes in membrane lipid content can either activate or silence relevant pathways. We have designed a synthetic fatty acid, 2-hydroxyoleic acid (2OHOA), that specifically activates sphingomyelin synthase (SGMS), thereby modifying the lipid content of cancer cell membranes and restoring lipid levels to those found in normal cells. In reverting, the structure of the membrane by activating SGMS, 2OHOA inhibits the RAS-MAPK pathway, which in turn fails to activate the CCND (Cyclin D)-CDK4/CDK6 and PI3K-AKT1 pathways. The overall result in SF767 cancer cells, a line that is resistant to apoptosis, is the sequential induction of cell cycle arrest, cell differentiation and autophagy. Such effects are not observed in normal cells (MRC-5) and thus, this specific activation of programmed cell death infers greater efficacy and lower toxicity to 2OHOA than that associated with temozolomide (TMZ), the reference drug for the treatment of glioma.     

Normalization of sphingomyelin levels by 2-hydroxyoleic acid induces autophagic cell death of SF767 cancer cells

The very high mortality rate of gliomas reflects the unmet therapeutic need associated with this type of brain tumor. We have discovered that the plasma membrane fulfills a critical role in the propagation of tumorigenic signals, whereby changes in membrane lipid content can either activate or silence relevant pathways. We have designed a synthetic fatty acid, 2-hydroxyoleic acid (2OHOA), that specifically activates sphingomyelin synthase (SGMS), thereby modifying the lipid content of cancer cell membranes and restoring lipid levels to those found in normal cells. In reverting, the structure of the membrane by activating SGMS, 2OHOA inhibits the RAS-MAPK pathway, which in turn fails to activate the CCND (Cyclin D)-CDK4/CDK6 and PI3K-AKT1 pathways. The overall result in SF767 cancer cells, a line that is resistant to apoptosis, is the sequential induction of cell cycle arrest, cell differentiation and autophagy. Such effects are not observed in normal cells (MRC-5) and thus, this specific activation of programmed cell death infers greater efficacy and lower toxicity to 2OHOA than that associated with temozolomide (TMZ), the reference drug for the treatment of glioma.     

The characteristics of the oxygen supply to the skeletal muscle of spontaneously hypertensive rats during starvation

Peculiarities of oxygen supply of a skeletal muscle under normal alimentary conditions and under conditions of alimentary starvation have been studied on spontaneously hypertensive rats (SHR) and normotensive rats (NTR). Calculations of oxygen permeability through hematoparenchymatous barrier (HPB) have shown that it is higher (by 26%) in SHR than in NTR. This difference can be a result of higher content of arachidonic acid and intensity of lipid peroxidation in phospholipids of plasma membranes. After 5-day starvation tension of O2 in skeletal muscle of SHR increased due to a decrease of oxygen demand and increase of oxygen permeability through HPR. The observed modification of fatty-acid spectrum of plasma membrane phospholipids and a sharp decrease of cholesterol content in them can be responsible for an increase of oxygen permeability through HPB.     

Differential effect of 2-hydroxyoleic acid enantiomers on protein (sphingomyelin synthase) and lipid (membrane) targets

The complex dual mechanism of action of 2-hydroxyoleic acid (2OHOA), a potent anti-tumor compound used in membrane lipid therapy (MLT), has yet to be fully elucidated. It has been demonstrated that 2OHOA increases the sphingomyelin (SM) cell content via SM synthase (SGMS) activation. Its presence in membranes provokes changes in the membrane lipid structure that induce the translocation of PKC to the membrane and the subsequent overexpression of CDK inhibitor proteins (e.g., p21^Cip1). In addition, 2OHOA also induces the translocation of Ras to the cytoplasm, provoking the silencing of MAPK and its related pathways. These two differential modes of action are triggered by the interactions of 2OHOA with either lipids or proteins. To investigate the molecular basis of the different interactions of 2OHOA with membrane lipids and proteins, we synthesized the R and S enantiomers of this compound. A molecular dynamics study indicated that both enantiomers interact similarly with lipid bilayers, which was further confirmed by X-ray diffraction studies. By contrast, only the S enantiomer was able to activate SMS in human glioma U118 cells. Moreover, the anti-tumor efficacy of the S enantiomer was greater than that of the R enantiomer, as the former can act through both MLT mechanisms. The present study provides additional information on this novel therapeutic approach and on the magnitude of the therapeutic effects of type-1 and type-2 MLT approaches. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

Protective effect of blackcurrant on liver cell membrane of rats intoxicated with ethanol

Chronic ethanol intoxication oxidative stress participates in the development of many diseases. Nutrition and the interaction of food nutrients with ethanol metabolism may modulate alcohol toxicity. One such compound is blackcurrant, which also has antioxidant abilities. We investigated the effect of blackcurrant as an antioxidant on the composition and electrical charge of liver cell membranes in ethanol-intoxicated rats. Qualitative and quantitative phospholipid composition and the presence of integral membrane proteins were determined by high-performance liquid chromatography. Electrophoresis was used to determine the surface charge density of the rat liver cell membranes. Ethanol intoxication is characterized by changes in cell metabolism that alter the structure and function of cell membrane components. Ethanol increased phospholipid levels and altered the level of integral proteins as determined by decreased phenylalanine, cysteine, and lysine. Ethanol significantly enhanced changes in the surface charge density of the liver cell membranes. Administration of blackcurrant to rats intoxicated with ethanol significantly protected lipids and proteins against oxidative modifications. It is possible that the beneficial effect of blackcurrant is connected with its abilities to scavenge free radicals and to chelate metal ions.     

Lipid composition and fluidity of liver plasma membranes from rats with chronic dietary iron overload

Liver plasma membranes isolated from rats with chronic dietary iron overload showed a large modification of their phospholipid fatty acid composition. Specifically, a significant decrease in polyunsaturated fatty acids and a parallel increase in saturated fatty acids was observed. This pattern was consistent with thein vivo occurrence of lipoperoxidative reactions in the liver plasma membranes. However, neither change in the cholesterol/phospholipid molar ratio nor in the lipid/protein ratio was detected. Direct measurement of the plasma membrane fluidity state by electron spin resonance spectrometry did not reveal any difference between control and iron-treated rats. These findings indicate that chronic dietary iron overload can induce lipid peroxidation of rat liver plasma membranes, but this event does not bring about modification in the physical state of the membrane.     

Change of synaptic membrane lipid composition and fluidity by chronic administration of lithium

The effect of chronic administration of lithium salts on the lipid composition and physical properties of the synaptosomal plasma membrane was examined in rat brain. The effect of lithium treatment has been studied on the fluorescence polarization of synaptosomal plasma membrane and artificial lipid vesicles and on the lipid composition of the membranes. Fluorescence polarization of lipophilic probes was used to study membrane lipid structure. Steady-state polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), a probe of the hydrophobic core, was significantly lower in plasma membranes from lithium-treated animals. Altered DPH polarization was due to a decrease in the order parameter of the probe. The lithium-treatment also changed the fluorescence of 1-anilino-8-naphthalene sulfonate (ANS), a probe that binds to the polar head group of the phospholipids and to proteins on the membrane surface. Synaptic plasma membranes from treated rats presented no significant changes on the cholesterol-to-phospholipid ratio, although the phospholipid class distribution was altered and the membrane phospholipid unsaturation increased. In summary, the neural plasma membranes became disorder after chronic lithium administration at therapeutic levels. This structural change may be due to changes in plasma membrane phospholipid distribution and to the degree of unsaturation of phospholipid fatty acids.     

Calcium and abnormal reactivity of vascular smooth muscle in hypertension

It has been well documented that vascular smooth muscle (VSM) reactivity, as well as calcium sensitivity in response to neurotransmitters is increased in a number of blood vessels in established hypertension. Regulation of VSM reactivity involves the interaction of neurotransmitters and blood-borne hormones with specific receptors on target cell membranes. This results in phospholipase-C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) and the generation of two second messengers: inositol 1,4,5 trisphosphate (IP3) and diacylglycerol (DAG) both of which act synergistically to produce muscle contraction. We will summarize recent findings in this review which suggest that in essentially hypertensive patients and spontaneously hypertensive rats (SHR), the activation of phospholipase C in response to hormones is increased. Further, we will discuss how increases in phospholipase C activation via GTP-binding proteins may explain the observed increases in Ca2+ influx through potential- and receptor-operated Ca2+ channels, increased activation of protein kinase-C and increased [Ca2+]i in hormone-stimulated blood platelets and VSM cells in the hypertensive state. In addition to these defects, a decrease in the plasma membrane Ca2+ pump and Ca2+-binding proteins has been demonstrated in hypertension. Thus, it appears that the defect in Ca2+ metabolism in the hypertensive vessels is multifocal. All these defects in Ca2+ metabolism together may lead to an increase in peripheral vascular resistance with a concomitant increase in blood pressure.     

Lipid peroxidation in purified plasma membrane fractions of rat liver in relation to the hepatoxicity of carbon tetrachloride

Preparations of rat liver sinusoidal plasma membrane have been tested for their ability to metabolize the hepatotoxin carbon tetrachloride (CCl4) to reactive free radicals in vitro and compared in this respect with standard preparations of rat liver microsomes. The sinusoidal plasma membranes were relatively free of endoplasmic reticulum-associated activities such as the enzymes of the cytochrome P450 system and glucose-6-phosphatase. CCl4 metabolism was measured as (i) covalent binding of [14C]-CCl4 to membrane protein, (ii) electron spin resonance spin-trapping of CCl3. radicals and (iii) CCl4-induced lipid peroxidation. By all of these tests, purified sinusoidal plasma membranes were found unable to metabolize CCl4. The fatty acid composition of the plasma membranes was almost identical to that of the microsomal preparation and both membrane fractions exhibited similar rates of the lipid peroxidation that was stimulated non-enzymically by gamma-radiation or incubation with ascorbate and iron. The absence of CCl4-induced lipid peroxidation in the plasma membranes seems to be due, therefore, to an absence of CCl4 activation rather than an inherent resistance to lipid peroxidation. We conclude that damage to the hepatocyte plasma membrane during CCl4 intoxication is not due to a significant local activation of CCl4 to CCl3. within that membrane.     

Insulin effect on the phospholipid organization and some enzyme activities of rat liver membrane fractions

1. The influence of insulin on rat liver membrane lipid composition, fluidity, some enzyme activities and asymmetry of microsomal phospholipids were investigated. 2. The total phospholipids and cholesterol were increased in microsomes and reduced in plasma membranes from insulin-treated rats. 3. Of all the investigated enzymes participating in the lipid metabolism, only the neutral sphingomyelinase activity was observed to be enhanced, whereas the ceramide-phosphatidylethanolamine (PE) synthetase and phospholipase A2 activities remained unchanged. 4. Insulin administration caused translocation of phosphatidylserine (PS) and PE to the outer leaflet and of phosphatidylinositol (PI) to the inner leaflet of microsomal membranes.     

Changes in hepatic protein expression in spontaneously hypertensive rats suggest early stages of non-alcoholic fatty liver disease

Hypertension is a systemic disorder affecting numerous physiological processes throughout the body. As non-alcoholic fatty liver disorder (NAFLD) is a common comorbidity of hypertension in humans, we hypothesized that molecular hepatic physiology would be altered in a model of genetic hypertension. Despite the broad use of the spontaneously hypertensive rat (SHR) model, little is known regarding how hypertension influences hepatic function under basal conditions. In order to determine whether hypertension induces changes in the hepatic protein expression suggestive of early stages of NAFLD, we compared the whole tissue proteome of livers from SHR and Wistar Kyoto (WKY) 16 week old rats using 2DGE and MALDI-TOF MS. Fifteen proteins were identified that display different levels of expression between the SHR and WKY livers: 50% of proteins have mitochondrial or anti-oxidant functions while 20% are involved in lipid metabolism. Quininoid dihydropterin reductase, sulfite oxidase, and glutathione-S-transferase mu 1 were all identified as either undergoing a difference in post-translation modification or a difference in protein abundance in SHR compared to WKY livers. As oxidative stress is a well described component of both NAFLD and hypertension in SHR, the identification of novel changes in protein expression provides possible mechanisms connecting these two pathologies in humans.     

Accumulation of raft lipids in T‐cell plasma membrane domains engaged in TCR signalling

Activating stimuli for T lymphocytes are transmitted through plasma membrane domains that form at T‐cell antigen receptor (TCR) signalling foci. Here, we determined the molecular lipid composition of immunoisolated TCR activation domains. We observed that they accumulate cholesterol, sphingomyelin and saturated phosphatidylcholine species as compared with control plasma membrane fragments. This provides, for the first time, direct evidence that TCR activation domains comprise a distinct molecular lipid composition reminiscent of liquid‐ordered raft phases in model membranes. Interestingly, TCR activation domains were also enriched in plasmenyl phosphatidylethanolamine and phosphatidylserine. Modulating the T‐cell lipidome with polyunsaturated fatty acids impaired the plasma membrane condensation at TCR signalling foci and resulted in a perturbed molecular lipid composition. These results correlate the accumulation of specific molecular lipid species with the specific plasma membrane condensation at sites of TCR activation and with early TCR activation responses.