Lipid fluidity of hepatocyte plasma membrane subfractions and their differential regulation by calcium

Rat hepatocyte plasma membranes were subfractionated by several methods into canalicular, sinusoidal and mixed contiguous plus sinusoidal membranes. Assessment of lipid fluidity by steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene and 12-(9-anthroyloxy)stearate indicates that the canalicular fraction is less fluid than the other membranes. Incubation with calcium decreases the fluidity of the sinusoidal and contiguous membranes by altering the lipid composition, an action which is not reversed by subsequent chelation of the cation. This effect of calcium is not observed in canalicular membranes.     

Calcium modulates the lipid dynamics of rat hepatocyte plasma membranes by direct and indirect mechanisms

Calcium ion decreases the motional freedom of lipid molecules in isolated rat hepatocyte plasma membranes and in sonicated dispersions (liposomes) of the membrane lipid. The decrease in lipid fluidity was monitored by estimation of the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. At least two processes are involved in the mode of action of the cation. The first is direct, i.e., observed on addition of calcium to the liposomes, relatively rapid, with a half-time of 10-15 at 37 degrees C, proportional to the calcium concentration in the range 0-4 mM, and readily reversed on addition of excess EDTA. The second mechanism is indirected and requires the presence of the membrane proteins. It occurs relatively slowly, with a half-time of 75 min at 37 degrees C, tends to plateau with a calcium half-saturation concentration of approximately 1 mM, is of greater magnitude than the direct effect, and cannot be reversed on chelation of calcium by EDTA. Moreover, the indirect effect is specific for Ca2+ as compared to other divalent cations and it results in changes in the lipid composition. Stimulation of phospholipase A activity is likely but does not account for the change in fluidity. The direct action of calcium is ascribed to binding to the lipid bilayer, whereas the indirect action probably results from modulation of membrane-bound enzymes which can alter the lipid composition. The effects of calcium on the membrane lipid fluidity may underly certain of its regulatory actions on membrane functions.     

The effects of cell proliferation on the lipid composition and fluidity of hepatocyte plasma membranes.

The fluorescence probe, 1,6-diphenyl-1,3,5-hexatriene, has been used to investigate the effects of controlled and uncontrolled growth on the dynamic properties of the lipid regions of hepatocyte plasma membranes. DPH was incubated with plasma membranes derived from quiescent and regenerating liver and Morris hepatoma 7777, and the resulting systems were studied by fluorescence polarization spectroscopy. Membranes from the rapidly growing hepatoma exhibited a significantly lower fluorescence polarization than observed in quiescent liver, suggesting the presence of a more fluid membrane lipid domain. Membranes from regenerating liver exhibited a time-dependent increase in membrane fluidity, reaching a maximum 12 h after growth stimulation. A close correspondence between membrane fluidity and the cholesterol-phospholipid ratio was also observed where a decrease in this ratio resulted in a more fluid lipid matrix. These results suggest that cell cycling, as observed in regenerating liver and Morris hepatoma 7777, results in significant increases in membrane fluidity, a property which may play an important regulatory role in various cell functions.     

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.     

Studies on regenerating liver and hepatoma plasma membranes--II. Membrane fluidity and enzyme activity

1. Rat hepatocyte plasma membranes isolated from Morris hepatoma 7288C, normal and regenerating liver were labelled with the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. 2. Steady-state fluorescence polarisation measurements indicated an increased fluidity of the membranes in the early stages of regeneration, returning to normal levels after 48 hr. 3. There was a decrease in hepatoma plasma membrane fluidity compared to normal hepatocytes. Changes in fluorescence polarisation with temperature (Arrhenius studies) indicate an increase in the lower critical temperature for the membrane lipid thermotropic transition of hepatoma compared to normal liver plasma membranes. 4. These changes in membrane lipid fluidity alter the activation of some intrinsic and extrinsic membrane bound enzymes.     

Fluidity and lipid composition of oat and rye shoot plasma membrane: effect of sterol perturbation by xenobiotics

Oat and rye plants were treated with either tetcyclacis (an experimental plant growth regulator), nuarimol (a fungicide) or gamma-ketotriazole (an experimental herbicide). These treatments reduced shoot growth and changed the lipid composition of the shoot plasma membranes. In oat, both tetcyclacis and nuarimol treatments increased plasma membrane cholesterol and increased the phosphatidylethanolamine/phosphatidylcholine (PE/PC) ratio, whereas gamma-ketotriazole treatment reduced cholesterol and the PE/PC ratio. In rye, all treatments reduced the PE/PC ratio. Generally, the sterol/phospholipid ratio was less in oat than in rye but the cholesterol/phospholipid ratio was greater. With all treatments in oat and rye, increases were observed in unsaturation of the phospholipid acyl chains. The fluidity of membranes was measured by steady-state fluorescence polarisation of the probe diphenylhexatriene; oat membranes were more fluid than rye. Membrane fluidity was greater in plasma membranes from plants treated with the xenobiotics than the controls. The results are discussed in the context of the effect of plasma membrane lipid composition on membrane fluidity, and it is concluded that there appears to be no overall simple relationship between membrane lipid composition and fluidity that holds for all treatments in both species.     

Cultured human skin fibroblasts modify their plasma membrane lipid composition and fluidity according to growth temperature suggesting homeoviscous adaptation at hypothermic (30 degrees C) but not at hyperthermic (40 degrees C) temperatures.

Mammalian cell metabolism is responding to changes in temperature. Body temperature is regulated around 37 degrees C, but temperatures of exposed skin areas may vary between 20 degrees C and 40 degrees C for extended periods of time without apparent disturbance of adequate cellular functions. Cellular membrane functions are depending from temperatures but also from their lipid environment, which is a major component of membrane fluidity. Temperature-induced changes of membrane fluidity may be counterbalanced by adaptive modification of membrane lipids. Temperature-dependent changes of whole cell- and of purified membrane lipids and possible homeoviscous adaptation of membrane fluidity have been studied in human skin fibroblasts cultured at 30 degrees C, 37 degrees C, and 40 degrees C for ten days. Membrane anisotropy was measured by polarized fluorescence spectroscopy using TMA-DPH for superficial and DPH for deeper membrane layers. Human fibroblasts were able to adapt themselves to hypothermic temperatures (30 degrees C) by modifying the fluidity of the deeper apolar regions of the plasma membranes as reported by changes of fluorescence anisotropy due to appropriate changes of their plasma membrane lipid composition. This could not be shown for the whole cells. At 40 degrees C growth temperature, adaptive changes of the membrane lipid composition, except for some changes in fatty acid compositions, were not seen. Independent from the changes of the membrane lipid composition, the fluorescence anisotropy of the more superficial membrane layers (TMA-DPH) increased in cells growing at 30 degrees C and decreased in cells growing at 40 degrees C.     

Erythrocyte membrane fluidity and changes in plasma lipid composition: a possible relationship in childhood obesity

We have studied plasma lipid patterns and erythrocyte membrane fluidity in 60 obese children and 20 normal children. Plasma levels of total cholesterol and associated low-density lipoproteins were significantly increased in 20 obese patients with respect to controls. A significant decrease in membrane fluidity, measured as an increase in the fluorescence polarization value of the probe 1,6-diphenyl-1,3,5-hexatriene, associated with an increase in the cholesterol/protein ratio has been shown in obese patients. The study of the correlation between erythrocyte membrane fluidity and plasma cholesterol has indicated that significant changes in fluidity and membrane lipid composition also occur in erythrocytes of obese patients with normal plasma lipid levels. These findings confirm that the erythrocyte membrane responds very early to modifications of plasma lipoproteins and suggest that in childhood obesity a modified transfer of cholesterol from plasma to erythrocyte membrane may take place.     

Isolation of Raja erinacea basolateral liver plasma membranes: characterization of lipid composition and fluidity.

Developing a method for isolating skate (Raja erinacea) basolateral liver plasma membranes, as well as characterizing the lipid composition and fluidity of these membranes, was the primary purpose of this study. Membranes were isolated using self-generating Percoll gradients. Marker enzyme studies indicate that this preparation is highly enriched in the basolateral domain of the liver plasma membrane and largely free of contamination by intracellular organelles or canalicular membranes. Further, these membranes contain the agency responsible for Na(+)-dependent alanine transport. This finding indicates that this membrane preparation will be useful for the study of skate liver plasma membrane transport processes. The lipid composition and fluidity (as assessed by the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene) of the skate basolateral liver plasma membrane shows little variation among preparations. Further, DPH anisotropy plotted as a function of temperature yields a straight line (r = 0.99) which indicates that there is no lipid phase change in these membranes from 4 degrees to 37 degrees C. The membrane preparation does contain substantial phospholipase A2 activity. The function of this enzyme is, in part, to modify membrane lipid composition and fluidity in response to temperature variations; therefore, this finding suggests that in situ lipid metabolizing enzymes may play a central role in the adaptation of skate basolateral liver plasma membranes to changes in the ambient temperature.     

SCP-2/SCP-x gene ablation alters lipid raft domains in primary cultured mouse hepatocytes

Although reverse cholesterol transport from peripheral cell types is mediated through plasma membrane microdomains termed lipid rafts, almost nothing is known regarding the existence, protein/lipid composition, or structure of these putative domains in liver hepatocytes, cells responsible for the net removal of cholesterol from the body. Lipid rafts purified from hepatocyte plasma membranes by a nondetergent affinity chromatography method were: i) present at 33 +/- 3% of total plasma membrane protein; ii) enriched in key proteins of the reverse cholesterol pathway [scavenger receptor class B type I (SR-B1), ABCA1, P-glycoprotein (P-gp), sterol carrier protein-2 (SCP-2)]; iii) devoid of caveolin-1; iv) enriched in cholesterol, sphingomyelin, GM1, and phospholipids low in polyunsaturated fatty acid and double bond index; and v) exhibited an intermediate liquid-ordered lipid phase with significant transbilayer fluidity gradient. Ablation of the gene encoding SCP-2 significantly altered lipid rafts to: i) increase the proportion of lipid rafts present, thereby increasing raft total content of ABCA1, P-gp, and SR-B1; ii) increase total phospholipids while decreasing GM1 in lipid rafts; iii) decrease the fluidity of lipid rafts, consistent with the increased intermediate liquid-ordered phase; and iv) abolish the lipid raft transbilayer fluidity gradient. Thus, despite the absence of caveolin-1 in liver hepatocytes, lipid rafts represented nearly one-third of the mouse hepatocyte plasma membrane proteins and displayed unique protein, lipid, and biophysical properties that were differentially regulated by SCP-2 expression.     

Membrane fluidity aspects in endocytosis; a study with the fluorescent probe trimethylamino-diphenylhexatriene in L929 cells

The fluorescent hydrophobic plasma membrane probe, trimethylamino-diphenylhexatriene (TMA-DPH) was previously shown to follow the plasma membrane throughout its internalization and recycling process and thus to behave as a marker for endo- and exocytosis in living cell systems. In this paper, we made use of these properties to investigate membrane fluidity effects associated with endocytosis in L929 cells. For that purpose we performed TMA-DPH fluorescence anisotrophy measurements which showed that endocytosis starts from particularly rigid regions of the plasma membrane (probably coated pits). The fluorescence anisotropy then continuously decreases to a lower limit corresponding to the membrane fluidity of the probe in the lysosomial membrane. Strikingly, the value of this limit is identical to the average anisotropy value in the peripheral membrane, which suggests that lysosomes and plasma membrane may have a similar phospholipidic composition and a possible common origin.     

Lipid organization regulates annexin A2 Ca(2+)-sensitivity for membrane bridging and its modulator effects on membrane fluidity

Annexin A2 (AnxA2) is a phospholipid binding protein that has been implicated in many membrane-related cellular functions. AnxA2 is able to bind different acidic phospholipids such as phosphatidylserine (PS) and phosphatidylinositol-4,5-bisphosphate (PI2P). This binding is mediated by Ca(2+)-dependent and Ca(2+)-independent mechanisms. The specific functions of annexin A2 related to these two phospholipids and the molecular mechanisms involved in their interaction remain obscure. Herein we studied the influence of lipid composition on the Ca(2+)-dependency of AnxA2-mediated membrane bridging and on membrane fluidity. Membrane models of ten different lipid compositions and detergent-resistant membranes from two cellular sources were investigated. The results show that the AnxA2-mediated membrane bridging requires 3 to 50 times less calcium for PS-membranes than for PI2P-membranes. Membrane fluidity was measured by the ratiometric fluorescence parameter generalized polarization method with two fluorescent probes. Compared to controls containing low phospholipid ligand, AnxA2 was found to reduce the membrane fluidity of PI2P-membranes twice as much as the PS-membranes in the presence of calcium. On the contrary, at mild acidic pH in the absence of calcium AnxA2 reduces the fluidity of the PS-membranes more than the PI2P-membranes. The presence of cholesterol on the bilayer reduced the AnxA2 capacity to reduce membrane fluidity. The presented data shed light on the specific roles of PI2P, PS and cholesterol present on membranes related to the action of annexin A2 as a membrane bridging molecule during exocytosis and endocytosis events and as a plasma membrane domain phospholipid packing regulator.     

Effect of calcium ions on plasma membrane structure

The structure of thymocyte's plasma membranes was studied as affected by calcium ions (0-1.126 mM). The fluorescence intensity of 1-anilinonaphthalene-8-sulphonate, the epimerization degree of pyrene and fluorescence anisotropy of membrane proteins were studied. The change of electrochemical properties of membranes, conformation of membrane proteins and lipid fluidity has been shown.     

Ordering of bulk membrane lipid or protein promotes activity of plasma membrane Mg2+ATPase

Treatment of liver plasma membranes with phospholipase A2 or high doses of concanavalin A enhances the activity of Mg2+ATPase assayed at temperatures greater than 30 degrees C. The effects of the two treatments are not additive. Both the removal of phospholipids and binding of the lectin increase the degree of polarization of fluorescence of the lipid-soluble fluorophores, diphenylhexatriene and beta-parinaric acid, suggesting that decreased lipid fluidity may activate Mg2+-ATPase. In fact modification of lipid fluidity by reconstitution of phospholipase-treated membranes with phosphatidylcholines of defined fatty acid composition or by addition of cis-vaccenic acid showed a strong inverse correlation between Mg2+ATPase activity and lipid fluidity as monitored by fluorescence polarization. However, despite the ability of concanavalin A to nonspecifically order membrane lipid, its effect on Mg2+ATPase is apparently not mediated in this manner because other enzyme-activating lectins such as Ricinus communis agglutinin and wheat germ agglutinin are without effect on lipid fluidity. The facts that lectins of lower valency than tetravalent native concanavalin A such as divalent succinyl concanavalin A are far less effective in activating the enzyme and that paraformaldehyde treatment also activates suggests that cross-linking of membrane proteins is responsible. Hence, the diminution in activity of this membrane enzyme due to the disordering effect of heat in the physiological temperature range can be counteracted by isothermally increasing the order of either membrane lipid or protein.     

Effect of membrane fatty acyl composition on LDL metabolism in Hep G2 hepatocytes

The mechanism by which dietary cis-unsaturated fatty acids lower plasma levels of low-density lipoprotein (LDL) cholesterol is unknown. Since plasma membrane incorporation of dietary cis-unsaturated fatty acids is known to alter the function of plasma membrane associated proteins, perhaps by increasing membrane fluidity, we examined LDL receptor function in Hep G2 hepatocytes that were unmodified, enriched with the cis-unsaturated fatty acids oleate or linoleate, or enriched with the saturated fatty acids stearate or palmitate. Hepatocytes enriched in cis-unsaturated fatty acids exhibited augmented LDL binding, uptake, and degradation in comparison to unmodified cells. In contrast, Hep G2 hepatocytes enriched in saturated fatty acids had decreased LDL binding, uptake, and degradation. Enrichment with oleate or linoleate resulted in a decrease in the calculated fatty acyl mole-weighted melting point of the plasma membrane and an increase in plasma membrane fluidity, as measured by the steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene incorporated into the plasma membrane. Conversely, stearate or palmitate enrichment resulted in an increased plasma membrane fatty acyl mole-weighted melting point and decreased plasma membrane fluidity. LDL binding, uptake, and degradation varied with plasma membrane fluidity in a highly correlated manner. Thus, one mechanism by which dietary cis-unsaturated fatty acids lower LDL cholesterol may possibly involve an alteration in membrane lipid composition or membrane fluidity that promotes enhanced LDL receptor function, thereby leading to increased hepatic clearance of LDL.     

Modulation of Na+-H+ exchange by ethinyl estradiol in rat colonic brush-border membrane vesicles

Prior studies by our laboratory have suggested that a relationship may exist between rat colonic brush-border membrane vesicular fluidity and Na+-H+ exchange. To further explore this possible relationship, in the present studies the effects of ethinyl estradiol (17 alpha-ethinyl-1,3,5-estratriene-3,17-beta-diol) administration subcutaneously (5 mg/kg body wt. per day) for 5 days, on rat colonic brush-border membrane fluidity and Na+-H+ exchange were examined. This treatment regimen has previously been shown to decrease the lipid fluidity of rat hepatic and rabbit small intestinal plasma membranes. In agreement with these prior studies, the present results demonstrate that this agent decreases the lipid fluidity of treated-rat colonic brush-border membranes compared to control membranes, as assessed by steady-state fluorescence polarization techniques using three different fluorophores. An increase in the cholesterol content and cholesterol/phospholipid molar ratio of treated-membranes appear to, at least partially, be responsible for the fluidity differences. Furthermore, examination of the kinetic parameters for amiloride-sensitive sodium-stimulated proton efflux in treated and control membrane vesicles, utilizing the pH-sensitive fluorescent dye, Acridine orange, revealed that ethinyl estradiol administration decreased the Vmax for this exchange mechanism, expressed in arbitrary fluorescence units, by approx. 25% but did not influence its Km for sodium. These data, therefore, lend further support to the contention that alterations in fluidity may modulate Na+-H+ exchange in rat colonic brush-border membrane vesicles.     

The influence of microtubule integrity on plasma membrane fluidity in L929 cells

The aim of this work was to examine the possible influence of the integrity of the microtubule network on the plasma membrane fluidity of L929 mouse fibroblasts. The L929 cell line was selected for the ease of culture and the stability of its characteristics. The cells were treated with colchicine, nocodazole and vinblastine, three microtubule-depolymerizing drugs, at various concentrations and for various times. Membrane fluidity was assessed from fluorescence depolarization measurements with the plasma membrane probe TMA-DPH. Each of the drugs induced a significant, dose-dependent decrease in fluorescence anisotropy. The effect levelled off (5-7% decrease) after approximately 90 min of treatment, and could be unambiguously interpreted as resulting from an increase in membrane fluidity. The cumulative action of the drugs did not significantly increase the effect. The effects of colchicine and nocodazole could be reversed by incubation in drug-free medium, but not that of vinblastine. The results are discussed in correlation with the kinetics of the three drugs interaction with tubulin or microtubules. It is concluded that the microtubule integrity contributed to the high plasma membrane lipidic order, but less than other factors, like the lipid composition and the cholesterol content.     

The influence of microtubule integrity on plasma membrane fluidity in L929 cells

The aim of this work was to examine the possible influence of the integrity of the microtubule network on the plasma membrane fluidity of L929 mouse fibroblasts. The L929 cell line was selected for the ease of culture and the stability of its characteristics. The cells were treated with colchicine, nocodazole and vinblastine, three microtubule-depolymerizing drugs, at various concentrations and for various times. Membrane fluidity was assessed from fluorescence depolarization measurements with the plasma membrane probe TMA-DPH. Each of the drugs induced a significant, dose-dependent decrease in fluorescence anisotropy. The effect levelled off (5-7% decrease) after ~ 90 min of treatment, and could be unambiguously interpreted as resulting from an increase in membrane fluidity. The cumulative action of the drugs did not significantly increase the effect. The effects of colchicine and nocodazole could be reversed by incubation in drug-free medium, but not that of vinblastine. The results are discussed in correlation with the kinetics of the three drugs interaction with tubulin or microtubules. It is concluded that the microtubule integrity contributed to the high plasma membrane lipidic order, but less than other factors, like the lipid composition and the cholesterol content.     

Regional differences in the lipid composition and fluidity of rat colonic brush-border membranes

The lipid composition and fluidity of brush-border membranes prepared from rat proximal and distal colonocytes were determined. Fluidity, as assessed by steady-state fluorescence polarization techniques using the fluorophores 1,6-diphenyl-1,3,5-hexatriene, DL-2(9-anthroyl)stearic acid and DL-12(9-anthroyl)stearic acid, was decreased in distal compared to proximal plasma membranes. This pattern was similar to that previously described for both antipodal plasma membranes in rat enterocytes of the small intestine. The decrease in fluidity of the distal as compared to the proximal membranes resulted from an increase in cholesterol content, cholesterol/phospholipid molar ratio and degree of saturation of the fatty acid residues in the distal membranes. The specific activities of total alkaline phosphatase and cysteine-sensitive alkaline phosphatase, enzymes previously shown to be functionally dependent on the physical state of the colonic brush-border membrane's lipid, were also significantly lower in distal as compared to proximal clonic plasma membranes. These studies, therefore, demonstrate that differences in the lipid fluidity, lipid composition and certain enzymatic activities exist in brush-border membranes prepared from rat proximal and distal colonocytes. The regional variation in rat colonic luminal membrane lipid fluidity and composition may, at least partially, be responsible for differences in these enzymatic activities as well as in sodium and water absorption along the length of this organ.     

Influence of cholesterol content on red cell membrane viscoelasticity and fluidity.

The purpose of this investigation was to correlate the viscoelastic properties and lipid fluidity of the red blood cell membrane to its lipid composition. The viscoelastic properties of human red cells that had been enriched or depleted in cholesterol were determined by the micropipette technique. The lipid fluidity of the outer and inner leaflets of the erythrocyte membrane was concurrently assessed by steady state fluorescence depolarization. The elastic modulus and the viscosity moduli of the erythrocyte membrane showed no significant differences between the cholesterol-modified and the control cells. Cholesterol enrichment decreased the lipid fluidity of the outer membrane leaflet alone, and cholesterol depletion increased the fluidity mainly of the inner leaflet.