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.     

Membrane fluidity changes are associated with the antiarrhythmic effects of docosahexaenoic acid in adult rat cardiomyocytes

Previous studies using neonatal rat cardiomyocytes have reported antiarrhythmic effects of long-chain polyunsaturated fatty acids (PUFAs). In this study, we examined the effects of the n-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) on the spontaneous contractile activity and membrane fluidity of adult rat ventricular myocytes. Cardiomyocytes were induced to contract spontaneously by continuous superfusion of a solution containing the arrhythmogenic agents isoproterenol (a beta-adrenergic receptor agonist) or lysophosphatidylcholine. The percentage of cardiomyocytes displaying spontaneous contractions induced by isoproterenol when pretreated with the saturated fatty acid docosanoic acid was 48.1 +/- 7.7%; the percentage for cardiomyocytes pretreated with DHA was 7.1 +/- 2.4% (P < 0.01). DHA significantly prevented lysophosphatidylcholine-induced spontaneous contractions (17.7 +/- 6.5%) compared with treatment with the saturated fatty acid stearic acid (78.0 +/- 7.3%, P < 0.01). The membrane fluidizing agent benzyl alcohol also significantly prevented spontaneous contractions in cardiomyocytes. Membrane fluidity was determined by steady-state fluorescence anisotropy (r(ss)) using the fluorescent probe N-((4-(6-phenyl-1,3,5-hexatrienyl)phenyl)propyl) trimethyl-ammonium p-toluene-sulfonate (TMAP-DPH). DHA and benzyl alcohol dose-dependently decreased the r(ss); however, saturated fatty acids were without effect. These results suggest that the antiarrhythmic mechanisms of the n-3 PUFAs such as DHA may involve changes in membrane fluidity.     

Termination of asynchronous contractile activity in rat atrial myocytes by n-3 polyunsaturated fatty acids

A protective effect of the n-3 polyunsaturated fatty acids (PUFAs) in preventing ventricular fibrillation in experimental animals and cultured cardiomyocytes has been demonstrated in a number of studies. In this study, a possible role for the n-3 PUFAs in the treatment of atrial fibrillation (AF) was investigated at the cellular level using atrial myocytes isolated from young adult rats as the experimental model. Electrically-stimulated, synchronously-contracting myocytes were induced to contract asynchronously by the addition of 10 M isoproterenol. Asynchronous contractile activity was reduced following acute addition of the n-3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) at 10 M, compared with no fatty acid addition (from 99.0 ±: 1.0% to 30.7 ± 5.2% (p < 0.05) for DHA and 23.8 ± 2.8% (p < 0.01) for EPA), while the saturated fatty acid, docosanoic acid (DA) and the methyl ester of DHA (DHA m.e.) did not exert a significant effect on asynchronous contractile activity. Asynchronous contractile activity was also reduced to 1.7 ± 1.7% in the presence of the membrane fluidising agent, benzyl alcohol (p < 0.001 vs no fatty acid addition). Cell membrane fluidity was determined by steady state fluorescence anisotropy using the fluorescent probe, TMAP-DPH. Addition of DHA, EPA or benzyl alcohol significantly increased sarcolemmal membrane fluidity (decreased anisotropy, r_ss) of atrial myocytes compared with no addition of fatty acid (control) (from r_ss = 0.203 ±0.004 to 0.159 ± 0.004 (p < 0.01) for DHA, 0.166 ± 0.001 (p < 0.01) for EPA and 0.186 ±0.003 (p < 0.05) for benzyl alcohol, while DA and DHA m.e. were without effect. It is concluded that the n-3 PUFAs exert anti-asynchronous effects in rat atrial myocytes by a mechanism which may involve changes in membrane fluidity.     

Red Blood Cell Membrane Fluidity in the Etiology of Multiple Sclerosis

Organisms adjust the order, or fluidity, of their cellular membranes in response to changes in their physiochemical environment by adjusting the lipid composition of their membranes. We investigated membrane fluidity using the phospholipid, fatty acid and cholesterol content of red blood cells (RBCs) from multiple sclerosis (MS) patients and correlated this with C-reactive protein (CRP) as well as with the severity of neurological outcome as measured by the Kurtzke Expanded Disability Status Scale (EDSS) and its Functional System Scores. The study group consisted of 31 patients with MS and 30 healthy control subjects. Phospholipids were determined using a colorimetric assay, fatty acids by gas chromatography, cholesterol by an enzymatic assay and CRP by a Beckman nephelometer. Cell membrane fluidity was calculated according to previously established formulae. RBC membrane fluidity as measured by the saturated to polyunsaturated fatty acid ratio was higher in patients than in controls (P = 0.04). The phosphatidylethanolamine saturated to polyunsaturated fatty acid ratio showed highly significant positive correlations with the EDSS and CRP < 5 μg/ml. CRP showed significant inverse correlations with the saturated nature but positive correlations with the ordered-crystalline-phase to liquid-crystalline-phase lipid ratio. In this study we show that membrane fluidity as measured by the relationship between membrane fatty acids, phospholipids and cholesterol is closely interrelated with inflammation and disease outcome in patients with MS. In conclusion, our findings suggest that the membrane lipid composition of patients with MS and, consequently, membrane fluidity are altered, which seems to be influenced by the inflammatory status.     

Diet-induced docosahexaenoic acid non-raft domains and lymphocyte function

Docosahexaenoic acid (DHA) is an n-3 polyunsaturated fatty acid (PUFA) that generally suppresses the function of T lymphocytes and antigen presenting cells (APCs). An emerging mechanism by which DHA modifies lymphocyte function is through changes in the organization of sphingolipid/cholesterol lipid raft membrane domains. Two contradictory models have been proposed to explain how DHA exerts its effects through changes in raft organization. The biophysical model, developed in model membranes, shows that DHA-containing phospholipids form unique non-raft membrane domains, that are organizationally distinct from lipid rafts, which serve to alter the conformation and/or lateral organization of lymphocyte proteins. In contrast, the cellular model on DHA and rafts shows that DHA suppresses lymphocyte function, in part, by directly incorporating into lipid rafts and altering protein activity. To reconcile opposing biophysical and cellular viewpoints, a major revision to existing models is presented herein. Based largely on quantitative microscopy data, it is proposed that DHA, consumed through the diet, modifies lymphocyte function, in part, through the formation of nanometer scale DHA-rich domains. These nano-scale domains disrupt the optimal raft-dependent clustering of proteins necessary for initial signaling. The data covered in this review highlights the importance of understanding how dietary n-3 PUFAs modify lymphocyte membranes, which is essential toward developing these fatty acids as therapeutic agents for treating inflammatory diseases.     

Effects of fatty acid unsaturation numbers on membrane fluidity and α-secretase-dependent amyloid precursor protein processing

Fatty acids may integrate into cell membranes to change physical properties of cell membranes, and subsequently alter cell functions in an unsaturation number-dependent manner. To address the roles of fatty acid unsaturation numbers in cellular pathways of Alzheimer's disease (AD), we systematically investigated the effects of fatty acids on cell membrane fluidity and α-secretase-cleaved soluble amyloid precursor protein (sAPP(α)) secretion in relation to unsaturation numbers using stearic acid (SA, 18:0), oleic acid (OA, 18:1), linoleic acid (LA, 18:2), α-linolenic acid (ALA, 18:3), arachidonic acid (AA, 20:4), eicosapentaenoic acid (EPA, 20:5), and docosahexaenoic acid (DHA, 22:6). Treatments of differentiated human neuroblastoma (SH-SY5Y cells) with AA, EPA and DHA for 24h increased sAPP(α) secretion and membrane fluidity, whereas those treatments with SA, OA, LA and ALA did not. Treatments with AA and DHA did not alter the total expressions of amyloid precursor protein (APP) and α-secretases in SH-SY5Y cells. These results suggested that not all unsaturated fatty acids but only those with 4 or more double bonds, such as AA, EPA and DHA, are able to increase membrane fluidity and lead to increase in sAPP(α) secretion. This study provides insights into dietary strategies for the prevention of AD.     

Pyrene eximer mapping in cultured fibroblasts by ratio imaging and time-resolved microscopy

The kinetics of pyrene eximer formation provide a measure of lateral diffusibility in bilayer membranes. Swiss 3T3 fibroblasts were labeled with pyrene, pyrenedecanoic acid (PDA) and 1,3-bis(1 pyrene) propane (BPP) by incubation in the presence of Pluronic F127. Single-cell emission spectra obtained by epifluorescence microscopy (excitation 350 nm) with photodiode array detection showed monomer (380-420 nm) and eximer (475 nm) peaks. The eximer-to-monomer fluorescence ratio (E/M) increased with increasing temperature and loading time. Time-resolved microscopy studies of fibroblasts labeled with PDA for 15 min gave monomer and eximer lifetimes of 101 and 78 ns, respectively, with a monomer-to-eximer conversion rate of 0.02 ns-1. E/M ratio images were obtained with a microchannel plate intensifier and CCD camera at 350-nm excitation and 405 +/- 5 nm (monomer) and greater than 470-nm (eximer) emission wavelengths. E/M ratios of PDA showed spatial variation across the cell with highest ratios at the peripheral plasma membrane. These results establish the methodology to label cells with pyrene eximer-forming probes and to image eximer distributions in membranes of intact cultured cells. Eximer-to-monomer fluorescence ratios are sensitive to maneuvers that alter the membrane physical state and should be of utility in examining the cellular regulation of membrane fluidity.     

Dimethylnitrosamine inhibits the glucagon-stimulated adenylate cyclase activity of rat liver plasma membranes and decreases plasma membrane fluidity

The effect of the hepatocarcinogen dimethylnitrosamine on rat liver plasma membrane adenylate cyclase activity and lipid fluidity was assessed. Glucagon-stimulated adenylate cyclase activity exhibited a complex response to increasing concentrations of dimethylnitrosamine, whereas fluoride-stimulated adenylate cyclase activity was progressively inhibited. Maximal inhibitory effects were observed at a concentration of 15 mM in both cases. The activity of detergent-solubilized adenylate cyclase was unaffected by dimethylnitrosamine. ESR analysis using a fatty acid spin probe showed that dimethylnitrosamine produced a marked, dose-dependent reduction in the fluidity of the plasma membrane with a maximal effect occurring at 20 mM. Dimethylnitrosamine also elevated the temperature at which the lipid phase separation occurred in rat liver plasma membranes, from 28 degrees C to 31 degrees C. The non-carcinogenic but structurally similar compound, dimethylamine hydrochloride neither inhibited adenylate cyclase nor decreased plasma membrane fluidity. It is suggested that the decrease in membrane fluidity, induced by dimethylnitrosamine, via its effects on membrane fluidity, could influence plasma membrane function and cellular regulation.     

Alteration of retinal rod outer segment membrane fluidity in a rat model of Smith-Lemli-Opitz syndrome

Smith-Lemli-Opitz syndrome (SLOS) is caused by an inherited defect in the last step in cholesterol (Chol) biosynthesis, leading to abnormal accumulation of 7-dehydrocholesterol and decreased Chol levels. Progressive retinal degeneration occurs in an animal model of SLOS, induced by treating rats with AY9944, a selective inhibitor of the enzyme affected in SLOS. Here we evaluated alterations in the biochemical and physical properties of retinal rod outer segment (ROS) membranes in this animal model. At 1 month of AY9944 treatment, there were modest alterations in fatty acid composition, but no significant differences in cis-parinaric acid (cPA) spectroscopic parameters in ROS membranes from treated versus control rats. However, at 3 months, ROS docosahexaenoic acid (DHA) content was dramatically reduced, and cPA fluorescence anisotropy values were decreased, relative to controls. Also, 1,6-diphenyl-1,3,5-hexatriene exhibited decreased rotational motion and increased orientational order in ROS membranes from 3 month-old AY9944-treated rats, relative to controls. No significant changes in protein:lipid ratios were observed; however, rhodopsin regenerability was compromised by 3 months of treatment. These findings are consistent with reduced ROS membrane fluidity in the SLOS rat model, relative to controls, primarily due to the dramatic reduction in membrane DHA levels, rather than altered sterol composition.     

A comparison of the effects of linolenic (18:3 omega 3) and docosahexaenoic (22:6 omega 3) acids on phospholipid bilayers

The class of long chain polyunsaturated fatty acids known as omega-3 are believed to be involved in prevention of a number of human afflictions. The mode of action for two of the most common omega-3 fatty acids, linolenic 18:3 delta 9,12,15 and docosahexaenoic 22:6 delta 4,7,10,13,16,19 (DHA), is not known. One suggestion is that they may be incorporated into membranes and there provide some specific function. Here we compare the effects of DHA and its metabolic precursor linolenic acid on the membrane properties of fluidity, fusion and permeability. The fatty acids were investigated as both free fatty acids and mixed chain 18:0, 18:3 and 18:0, 22:6 phosphatidylcholines (PCs). Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and a series of anthracene stearic acid probes indicates 20 mol% incorporation of either fatty acid into dipalmitoylphosphatidylcholine bilayers broadens and depresses the temperature of the phase transition, but has almost no effect on fluidity in the liquid crystalline state. Similar fluidity was also observed in the liquid crystalline bilayers of the mixed chain PCs using the same set of fluorescent fatty acid probes. In contrast, DHA as a free fatty acid or as part of a mixed chain PC, causes a much greater enhancement than linolenic acid of the rates of fusion and permeability as monitored by fluorescence resonance energy transfer and aqueous compartment mixing (fusion) and by lipid vesicle swelling in isotonic erythritol, (permeability). These experiments establish a clear distinction between the effects of linolenic acid and DHA in membranes.     

Exploration of Lipid Metabolism in Relation with Plasma Membrane Properties of Duchenne Muscular Dystrophy Cells: Influence of L-Carnitine

Duchenne muscular dystrophy (DMD) arises as a consequence of mutations in the dystrophin gene. Dystrophin is a membrane-spanning protein that connects the cytoskeleton and the basal lamina. The most distinctive features of DMD are a progressive muscular dystrophy, a myofiber degeneration with fibrosis and metabolic alterations such as fatty infiltration, however, little is known on lipid metabolism changes arising in Duchenne patient cells. Our goal was to identify metabolic changes occurring in Duchenne patient cells especially in terms of L-carnitine homeostasis, fatty acid metabolism both at the mitochondrial and peroxisomal level and the consequences on the membrane structure and function. In this paper, we compared the structural and functional characteristics of DMD patient and control cells. Using radiolabeled L-carnitine, we found, in patient muscle cells, a marked decrease in the uptake and the intracellular level of L-carnitine. Associated with this change, a decrease in the mitochondrial metabolism can be seen from the analysis of mRNA encoding for mitochondrial proteins. Probably, associated with these changes in fatty acid metabolism, alterations in the lipid composition of the cells were identified: with an increase in poly unsaturated fatty acids and a decrease in medium chain fatty acids, mono unsaturated fatty acids and in cholesterol contents. Functionally, the membrane of cells lacking dystrophin appeared to be less fluid, as determined at 37°C by fluorescence anisotropy. These changes may, at least in part, be responsible for changes in the phospholipids and cholesterol profile in cell membranes and ultimately may reduce the fluidity of the membrane. A supplementation with L-carnitine partly restored the fatty acid profile by increasing saturated fatty acid content and decreasing the amounts of MUFA, PUFA, VLCFA. L-carnitine supplementation also restored muscle membrane fluidity. This suggests that regulating lipid metabolism in DMD cells may improve the function of cells lacking dystrophin.     

Docosahexaenoic Acid Is Required for the Survival of Rat Retinal Photoreceptors In Vitro

Abstract: The effect of docosahexaenoic acid (DHA) on neuronal survival was studied in cultured cells isolated from newborn rat retina. In vivo, the content of DHA in the retina increased nearly fourfold from days 2 to 12 after birth, whereas in retinal cells in culture it remained constant. Unlike amacrine cells, the photoreceptor cells in control cultures underwent a selective degeneration, starting at day 7, that led to their massive death by day 11. The addition of DHA at day 7 led to its active incorporation by the cultures, increasing from 6 to 21% of total fatty acids in cell lipids, and completely prevented photo-receptor cell death. When other fatty acids were tested, both neuronal fatty acid composition and photoreceptor death were the same as in control cultures. These results indicate that DHA is specifically required for the survival of retinal photoreceptors.     

Lipid alterations in human colon epithelial cells induced to differentiation and/or apoptosis by butyrate and polyunsaturated fatty acids

The present study highlights the important association between lipid alterations and differentiation/apoptotic responses in human colon differentiating (FHC) and nondifferentiating (HCT-116) cell lines after their treatment with short-chain fatty acid sodium butyrate (NaBt), polyunsaturated fatty acids (PUFAs), and/or their combination. Our data from GC/MS and LC/MS/MS showed an effective incorporation and metabolization of the supplemented arachidonic acid (AA) or docosahexaenoic acid (DHA), resulting in an enhanced content of the respective PUFA in individual phospholipid (PL) classes and an altered composition of the whole cellular fatty acid spectrum in both FHC and HCT-116 cells. We provide novel evidence that NaBt combined with PUFAs additionally modulated AA and DHA cellular levels and caused their shift from triacylglycerol to PL fractions. NaBt increased, while AA, DHA and their combination with NaBt decreased endogenous fatty acid synthesis in FHC but not in HCT-116 cells. Fatty acid treatment also altered membrane lipid structure, augmented cytoplasmic lipid droplet accumulation, reactive oxygen species (ROS) production and dissipation of the mitochondrial membrane potential. All these parameters were significantly enhanced by combined NaBt/PUFA treatment, but only in FHC cells was this accompanied by highly increased apoptosis and suppressed differentiation. Moreover, the most significant changes of ROS production, differentiation and apoptosis among the parameters studied, the highest effects of combined NaBt/PUFA treatment and a lower sensitivity of HCT-116 cells were confirmed using two-way ANOVA. Our results demonstrate an important role of fatty acid-induced lipid alterations in the different apoptotic/differentiation response of colon cells with various carcinogenic potential.     

Adaptive Changes in Membrane Lipids of Barophilic Bacteria in Response to Changes in Growth Pressure

The lipid compositions of barophilic bacterial strains which contained docosahexaenoic acid (DHA [22:6n-3]) were examined, and the adaptive changes of these compositions were analyzed in response to growth pressure. In the facultatively barophilic strain 16C1, phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) were major components which had the same fatty acid chains. However, in PE, monounsaturated fatty acids such as hexadecenoic acid were major components, and DHA accounted for only 3.7% of the total fatty acids, while in PG, DHA accounted for 29.6% of the total fatty acids. In response to an increase in growth pressure in strain 16C1, the amounts of saturated fatty acids in PE were reduced, and these decreases were mainly balanced by an increase in unsaturated fatty acids, including DHA. In PG, the decrease in saturated fatty acids was mainly balanced by an increase in DHA. Similar adaptive changes in fatty acid composition were observed in response to growth pressure in obligately barophilic strain 2D2. Furthermore, these adaptive changes in response were also observed in response to low temperature in strain 16C1. These results confirm that the general shift from saturated to unsaturated fatty acids including DHA is one of the adaptive changes in response to increases in pressure and suggest that DHA may play a role in maintaining the proper fluidity of membrane lipids under high pressure.     

Changes in tissue and mitochondrial membrane composition during rapid growth, maturation and aging in rainbow trout, Oncorhynchus mykiss

Membrane compositions, particularly of mitochondria, could be critical factors in the mechanisms of growth and aging processes, especially during phases of high oxidative stress that result in molecular damage. In the present study, liver and mitochondrial membrane phospholipid (PL) compositions were analyzed in rainbow trout during its four first years of life, a period characterized by rapid growth and high oxidative stress. Specifically, farmed fish of three ages (1-, 2- and 4-years) were studied, and PL compositions of whole liver and liver mitochondria, and fatty acid compositions of individual PL classes were determined. Liver mitochondrial membranes showed a PL composition different to that of the whole tissue suggesting adaptation of cell and subcellular membranes to specific functions. Individual PL had characteristic fatty acid compositions that were similar in whole liver and mitochondrial membranes. Whole liver and mitochondria showed increased lipid peroxidation with age along with changes in membrane PL fatty acid compositions. Most PL classes showed similar changes in fatty acid composition among the age groups, with reduced proportions of docosahexaenoic acid (DHA) and, generally, concomitantly increased levels of monounsaturated fatty acids, which together resulted in reduced peroxidation index (PIn). However, total polyunsaturated fatty acid (PUFA) content did not change significantly with age due to increased eicosapentaenoic acid, docosapentaenoic acid and, in most PL, increased n−6 PUFA. These results suggest there may be oxidation of PL DHA with compensatory mechanisms to maintain membrane fluidity and function. However, modification of fatty acid composition of specific PLs, such as cardiolipin, could affect the electron transport chain efficiency and propagate the oxidative reaction throughout the cell. In addition, both the content and fatty acid composition of sphingomyelin, which has been suggested as a possible mediator of cell dysfunction and apoptosis, changed with age differently to the other PL classes. Moreover, these changes showed different trends between mitochondria and whole liver. These data suggest there is marked oxidative stress associated with rapid growth and maturation in rainbow trout. Changes observed in membrane lipids point to their possible participation in the processes involved in this species response to oxidative stress and damage accumulation rate.     

Liposome-incorporated DHA increases neuronal survival by enhancing non-amyloidogenic APP processing

The fluidity of neuronal membranes plays a pivotal role in brain aging and neurodegeneration. In this study, we investigated the role of the omega-3 fatty acid docosahexaenoic acid (DHA) in modulation of membrane fluidity, APP processing, and protection from cytotoxic stress. To this end, we applied unilamellar transfer liposomes, which provided protection from oxidation and effective incorporation of DHA into cell membranes. Liposomes transferring docosanoic acid (DA), the completely saturated form of DHA, to the cell cultures served as controls. In HEK-APP cells, DHA significantly increased membrane fluidity and non-amyloidogenic processing of APP, leading to enhanced secretion of sAPPα. This enhanced secretion of sAPPα was associated with substantial protection against apoptosis induced by ER Ca(2+) store depletion. sAPPα-containing supernatants obtained from HEK-APP cells exerted similar protective effects as DHA in neuronal PC12 cells and HEK293 control cells. Correlating to further increased sAPPα levels, supernatants obtained from DHA-treated HEK-APP cells enhanced protection, whereas supernatants obtained from DHA-treated HEK293 control cells did not inhibit apoptosis, likely due to the low expression of endogenous APP and negligible sAPPα secretion in these cells. Further experiments with the small molecule inhibitors LY294002 and SP600125 indicated that sAPPα-induced cytoprotection relied on activation of the anti-apoptotic PI3K/Akt pathway and inhibition of the stress-triggered JNK signaling pathway in PC12 cells. Our data suggest that liposomal DHA is able to restore or maintain physiological membrane properties, which are required for neuroprotective sAPPα secretion and autocrine modulation of neuronal survival.     

Docosahexaenoic acid but not eicosapentaenoic acid withstands dietary cholesterol-induced decreases in platelet membrane fluidity

To determine the differenetial effects of docosahexaenoic (DHA) and eicosapentaenoic (EPA) acid on platelet membrane fluidity under hypercholesterolemic conditions. DHA and EPA were orally administered (300 mg/kg body weight^.day) to hypercholesterolemic rats for 12 weeks. Membrane fluidity, evaluated by fluorescence polarization of nonpolar 1,6-diphenyl-1,3,5-hexatriene (DPH), of the platelets of high cholesterol (HC; 1%)-fed rats decreased significantly compared with that of the platelets of normocholesterolemic rats. In HC-fed rats, dietary administration of DHA, unlike that of EPA, significantly increased platelet membrane fluidity. A high cholesterol diet significantly increased platelet aggregation, compared with the platelet aggregation of normocholesterolemic rats. DHA administration significantly decreased the aggregation, whereas EPA had no effect. Levels of EPA in the platelets of the EPA-fed HC rats and those of DHA in the platelets of the DHA-fed HC rats increased by 482 and 174%, respectively, compared with those in the platelets of the HC-fed rats. The unsaturation index and the ratio of saturated to (poly)unsaturated fatty acid of the platelet membrane increased only in the DHA-fed rats. The phospholipid content in platelet membranes remained unaltered in all groups, whereas the cholesterol content decreased significantly in DHA-fed rats, resulting in a significant decrease in the cholesterol/phospholipid molar ratio only in the platelet membranes of DHA-fed rats. These results suggest that DHA is a more potent membrane-fluidizer than EPA in withstanding cholesterol-induced decreases in platelet membrane fluidity and a stronger ameliorative modulator of platelet hyperaggregation.     

Lipid fluidity directly modulates the overall protein rotational mobility of the Ca-ATPase in sarcoplasmic reticulum

We have developed a quantitative and relatively model-independent measure of lipid fluidity using EPR and have applied this method to compare the temperature dependence of lipid hydrocarbon chain fluidity, overall protein rotational mobility, and the calcium-dependent enzymatic activity of the Ca-ATPase in sarcoplasmic reticulum. We define membrane lipid fluidity to be T/eta, where eta is the viscosity of a long chain hydrocarbon reference solvent in which a fatty acid spin label gives the same EPR spectrum (quantitated by the order parameter S) as observed for the same probe in the membrane. This measure is independent of the reference solvent used as long as the spectral line shapes in the membrane and the solvent match precisely, indicating that the same type of anisotropic probe motion occurs in the two systems. We argue that this empirical measurement of fluidity, defined in analogy to the macroscopic fluidity (T/eta) of a bulk solvent, should be more directly related to protein rotational mobility (and thus to protein function) than are more conventional measures of fluidity, such as the rate or amplitude of rotational motion of the lipid hydrocarbon chains themselves. This new definition thus offers a fluidity measure that is more directly relevant to the protein's behavior. The direct relationship between this measure of membrane fluidity and protein rotational mobility is supported by measurements in sarcoplasmic reticulum. The overall rotational motion of the spin-labeled Ca-ATPase protein was measured by saturation-transfer EPR. The Arrhenius activation energy for protein rotational mobility (11-12 kcal/mol/degree) agrees well with the activation energy for lipid fluidity, if defined as in this study, but not if more conventional definitions of lipid fluidity are used. This agreement, which extends over the entire temperature range from 0 to 40 degrees C, suggests that protein mobility depends directly on lipid fluidity in this system, as predicted from hydrodynamic theory. The same activation energy is observed for the calcium-dependent ATPase activity under physiological conditions, suggesting that protein rotational mobility (dependent on lipid fluidity) is involved in the rate-limiting step of active calcium transport.     

Docosahexaenoic Acid Is a Major n-3 Polyunsaturated Fatty Acid in Bovine Retinal Microvessels

Abstract: The aim of this study was to purify microvessels from bovine retina and also to cultivate bovine retinal endothelial cells (BRECs) or intramural pericytes, to determine their fatty acid composition. Microvessels were obtained after Dounce homogenization of the retina followed by centrifugation on albumin cushion and finally microvessels in the pellet were trapped on a 100-µm nylon filter. Contamination of microvessel preparations by neuronal tissue, assessed after both microscopic examination and western blotting with a monoclonal antibody raised against rhodopsin, was minor. In the entire bovine retina, docosahexaenoic acid (DHA) represented 23.3% of the total fatty acids and there was about three times less arachidonic acid (AA) (8.2%) than DHA. In contrast, DHA and AA levels were almost equivalent in the retinal microvessels with ∼10% of total fatty acids. When compared with intact microvessels, the DHA proportion of confluent monolayers of both BRECs or pericytes in primary cultures dropped to ∼2% of the total fatty acids, whereas AA was unchanged. Culture medium supplementation with unesterified DHA (10 µ M ) restored the DHA proportion of BRECs close to the microvascular value at the expense of linoleic acid without affecting AA very much. In contrast, DHA supplementation in pericytes increased the DHA proportion of these cells at the expense of AA. In conclusion, DHA of intact microvessels represented 10% of the total fatty acids, which was close to the AA proportion. Mild DHA supplementation of BRECs or pericytes in primary cultures restored their DHA proportion to the original microvessel value. This high percentage of polyunsaturated fatty acids in retinal microvessels should allow us to test the hypothesis that oxidation products derived from these fatty acids may be involved in the pathogenic process leading to diabetic retinopathy.     

Eicosapentaenoic acid and docosahexaenoic acid effects on tumour mitochondrial metabolism, acyl CoA metabolism and cell proliferation

In order to investigate the effects of high-fat diets rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), Wistar rats bearing subcutaneous implants of the Walker 256 tumour were fed pelleted chow containing low DHA/EPA or high DHA/EPA. The presence of n-3 polyunsaturated fatty acids (PUFAs) led to a marked suppression (35-46%) of tumour growth over a 12 day period. Both the whole tumour homogenate and the Percoll-purified mitochondrial fraction presented significant changes in fatty acid composition. The levels of EPA increased in both n-3 dietary groups while the levels of DHA increased only in the high DHA/EPA group, in comparison with the control chow-fed group. The presence of n-3 PUFAs led to an increase in mitochondrial acyl CoA synthetase activity, but neither the cytoplasmic acyl CoA content nor the n-3 fatty acid composition of the cytoplasmic acyl CoAs was altered by the diet. The content of thiobarbituric acid-reactive substances (TBARS) was increased in the low DHA/EPA group but was unchanged in the high DHA/EPA group. In vitro studies with the Walker 256 cell line showed a 46% decrease in cell growth in the presence of either EPA or DHA which was accompanied by a large decrease in the measured mitochondrial membrane potential. The TBARS content was increased only in the EPA-exposed cells. Cell cycle analysis identified a decrease in G0-G1 phase cells and an increase in G2-M phase cells and apoptotic cells, for both EPA and DHA-exposed cells. The data show that the presence of n-3 PUFAs in the diet is able to significantly after the growth rate of the Walker 256 tumour. The involvement of changes in mitochondrial membrane composition and membrane potential have been indicated for both EPA and DHA, while changes in lipid peroxidation have been identified in the presence of EPA but not of DHA.