Bulk Lipid Fluidity Research Articles

Abstract 16909: Eicosapentaenoic Acid and Docosahexaenoic Acid Have Competing Effects on Membrane Lipid Dynamics Due to Differences in Structure

Background: Omega-3 fatty acids (n3-FAs) incorporate into vascular cell membranes where they modulate protein function and signal transduction. The n3-FA eicosapentaenoic acid (EPA) delivered in ethyl ester form (icosapent ethyl) reduced a broad range of cardiovascular events in high-risk patients (REDUCE-IT). This was not observed with mixed n3-FA formulations containing docosahexaenoic acid (DHA) in STRENGTH and other trials. These discordant outcomes indicate DHA may counter certain EPA effects due to its additional carbons and double bond. We compared these n3-FA effects on membrane lipid dynamics and width, key determinants of protein function. Methods: Membrane lipid fluidity was measured by fluorescence anisotropy approaches in large unilamellar vesicles (LUVs) of 100 nm diameter containing 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC) and 50 mol% cholesterol with EPA and/or DHA (0-10 mol%). LUVs were treated with the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), at 1 mol% and evaluated using a spectrofluorometer with a polarization accessory. Membrane width ( i.e ., unit cell periodicity) of the samples was determined by small angle x-ray scattering (SAXS). Results: DHA alone increased bulk lipid fluidity as indicated by a dose-dependent reduction in apparent rotational correlation time (ARCT); at 10 mol%, there was a ~20% (p<0.05) reduction from ~19 to ~16 nsec compared to control (untreated membranes). By contrast, EPA had no significant disordering effect except when combined at equimolar levels; the EPA/DHA combination showed a reduction in ARCT to ~18 nsec at 10 mol% that was different from EPA alone (p<0.05). Consistent with these findings, SAXS analysis showed DHA containing membranes had a reduced width compared to EPA by 5% or 3 Å (p<0.05) due to increased trans-gauche isomerizations in the phospholipid acyl chains. Conclusion: EPA maintained membrane fluidity while DHA increased fluidity and thereby reduced membrane width due to its additional carbons and double bond. The EPA/DHA combination increased membrane fluidity compared to control and EPA alone. The disordering effects of DHA may counter EPA stabilizing effects and explain discordant clinical outcomes with different n3-FA formulations.

Amyloid beta-peptides increase annular and bulk fluidity and induce lipid peroxidation in brain synaptic plasma membranes.

Amyloid beta-peptides (A beta) may alter the neuronal membrane lipid environment by changing fluidity and inducing free radical lipid peroxidation. The effects of A beta(1-40) and A beta(25-35) on the fluidity of lipids adjacent to proteins (annular fluidity), bulk lipid fluidity, and lipid peroxidation were determined in rat synaptic plasma membranes (SPM). A fluorescent method based on radiationless energy transfer from tryptophan of SPM proteins to pyrene and pyrene monomer-eximer formation was used to determine SPM annular fluidity and bulk fluidity, respectively. Lipid peroxidation was determined by the thiobarbituric acid assay. Annular fluidity and bulk fluidity of SPM were increased significantly (p < or = 0.02) by A beta(1-40). Similar effects on fluidity were observed for A beta(25-35) (P < or = 0.002). Increased fluidity was associated with lipid peroxidation. Both A beta peptides significantly increased (p < or = 0.006) the amount of malondialdehyde in SPM. The addition of a water-soluble analogue of vitamin E (Trolox) inhibited effects of A beta on lipid peroxidation and fluidity in SPM. The fluidizing action of A beta peptides on SPM may be due to the induction of lipid peroxidation by those peptides. A beta-induced changes in neuronal function, such as ion flux and enzyme activity, that have been reported previously may result from the combined effects of lipid peroxidation and increased membrane fluidity.

Changes in plasma membrane fluidity of Bryonia dioica internodes during thigmomorphogenesis

Fluidity changes in plasma membrane (PM) lipid extracts or native membranes isolated from Bryonia dioica internodes after a mechanical stimulation were monitored by steady-state fluorescence polarization with 1,6-diphenyl-1,3,5-hexatriene as a probe. The signal was shown to rapidly induce an increase in the bulk lipid fluidity. This event was closely related to a relative enrichment in some phospholipid species (PC, PG and PS) as well as a significant increase in the unsaturation index of total fatty acyl chains. Free sterols and protein content did not appear to be involved into this process. After 48 h, lipids from rubbed internodes became less fluid than PM lipids from control internodes.

Halothane regulates G-protein-dependent phospholipase C activity in turkey erythrocyte membranes

The ability of halothane to stimulate phospholipase C (PLC) was examined in turkey erythrocyte membranes prepared from [3H]inositol-labeled turkey erythrocytes by measuring [3H]inositol phosphate formation ([3H]InsP) in the presence and absence of G-protein activation. In the presence of guanosine 5'-3-O-(thio)triphosphate) (GTP gamma S), halothane (0.5-10 mM) caused a dose-dependent activation of PLC. The EC50 value for halothane-induced PLC activation was 2.8 +/- 0.3 mM. Halothane (0.1-30 mM) had no effect on PLC activity in the absence of G-protein activation and did not affect Ca(2+)-dependent PLC activity. The activation of PLC by GTP gamma S occurred after an initial lag period of 60 s which was followed by a linear increase in [3H]InsP. Halothane dose-dependently decreased the lag period for GTP gamma S-induced PLC activation (minimal value 15 s) and increased the rate of [3H]InsP formation at all time points following this lag. As a result, halothane shifted the EC50 value for GTP gamma S-induced PLC activation to the left (4-fold) and increased its maximal response. Halothane also caused a dose-dependent activation of PLC in the presence of AlF4-. Half-maximal stimulation of AlF4(-)-activated PLC occurred with an EC50 value of 2.9 +/- 0.4 mM halothane, which is similar to the halothane dose giving half-maximal stimulation of PLC in the presence of GTP gamma S. At low doses (0.1-0.3 mM) halothane inhibited both isoproterenol- and adenosine 5'-O-(2-thiodiphosphate) (ADP beta S)-induced [3H]InsP formation, whereas at higher concentrations it stimulated PLC independent of the presence of these agonists. At concentrations chosen to reflect their different membrane/buffer partition coefficients, both hexanol (5 mM) and benzyl alcohol (20 mM) fluidized turkey erythrocyte membranes to the same degree as halothane (5 mM). However, these agents had no effect on GTP gamma S- or AlF(4-)-induced PLC activity, indicating that halothane-induced PLC activation was not secondary to changes in bulk lipid fluidity properties. Halothane also stimulated [3H]inositol bisphosphate and [3H]inositol trisphosphate formation in intact erythrocytes. These data demonstrate that the anesthetic halothane can stimulate G-protein-dependent PLC activity and modify the responsiveness of this signaling system to activation by receptor-linked agonists.

Membrane deterioration during postharvest senescence of broccoli florets: modulation by temperature and controlled atmosphere storage

Abstract When freshly cut heads of broccoli ( Brassica oleracea L.) are stored in air at 23°C or 10°C, the florets rapidly senesce. Chlorophyll levels decline by 80–90% within 4 days at 23°C and within 10 days at 10°C, and under the same storage conditions microsomal phospholipid phosphate and fatty acids in the florets decline by 70–90%. The breakdown of membrane phospholipids is accompanied by a large decrease (… 35%) in bulk lipid fluidity of the membranes. These changes are temperature-dependent inasmuch as they are completely inhibited during storage for comparable periods in air at 5°C. Also, the potential of microsomal membranes isolated from florets to degrade exogenous, radiolabelled phosphatidylcholine increases during storage in air at 23°C or 10°C, but not at 5°C. Storage at 5°C or 10°C in a controlled atmosphere (5% CO 2 , 3% O 2 , 92% N 2 ) at … 80% relative humidity strongly inhibits loss of chlorophyll and the change in microsomal bulk lipid fluidity reflecting phospholipid breakdown. Thus reduced integrity of thylakoids and membrane phospholipid degradation appear to be inherent features of broccoli floret senescence that are sensitive, directly or indirectly, to ambient CO 2 and O 2 concentrations. These observations confirm the potential of using controlled atmospheres to enhance the storability of broccoli and identify physiological and biochemical parameters of floret senescence that can be used to establish optimum controlled atmosphere conditions.

Evaluation of age-related changes of physicochemical properties and functional activity of rat adipose plasma membranes and their possible relationship

Studies were carried out to evaluate structural state of adipose plasma membranes (PM) from mature adult and aged Wistar rats and its possible relation to functional changes in aging. PM lipids were probed by fluorescence of 1,6-diphenyl-1,3,5-hexatriene (DPH) and pyrene. The data on DPH anisotropy, pyrene excimerization and induction resonance energy transfer (IRET) from PM proteins to pyrene provided the evidence for age-dependent decrease in PM lipid phase fluidity inclusive of that of annular lipid. Interaction between PM lipids and integral proteins also changed in aging. The observed structural changes were due to age-related lipid compositional modification. Evidence for this conclusion was provided by the following data: (i) DPH anisotropy was increased in aging both in PM and liposomes from the same PM lipids; (ii) both saturation/unsaturation fatty acid ratio and relative content of phosphatidylethanolamine (PE) increased in aged PM, whereas cholesterol/phospholipid and lipid/protein ratios were age-independent; (iii) PM polypeptide composition remained practically unchanged during aging. Age-related changes of the PM structure were accompanied by a twofold decrease of high affinity insulin binding sites and elevation of their affinity to insulin. It was suggested that age-related changes of physicochemical properties of PM lipid phase might affect protein molecular conformation and function through either changes in bulk lipid fluidity or through specific lipid-protein interactions.

Alterations in membrane protein conformation in response to senescence-related changes

Measurements of diphenylhexatriene fluorescence polarization in smooth microsomal membranes isolated from senescing bean cotyledons have indicated that membrane bulk lipid fluidity decreases by ca two-fold as the cotyledons age. This change in fluidity can be largely attributed to an increase in relative sterol concentration (from ca 7 to ca 45 mol% with respect to phospholipid) and is accompanied by a change in the average conformation of membrane proteins detectable by spin labelling with 3-maleimido-PROXYL. When the sterol concentration of smooth microsomal membranes from young cotyledons was increased in vitro by treatment with cholesteryl hemisuccinate, the membrane lipid fluidity was reduced to a value comparable to that for corresponding membranes from senescent cotyledons. The cholesteryl hemisuccinate treatment also induced conformational changes in the membrane proteins, but did not alter protein composition as shown by SDS-PAGE. The observations collectively indicate that the sterol-induced decrease in bulk lipid fluidity of smooth microsomal membranes accompanying senescence is of sufficient magnitude to alter membrane protein conformation. These changes in protein conformation may also reflect sterol-induced protein aggregation, which is known to occur at relative sterol concentrations in excess of ca 27 mol% with respect to phospholipid.

Extrahepatic Cell Membrane Lipid Abnormalities and Cellular Dysfunction in Liver Disease

Liver disease is associated with characteristic changes in the lipid composition of the surface coat of plasma lipoprotein particles. Cholesterol and lecithin molecules accumulate as hepatic secretion of lecithin-cholesterol acyltransferase decreases, and the arachidonate content, the precursor for eicosanoid production, is also reduced. By exchange and equilibration processes, such abnormal circulating lipoproteins should tend to induce corresponding changes in cell membrane lipid composition; studies in both human and experimental liver disease confirm that this does occur and that it is wide-spread. The correct functioning of membrane proteins, which serve as receptors or are responsible for enzymatic and transport processes, is most commonly dependent on the fluidity of their lipid bilayer matrix. Because cholesterol enrichment of biomembranes reduces bulk lipid fluidity, it can be predicted that extrahepatic membrane dysfunction might be a general feature of severe liver disease. This concept is supported by increasing experimental evidence and, as a consequence, it is proposed that many of the cellular disturbances and metabolic abnormalities accompanying hepatic disease result from, or are exacerbated by, lipoprotein-induced changes in membrane lipid composition and function. Importantly, this mechanism also suggests that drugs which can fluidise membranes, such as S-adenosyl-L-methionine (SAMe), might help ameliorate cellular dysfunction.

Heinz bodies do not modify the membrane characteristics of common marmoset (Callithrix jacchus) erythrocytes

The possibility was examined that the membrane function of erythrocytes obtained from healthy common marmosets (Callithrix jacchus) was modified by the presence in the cells of Heinz bodies. No significant differences were found in erythrocyte endogenous free malonyl dialdehyde (MDA) or reduced glutathione (GSH) between normal human erythrocytes and marmoset erythrocytes containing Heinz bodies. Membrane fluorescent chromolipids, surface charge and thiol levels were similar in both species but average membrane bulk lipid fluidity was slightly elevated in the marmosets. It was concluded that, in contrast to the situation in human erythrocytes, the presence of Heinz bodies in red cells of marmosets does not adversely affect the properties of the membrane.

Biochemical localization of hepatic surface-membrane Na+,K+-ATPase activity depends on membrane lipid fluidity.

Membrane proteins of transporting epithelia are often distributed between apical and basolateral surfaces to produce a functionally polarized cell. The distribution of Na+,K+-ATPase [ATP phosphohydrolase (Na+/K+-transporting), EC 3.6.1.37] between apical and basolateral membranes of hepatocytes has been controversial. Because Na+,K+-ATPase activity is fluidity dependent and the physiochemical properties of the apical membrane reduces its fluidity, we investigated whether altering membrane fluidity might uncover cryptic Na+,K+-ATPase in bile canalicular (apical) surface fractions free of detectable Na+,K+-ATPase and glucagon-stimulated adenylate cyclase activities. Apical fractions exhibited higher diphenylhexatriene-fluorescence polarization values when compared with sinusoidal (basolateral) membrane fractions. When 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate (A2C) was added to each fraction, Na+,K+-ATPase, but not glucagon-stimulated adenylate cyclase activity, was activated in the apical fraction. In contrast, further activation of both enzymes was not seen in sinusoidal fractions. The A2C-induced increase in apical Na+,K+-ATPase approached 75% of the sinusoidal level. Parallel increases in apical Na+,K+-ATPase were produced by benzyl alcohol and Triton WR-1339. All three fluidizing agents decreased the order component of membrane fluidity. Na+,K+-ATPase activity in each subfraction was identically inhibited by the monoclonal antibody 9-A5, a specific inhibitor of this enzyme. These findings suggest that hepatic Na+,K+-ATPase is distributed in both surface membranes but functions more efficiently and, perhaps, specifically in the sinusoidal membranes because of their higher bulk lipid fluidity.

Membrane Deterioration in Senescing Carnation Flowers

The lipid fluidity of microsomal membranes from the petals of cut carnation flowers decreases as the flowers senesce. A comparable change in fluidity was induced by in vitro aging of microsomal membranes from young flowers under conditions in which membranous lipoxygenase-like activity was active. There was no change in fluidity when the membranes were aged in the presence of inhibitors of lipoxygenase or were heat-denatured prior to aging. Membranes from naturally senesced flowers and membranes that had been aged in vitro both sustained an increase in saturated:unsaturated fatty acid ratio that accounted for the decrease in lipid fluidity, and in both instances there was evidence for depletion of the unsaturated fatty acids, linoleic acid, and linolenic acid, which are substrates for lipoxygenase. Loss of lipid phosphate reflecting breakdown of membrane phospholipids preceded the depletion of unsaturated fatty acids attributable to the lipoxygenase-like activity. The data have been interpreted as indicating that fatty acid substrates for membrane-associated lipoxygenase-like activity are made available by the initiation of phospholipid degradation, and that the utilization of these substrates results in a selective depletion of unsaturated fatty acids from the membrane and an ensuing decrease in bulk lipid fluidity.

Pentachlorophenol alters the molecular organization of membranes in mammalian cells.

Biophysical measurements of motion parameters for fluorescent and paramagnetic probes partitioned into membranes of mammalian cells have indicated that the lipid bilayers of the membranes become destabilized as a result of treatment with sublethal doses of pentachlorophenol. Decreases in bulk lipid fluidity of up to 50%, which can be attributed to perturbation of the bilayer by the toxicant, were observed for purified plasma membrane and for preparations of total cell membrane. Spin labels that probe the hydrophobic interior and headgroup regions, respectively, of bilayers indicated that pentachlorophenol does not assume an amphipathic orientation in the membrane, but rather it partitions into the hydrophobic interior of the bilayer. Arrhenius plots of motion parameters were continuously linear over temperatures ranging from 20 to 55°C for both treated and control cells, indicating that pentachlorophenol does not induce lipid phase transitions. Treatment with sublethal doses of pentachlorophenol also induced a decline in levels of phospholipid phosphate per cell that can be attributed to a selective action of phospholipase C. Notwithstanding these changes, the cells remained viable judging from their ability to exclude trypan blue and thus the deleterious effects of pentachlorophenol on membranes can be interpreted as sublethal damage.

Membrane lipid composition and cellular function.

Membrane fatty acid composition, phospholipid composition, and cholesterol content can be modified in many different kinds of intact mammalian cells. The modifications are extensive enough to alter membrane fluidity and affect a number of cellular functions, including carrier-mediated transport, the properties of certain membrane-bound enzymes, binding to the insulin and opiate receptors, phagocytosis, endocytosis, depolarization-dependent exocytosis, immunologic and chemotherapeutic cytotoxicity, prostaglandin production, and cell growth. The effects of lipid modification on cellular function are very complex. They often vary from one type of cell to another, and they do not exert a uniform effect on all processes in a single cell line. Therefore, it is not yet possible to make any generalizations or to predict how a given system will respond to a particular type of lipid modification. Many of the functional responses probably are caused directly by the membrane lipid structural changes, which affect either bulk lipid fluidity or specific lipid domains. The conformation or quaternary structures of certain transporters, receptors, and enzymes probably are sensitive to changes in the structure of their lipid microenvironment, leading to changes in activity. Prostaglandin production is modulated by the availability of substrate fatty acids stored in the membrane phospholipids, but the underlying chemical mechanism still involves a change in membrane lipid structure. While this is the most likely mechanism, the possibility that the membrane lipid compositional change is an independent event that occurs concurrently but is not causally related to the functional perturbations also must be considered.

Role of bulk lipid fluidity in the thermal adaptation of Dictyostelium discoideum thermotaxis

Thermotactic responses of Dictyostelium discoideum, strain AX-2, were examined and found to be similar to those of strain NC-4. These responses, like those of strain NC-4, were dependent on the temperature at which the amoebae grew and developed into slugs. The lipid fluidity of strain AX-2 was found to be independent of development temperature. Consequently, the hypothesis that a change in bulk lipid fluidity is the mechanism for the adaptation of D. discoideum thermotaxis appears to be untenable.

Alterations in erythrocyte membrane fluidity by phenylhydrazine-induced peroxidation of lipids

The addition of phenylhydrazine (.05 – 0.5 mM) to hemoglobin-free human erythrocyte membranes results in the peroxidation of endogenous phospholipids as measured by the thiobarbaturic acid reaction. The incorporation of 1,6-diphenyl-1,3-5-hexatriene into these membranes revealed a decrease in bulk lipid fluidity. Additionally, the fluorescence intensity of 1-anilino-8-naphthalene sulfonate was decreased and red-shifted after phenylhydrazine treatment of the membranes. The results obtained are consistent with the view that changes in the physical state of plasma membranes subsequent to the peroxidation of membrane lipids may be a determinant of the mechanical properties of drug-treated as well as aging cells.

Isolation and characterization of the plasma membranes of cultured lymphoblasts from patients with cystic fibrosis and normal individuals

Plasma membranes were isolated from two cystic fibrosis lymphoblastoid cell lines and two age and sex-matched normal cell lines after controlled cell disruption. The preparations were enriched approx. 20-fold in plasma membrane markers and were essentially free of markers for other organelles except the Golgi-localized enzyme, galactosyltransferase. Sodium dodecyl sulphate polyacrylamide gel electrophoresis showed virtually identical polypeptide profiles after staining with Coomassie blue. Membranes from normal cells contained 0.49–0.67 μmol of total phospholipid/mg membrane protein and those from cystic fibrosis cells, 0.53–0.67 μmol. The cholesterol content was 0.35–0.41 μmol/mg protein for normal, and 0.43–0.49 μmol for cystic fibrosis cell membranes. The proportions of individual phospholipid classes and their fatty acid compositions as well as that of the total membrane lipid also did not differ significantly in the two cell types. The bulk lipid fluidity as reflected by the fluorescence polarization of β-parinaric acid and diphenylhexatriene was also not changed. The carbohydrate composition of cell surface glycopeptides released with papain was not consistently different with the normal and cystic fibrosis cells. The quantity of fucose in intact plasma membranes was also quantitated enzymatically and found to be very similar in both cell types.