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.     

A dietary regimen alters hepatocyte plasma membrane lipid fluidity and ameliorates ethinyl estradiol cholestasis in the rat

The beta-adrenergic receptor mediating the inhibition of sterol synthesis by catecholamines in freshly isolated human mononuclear leukocytes was defined pharmacologically by using selective beta 1- and beta 2-agonists and -antagonists. Incubation of cells for 6 h in a medium containing lipid-depleted serum resulted in a 3-fold increase in the incorporation of [14C]acetate or tritiated water into sterols. The beta-agonist (-)-isoproterenol was approximately equipotent with (-)-epinephrine and (-)-norepinephrine in suppressing sterol synthesis, yielding a sigmoidal log-dose-effect curve. Accordingly, the effects of the catecholamines were reversed by the beta-antagonist (+/-)-propranolol. The beta 2-agonists terbutaline and salbutamol inhibited sterol synthesis by 42 and 26%, respectively, at a concentration of 0.1 mmol/l. Contrary to that, the beta 1-agonists prenalterol and dobutamine had no effect. In accordance with the influence of the agonists, the beta 2-antagonist butoxamine, but not the beta 1-antagonists atenolol, metoprolol and practolol, reversed the catecholamine action on sterol synthesis. The results provide evidence that catecholamines may regulate sterol synthesis by stimulating beta 2-adrenergic receptors.     

Variations in dietary triacylglycerol saturation alter the lipid composition and fluidity of rat intestinal plasma membranes

Rats were maintained on nutritionally complete diets enriched in unsaturated (corn oil) or saturated (butter fat) triacylglycerols. After 6 weeks, significant differences in the lipid composition and fluidity of a number of intestinal membranes were observed. The corn oil diet (enriched mainly in linoleic acid) increased the overall unsaturation of the acyl chains and enhanced the lipid fluidity, as assessed by the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, of enterocyte microvillus and basolateral membranes and of colonocyte basolateral membranes. Concomitantly, the cholesterol content and the cholesterol/phospholipid molar ratio were increased in the microvillus but not in the basolateral membranes. The increased cholesterol in ileal microvillus membranes can result from enhanced cellular biosynthesis, since ileal slices from rats fed the unsaturated diet incorporated [14C]octanoate more rapidly into digitonin-precipitable sterol. Increased fluidity of the enterocyte microvillus and basolateral membranes, respectively, enhanced the enzyme specific activities of p-nitrophenylphosphatase and (Na+ + K+)-dependent adenosine triphosphatase. The results indicate that the lipid composition, fluidity and enzyme activities of intestinal plasma membranes can be altered by dietary means. Moreover, rat enterocytes possess regulatory mechanisms which modulate the cholesterol content of the microvillus membranes so as to mitigate changes in lipid fluidity.     

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.     

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.     

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.     

Dexamethasone-induced alterations in lipid composition and fluidity of rat proximal-small-intestinal brush-border membranes.

A series of experiments were conducted to examine the possible effects of subcutaneous administration of the synthetic glucocorticoid dexamethasone (100 micrograms/day per 100 g body wt.) on the lipid fluidity and lipid composition of rat proximal-small-intestinal brush-border membranes. After 4 days of treatment, membranes and their liposomes prepared from treated animals possessed a greater fluidity than did their control (diluent, 0.9% NaCl) counterparts, as assessed by steady-state fluorescence-polarization techniques using several different fluorophores. Examination of the effects of temperature on the anisotropy values of 1,6-diphenylhexa-1,3,5-triene, using Arrhenius plots, moreover, revealed that the mean break-point temperatures of the treated preparations were approx. 3-4 degrees C lower than those of their control-preparation counterparts. Changes in the sphingomyelin/phosphatidylcholine (PC) molar ratio as well as in certain of the fatty acids of the PC fraction of treated membranes, secondary to alterations in membrane PC levels and in lysophosphatidylcholine acyltransferase activities respectively, were also noted after dexamethasone administration. These compositional alterations appeared to be responsible, at least in part, for the differences in fluidity noted between treated and control plasma membranes. These results therefore demonstrate that dexamethasone administration can modulate the lipid fluidity and lipid composition of rat proximal-small-intestinal brush-border membranes.     

Effects of lindane on fluidity and lipid composition in rat renal cortex membranes.

The influence of lindane upon dynamic properties of plasma membranes from rat renal cortex has been investigated using a fluorescence polarization technique. Preincubation with lindane increased membrane fluidity in a manner that is dose-dependent. This increase was higher in brush border membranes than in basolateral membranes. However, a significant decrease of the membrane fluidity was found in brush border membranes when rats were injected with lindane for 12 days. A possible solution to this difference could involve a resistance to membrane disordering by lindane through a regulatory mechanism that would balance the amount of cholesterol and phospholipid classes in the renal cortex membranes of lindane-injected rats.     

Membrane fluidity and lipid composition of rat small intestinal brush-border membranes during postnatal maturation

Fluidity and lipid composition of rat small intestinal brush-border membranes (BBM) were studied during maturation in five age groups: newborns, sucklings (1-3 weeks), weaned (4-6 weeks), juveniles (8-10 weeks), and adults (12 weeks). Brush-border membrane fluidity was measured by steady-state fluorescence polarization. Fluorescent probes used were: 1,6-diphenyl-1,3,5-hexatriene, 1-(4-trimethylammonium)phenyl)-6-phenyl-1,3,5-hexatriene, and a set of n-(9-anthroyloxy) fatty acids. Fluorescence anisotropy measured with all fluorophores was increased in adult versus newborn rats (P less than 0.004). The weight ratio of saturated to cis-unsaturated fatty acids increased from birth to the suckling age (P less than 0.0004). The cholesterol to phospholipid molar ratio increased from birth to the weaned age (P less than 0.0001). Cholesterol to protein ratio and phospholipid to protein ratio decreased after the weaned age (P less than 0.004). The results not only describe maturational changes of brush-border membranes but also give a better understanding of the correlations between biophysical and biochemical data in biological membranes.     

Dexamethasone influences the lipid fluidity, lipid composition and glycosphingolipid glycosyltransferase activities of rat proximal-small-intestinal Golgi membranes.

Experiments were performed to examine the effects of subcutaneous administration of the synthetic glucocorticoid dexamethasone (100 micrograms/day per 100 g body wt.) on the lipid fluidity, lipid composition and glycosphingolipid glycosyltransferase activities of rat proximal-small-intestinal Golgi membranes. After 4 days of treatment, Golgi membranes and liposomes prepared from treated rats were found to possess a greater fluidity than their control (diluent or 0.9% NaCl) counterpart, as assessed by steady-state fluorescence-polarization techniques using three different fluorophores. Moreover, analysis of the effects of temperature on the anisotropy values of 1,6-diphenylhexa-1,3,5-triene, using Arrhenius plots, demonstrated that the mean break-point temperatures of treated preparations were 4-5 degrees C lower than those of control preparations. Changes in the fatty acyl saturation index and double-bond index of treated membranes, secondary to alterations in stearic acid, linoleic acid and arachidonic acid, at least in part, appeared to be responsible for the differences in fluidity noted between treated and control Golgi membranes. Concomitant with these fluidity and lipid-compositional alterations, treated membranes possessed higher specific activities of UDP-galactosyl-lactosylceramide galactosyltransferase and CMP-N-acetylneuraminic acid:lactosylceramide sialyltransferase than their control counterparts. Experiments utilizing benzyl alcohol, a known fluidizer, furthermore suggested that the fluidity alteration induced by dexamethasone may be responsible for the increased activity of the former, but not the latter, glycosphingolipid glycosyltransferase.     

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.     

Ethinylestradiol administration selectively alters liver sinusoidal membrane lipid fluidity and protein composition

Administration of high-dose ethinylestradiol to rats decreases bile flow, Na,K-ATPase specific activity, and liver plasma membrane fluidity. By use of highly purified sinusoidal and bile canalicular membrane fractions, the effect of ethinylestradiol administration on the protein and lipid composition and fluidity of plasma membrane fractions was examined. In sinusoidal fractions, ethinylestradiol (EE) administration decreased Na,K-ATPase activity (32%) and increased activities of alkaline phosphatase (254%), Mg2+-ATPase (155%), and a 160-kDa polypeptide (10-fold). Steady-state and dynamic fluorescence polarization was used to study membrane lipid structure. Steady-state polarization of diphenylhexatriene (DPH) was significantly higher in canalicular compared to sinusoidal membrane fractions. Ethinylestradiol (5 mg/kg per day for 5 days) selectively increased sinusoidal polarization values. Similar changes were demonstrated with the probes 2- and 12-anthroyloxystearate. Time-resolved fluorescence polarization measurements indicated that EE administration for 5 days did not change DPH lifetime but increased the order component (r infinity) and decreased the rotation rate (R). However, 1 and 3 days after EE administration and with low doses (10-100 micrograms/kg per day for 5 days) the Na,K-ATPase, bile flow, and order component were altered, but the rotation rate was unchanged. Vesicles prepared from total sinusoidal membrane lipids of EE-treated rats, as well as phospholipid vesicles, demonstrated increased DPH polarization, as did intact plasma membrane fractions. Liver plasma membrane fractions showed no change in free cholesterol or cholesterol/phospholipid molar ratio, while esterified cholesterol content was increased with high-dose but not low-dose ethinylestradiol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rat proximal small intestinal Golgi membranes: lipid composition and fluidity

The present studies were conducted to examine and characterize the lipid composition and physical state of the membrane lipids of rat proximal small intestinal Golgi membranes. Golgi membranes were purified from isolated enterocytes; lipids were extracted from these membranes and analyzed by thin-layer and gas-liquid chromatography. The 'static' and 'dynamic' components of fluidity of Golgi membranes and their liposomes were assessed by steady-state fluorescence polarization techniques utilizing r infinity and S values of 1,6-diphenyl-1,3,5-hexatriene and r values of DL-2-(9-anthroyl)- and DL-12-(9-anthroyl)stearic acid, respectively. Additional studies were also performed on these membranes, using benzyl and methyl alcohol, to examine the relationship between alterations in lipid fluidity and glycosphingolipid glycosyltransferase activities. The results of these studies demonstrated that: (1) the principal phospholipids and neutral lipids of intestinal Golgi membranes, respectively, were phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, and unesterified cholesterol and fatty acids; (2) the major fatty acids of Golgi membranes were palmitic (16:0), stearic (18:0), linoleic (18:2), arachidonic (20:4) and oleic (18:1) acids; (3) fluorescence polarization studies using diphenylhexatriene detected a thermotropic transition at 24-26 degrees C in Golgi membranes and liposomes prepared from lipid extracts of these membranes; (4) benzyl alcohol (25 and 50 mM) but not methyl alcohol (50 mM) significantly increased the fluidity of these membranes; and (5) at these same concentrations, benzyl alcohol was also found to increase significantly the specific activity of UDP-galactosyllactosylceramide galactosyltransferase but not CMP-acetylneuraminic acid: lactosylceramide sialyltransferase. Methyl alcohol was not found to influence either enzyme's activity in these membranes.     

Catalytic hydrogenation of fatty acyl chains in plasma membranes; effect on membrane lipid fluidity and expression of cell surface antigens

Optimal reaction conditions were established for hydrogenation of plasma membranes of living murine GRSL leukemia cells, using the water-soluble catalyst Pd(QS)2 (QS, sulphonated alizarine; C14H6O7NaS). Under these conditions more than 80% of the cells remained viable. Analysis by gas chromatography revealed that hydrogenation occurred predominantly in the 18:2, 20:4 and 22:6 fatty acyl chains of the membrane phospholipids. Under the same conditions hydrogenation was also performed in purified plasma membranes from GRSL cells and from rat liver, and in liposomes prepared from the total lipid extracts of these membranes. Hydrogenation increased the lipid structural order parameter in the membranes, as measured by fluorescence polarization. This increase was more pronounced in the liposomes (46%) than in the plasma membranes (17-25%). Hydrogenation increased the expression of a 15 kDa antigen on the surface of viable GRSL cells, as measured in a Fluorescence Activated Cell Sorter, using monoclonal antibodies. The expression of four other antigens, among which H-2k, was not or much less affected by this treatment.     

The fluidity of plasma membranes from ethanol-treated rat liver

Summary Male Wistar rats were maintained for 35–40 days on a liquid diet containing 36% of calories as ethanol. Ethanol was replaced by carbohydrates in the isocaloric diet fed to control animals. The effect of ethanol consumption has been studied on the fluorescence polarization of rat liver plasma membranes and artificial lipid vesicles and on the lipid composition of the membranes. Fluorescence polarization in both membranes and vesicles was determined using DPH and TMA-DPH as fluorescence markers; from these data, the polarization term (r_o/r–1)^–1 and flow activation energy (E) were calculated. The ethanol consumption induces a more fluid environment within the membrane core of liver plasma membranes; the ethanol-fed rat membranes are more resistant to the in vitro effect of ethanol disordering the membrane structure. Vesicles obtained with lipids from either control membranes or ethanol-fed rat membranes were treated with ethanol and the changes in polarization paralleled to those exhibited by the membranes. The absence of phase transitions and of E changes was also shown in temperature-dependence studies. The lower cholesterol content found in ethanol-fed rat plasma membranes might be responsible for observed variations in the microviscosity.Abbreviation OG octyl -D-glucopyranoside     

Alpha-adrenergically mediated changes in membrane lipid fluidity and Ca2/ binding in isolated rat liver plasma membranes

Noradrenaline (0.1-5 microM, in the presence of 5 microM propranolol to block beta-receptors), ATP (100 microM) and angiotensin II (0.1 microM), which are thought to increase cytosolic Ca2+ concentration by mobilizing Ca2+ from internal stores, increased the lipid fluidity as measured by diphenylhexatriene fluorescence polarization in plasma membranes isolated from rat liver. The effect of noradrenaline was dose-dependent and blocked by the alpha-antagonists phenoxybenzamine (50 microM) and phentolamine (1 microM). The response to a maximal dose of noradrenaline (5 microM) and that to ATP (100 microM) were not cumulative, suggesting that both agents use a common mechanism to alter the membrane lipid fluidity. In contrast, the addition of noradrenaline (5 microM) along with the foreign amphiphile Na+-oleate (1-30 microM) resulted in an increase in membrane lipid fluidity which was equivalent to the sum of individual responses to the two agents. In the absence of Mg2+, reducing free Ca2+ concentration by adding EGTA increased membrane lipid fluidity and abolished the effect of noradrenaline, suggesting that Ca2+ is involved in the mechanism by which the hormone exerts its effect on plasma membranes. Noradrenaline (5 microM) and angiotensin II (0.1 microM) also promoted a small release of 45Ca2+ (16 pmol/mg membrane proteins) from prelabelled plasma membranes. The effect of noradrenaline was suppressed by the alpha-antagonist phentolamine (5 microM). It is proposed that noradrenaline, via alpha-adrenergic receptors and other Ca2+ -mobilizing hormones, increases membrane lipid fluidity by displacing a small pool of Ca2+ bound to phospholipids, removing thus the mechanical constraints brought about by this ion.     

Lipid composition and fluidity of rat enterocyte basolateral membranes regional differences

The lipid composition and fluidity of basolateral membranes prepared from the mucosa of the proximal, middle and distal thirds of the rat small intestine were determined. Fluidity, as assessed by the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene and a series of anthroyloxy fatty acid derivatives, is decreased in the distal third as compared to the proximal segments. This pattern is similar to that described previously for microvillus membranes. The decrease in fluidity of the distal as compared to the proximal membranes results from an increase in cholesterol content, cholesterol/phospholipid molar ratio and degree of saturation of the fatty acid residues. In the middle and distal thirds of the gut, the degree of saturation of the fatty acid residues is higher in microvillus as compared to basolateral membranes, accounting in part for the characteristically lower fluidity of the luminal membranes. The specific activity of the basolateral membrane (Na⁺ + K⁺)-dependent adenosine triphosphatase is significantly lower in the distal as compared to the proximal and middle thirds of the intestinal mucosa. Studies of the binding of [³H]ouabain indicate that this pattern results from fewer enzyme sites in the distal membranes.