Cholesterol uptake is dependent on membrane fluidity in mycoplasmas

The transfer of elaidate-enriched Acholeplasma laidlawii cells in culture from 37°C to 4°C virtually arrested exogenous cholesterol incorporation into the cell membrane. Cholesterol uptake continued, though at a slower rate, in oleate-enriched A. laidlawii cells undergoing similar temperature shift-down. It is concluded that the incorporation of exogenous cholesterol into the cell membrane of living mycoplasmas is rapid when the membrane lipid bilayer is in the liquid-crystalline state and very slow when the lipid bilayer is in the gel state.     

Oligomerization of Clostridium perfringens epsilon-toxin is dependent upon membrane fluidity in liposomes

Clostridium perfringens epsilon-toxin binds to receptors on MDCK cells and forms a heptamer in membranes. The mechanism behind the oligomerization of epsilon-toxin was studied using carboxyfluorescein (CF)-loaded liposomes composed of various phosphatidylcholines (PCs). The toxin caused CF to leak from liposomes in a dose-dependent manner. The toxin-induced leakage of CF, binding of the toxin to liposomes, and formation of a functional oligomer increased as the phase-transition temperature (Tm) of the PC used in the liposomes decreased. Surface plasmon resonance analysis using an HPA sensorchip (BIAcore) also revealed that the binding of the toxin to liposomes increased with a decrease in the Tm of the PC used in liposomes. The oligomer that was formed in 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID)-treated liposomes was labeled, indicating that it inserts into a hydrophobic region. Furthermore, the rate of epsilon-toxin-induced CF leakage was enhanced by treatment with phosphatidylethanolamine or diacylglycerol, which is known to favor a lamellar-to-inverted hexagonal (L-H) phase transition. We show that membrane fluidity in the liposome plays an important role in the binding of the toxin to liposomes, insertion into the hydrophobic region in the bilayer of liposomes, and the assembly process in the bilayer.     

Electron paramagnetic resonance studies of membrane fluidity in ozone-treated erythrocytes and liposomes

Doxyl stearate spin probes which differed in the attachment of the nitroxide free radical to the fatty acid have been used to study membrane fluidity in ozone-treated bovine erythrocytes and liposomes. Analysis of EPR spectra of spin labels incorporated into lipid bilayer of the erythrocyte membranes indicates an increase in the mobility and decrease in the order of membrane lipids. In isolated erythrocyte membranes (ghosts) the most significant changes were observed for 16-doxylstearic acid. In intact erythrocytes statistically significant were differences for 5-doxylstearic acid. The effect of ozone on liposomes prepared from a lipid extract of erythrocyte lipids was marked in the membrane microenvironment sampled by all spin probes. Ozone apparently leads to alterations of membrane dynamics and structure but does not cause increased rigidity of the membrane.     

Increase in membrane fluidity in liposomes and plant protoplasts upon osmotic swelling

Osmotic gradient across the membrane of nonsonicated liposomes and rose petal protoplasts are shown to induce swelling. Concomitantly, the lipid fluidity as measured by fluorescence depolarization is increased, probably due to increase in molar free volume. It is suggested that osmotic swelling can affect cell physiology via changes in membrane fluidity.     

Involvement of erythrocyte skeletal proteins in the modulation of membrane fluidity by phenothiazines

The effects of phenothiazines (chlorpromazine, chlorpromazine sulfoxide, and trifluoperazine) and antimitotic drugs (colchicine and vinblastine) on the erythrocyte membrane have been investigated. Chlorpromazine and trifluoperazine induced a dose-dependent increase in the freedom of motion of stearic acid spin-labels bound to both intact erythrocytes and ghosts, but did not affect the freedom of motion of stearic acids bound to vesicles depleted of spectrin and actin or of ghosts resealed with anti-spectrin antibodies. Further, chlorpromazine and trifluoperazine were able to eliminate a protein 4.1 dependent membrane thermal transition detected by stearic acid spin-labels at 8.5 +/- 1.5 degrees C. Antimitotic drugs and chlorpromazine sulfoxide did not change either the freedom of motion of stearic acid spin-labels or the 8.5 degrees C membrane thermal transition. Results indicate the involvement of skeletal proteins as possible membrane target sites of biologically active phenothiazines and suggest that the control of stearic acid spin-label freedom of motion is mediated by the spectrin-actin network and the proteins that link the skeletal network to the membrane.     

Increase in membrane fluidity in liposomes and plant protoplasts upon osmotic swelling.

Osmotic gradient across the membrane of nonsonicated liposomes and rose petal protoplasts are shown to induce swelling. Concomitantly, the lipid fluidity as measured by fluorescence depolarization is increased, probably due to increase in molar free volume. It is suggested that osmotic swelling can affect cell physiology via changes in membrane fluidity.     

Preparation of EGF labeled liposomes and their uptake by hepatocytes

The preparation of epidermal growth factor (EGF) bearing liposomes (EGF-liposomes) and their uptake by rat hepatocytes were described. EGF was bound to the liposomal surface by the disulfide bridge linkage using a heterobifunctional cross-linking reagent, N-hydroxysuccinimidyl-3-(2-pyridyl-dithio) propionate. EGF-liposomes were taken up in a significant amount by rat hepatocytes in an EGF receptor mediated manner, and their uptake was dependent on the amount of labeled EGF coupled to the liposomal surface. These results raise the possibility that the EGF-liposomes may be an effective drug carrier to the cells having EGF-receptors like some cancer cells.     

Alcohols inhibit adipocyte basal and insulin-stimulated glucose uptake and increase the membrane lipid fluidity

Benzyl alcohol and ethanol, at aqueous concentrations that cause local anesthesia of rat sciatic nerve, affect structural and functional properties of rat adipocytes. The data strongly suggest that structurally-intact membrane lipids are required for the proper cellular uptake of glucose and for the physiologic response of adipocytes to insulin. The structure of adipocyte membrane lipids was examined with the spin label method. Isolated adipocyte ‘ghost’ membranes were labeled with the 5-nitroxide stearate spin probe I(12,3). Order parameters that are sensitive to the fluidity of the lipid environment of the incorporated probe were calculated from ESR spectra of labeled membranes. Benzyl alcohol and ethanol dramatically increased the fluidity of the adipocyte ghost membrane, as indicated by decreases in the polarity-corrected order parameter S. This concentration-dependent fluidization commenced at approx. 10 mM benzyl alcohol and progressively increased at all higher concentrations tested (up to 107 mM). S decreased approx. 5.7% at 40 mM benzyl alcohol, a change in S comparable in magnitude to that induced by a 6°C increase in the incubation temperature. Benzyl alcohol and ethanol inhibited basal glucose uptake in adipocytes and uptake maximally stimulated by insulin. Temperature-induced increases in membrane fluidity, detected with 1(12,3), that closely paralleled the fluidity effects of alcohols were associated only with increases in basal and insulin-stimulated glucose uptake. The contention that the membrane lipid fluidity plays a role in insulin action needs further study.     

Involvement of serum mannan binding proteins and mannose receptors in uptake of mannosylated liposomes by macrophages

The roles of serum mannan binding protein (MBP) and the mannose receptor in the cellular uptake of mannosylated liposomes (Man-liposomes) by macrophages were studied. Man-liposomes were prepared by incorporating cholesten-5-yloxy-N-(4-((1-imino-2-beta-D-thiomannosylethyl)amino)butyl)formamide (Man-C4-Chol) into small unilamellar long circulating liposomes consisting of cholesterol (Chol) and distearoyl phosphatidylcholine (DSPC). In the in vitro cellular uptake study with cultured mouse peritoneal macrophages, [(3)H]Man-liposomes were taken up to a great extent, whereas no significant uptake was observed for [(3)H]cholesterol and DSPC liposomes without Man-C4-Chol (Bare-liposomes). The uptake of [(3)H]Man-liposomes was dose- and temperature-dependent and inhibited by an excess of mannosylated bovine serum albumin, suggesting their specific uptake via membrane mannose receptor-mediated endocytosis. Furthermore, it was demonstrated that (111)In-MBP binds strongly to Man-liposomes based on the recognition of Man-C4-Chol and markedly enhanced their uptake by macrophages. These results are supported by confocal laser microscopic images. In addition, in vivo hepatic uptake of (111)In-MBP was enhanced by Man-liposomes. On the other hand, the uptake of Man-liposomes was significantly reduced by preincubation with serum and further with MBP-depleted serum suggesting inhibitory effects of serum proteins such as albumin on mannose receptor-mediated endocytosis. The involvement of serum-type MBP and membrane mannose receptors in the uptake of Man-liposomes is thus suggested.     

Membrane fluidity and myotonia: Effects of cholesterol and desmosterol on erythrocyte membrane fluidity in rats with 20,25-diazacholesterol-induced myotonia and on phospholipid liposomes

Previous spin-label and electromyographic experiments with rats fed 20,25-diazacholesterol, an inhibitor of the biosynthetic conversion of desmostero[ to cholesterol, demonstrated an increased erythrocyte membrane fluidity and myotonia, a prolonged muscle contraction upon stimulation. The current studies with rats showed normal erythrocyte fluidity in animals fed 20,25-diazacholesterol but maintained on a high-cholesterol diet and no myotonia. Studies of model membrane systems composed of phospholipid vesicles containing desmosterol, cholesterol, or both demonstrated that desmosterol increased membrane lipid fluidity relative to cholesterol, suggesting that in 20,25-diazacholesterol-induced myotonia, in which desmosterot accounts for 85% of the plasma sterol, the increased membrane fluidity previously observed in erythrocytes and sarcolemma m this animal model of human congenital myotonia may be due to desmosterol.     

Gadolinium chloride-induced shifts in intrahepatic distributions of liposomes

Intravenously administered gadolinium chloride caused only a slight decrease in the rate of elimination of small unilamellar liposomes from the blood and had no influence on the total hepatic uptake of these vesicles, but did alter their intrahepatic distribution substantially. Uptake by the non-parenchymal cells was substantially decreased, whereas uptake by the parenchymal cells showed a concomitant increase. Our earlier observations (Roerdink et al. (1981) Biochim. Biophys. Acta 677, 79-89) on the effect of lanthanides on the in vivo distribution of multilamellar liposomes have been extended, in that we demonstrate, in addition to the drop in elimination rate from the blood and in the over-all hepatic uptake, a shift of liposome distribution within the Kupffer cell population. While the larger Kupffer cells, which normally take up a major fraction of an injected liposome dose, were strongly inhibited in liposome uptake, the more numerous small macrophages showed a 3-4-fold increase in uptake.     

Involvement of outer membrane proteins in enterochelin-mediated iron uptake in Escherichia coli.

Escherichia coli K-12 grown in iron-deficient media contained a large amount of outer membrane proteins O-2a, O-2b, and O-3, while cells grown in iron-supplemented media contained far smaller amounts of these proteins. The iron uptake by the iron-deficient cells was significantly stimulated in the presence of enterochelin, while that by the iron-rich cells was not. The outer membrane isolated from cells grown in the iron-deficient media showed enterochelin-stimulated binding of iron, while the outer membrane from iron-rich cells and cytoplasmic membranes from both types of cells did not show such binding activity. The amount of iron bound by the outer membrane was almost equivalent to the amount of O-2a, O2b, or O-3, irrespective of the amount of these proteins in the outer membrane, which is controlled by the amount of iron in the medium. Small particles rich in these proteins were prepared from cells by EDTA extraction. The particles were active in enterochelin-mediated iron binding and the amount of iron bound was equivalent to the amount of each of these proteins in the particles. Although the outer membrane of E. coli B was as active in iron binding as that of E. coli K-12, it did not possess an appreciable amount of O-2a. Gel electrophoretic analysis revealed that 9-2b and 9-3 were identical with the proteins missing mutants feuB and feuA, respectively.     

Steroid hormone-induced effects on membrane fluidity and their potential roles in non-genomic mechanisms

Steroid hormones are lipophilic suggesting they intercalate into the bilayer of target cell plasma membranes, potentially altering the fluidity and function of the membrane. The present study measured the effects of steroidal exposure on both phospholipid fluidity and integral protein mobility. Studies were performed on the effects of a variety of steroids on phosphatidylcholine liposomes, synaptosomal plasma membranes and sarcoplasmic reticulum membranes. Progesterone decreased the lipid fluidity, whereas testosterone had no effect on lipid movement. The estrogen, 17 beta-estradiol, an aromatised metabolite of testosterone, increased lipid mobility. In each case, the steroid action was concentration-dependent. The steroids all increased the activity of the Ca2+ ATPase of SR membrane, in keeping with their effects on this enzyme's aggregation state. The results suggest that, although lipid fluidity is a factor influencing protein activity, their mobility within the bilayer is the primary determinant of enzyme activity in the membrane for most proteins.     

Calcium and magnesium induced changes in the relative fluidity of phosphatidylcholine liposomes

The effect of Ca²⁺ and Mg²⁺ on relative fluidity of phosphatidylcholine liposomes was studied by measuring the degree of chlorophyll fluorescence polarization. An increase in the degree of fluorescence polarization was observed on incubation of liposomes with different concentrations of Ca²⁺ or Mg²⁺. The results have been interpreted on the basis of increase in the size of liposomes which could be brought about by calcium or magnesium induced fusion of small unilamellar liposomes to form larger vesicles. Fusion of liposomes has also been confirmed by the experiments on efficiency of energy transfer from chlorophyll b to chlorophyll a, and transmission electron microscopy of liposomes before and after incubation with Ca²⁺ and Mg²⁺.     

Correlative changes in sucrose uptake, ATPase activity and membrane fluidity in carnation petals during senescence

Apparent sucrose uptake. ATPase activity and membrane fluidity changes were studied during the development and senescence of carnation flowers ( Dianthus caryophyllus L., cv. Cerise Royallette). Typical changes associated with senescence of a cut flower, such as respiration, ethylene production and fresh weight, were measured. Concomitant with a rise in respiration and ethylene production and a decline in fresh weight, a sharp decrease in apparent sucrose uptake was observed. Sucrose uptake was pH dependent (pH optimum, 5.5) and influenced by membrane integrity. Apparently, the activity of ATPase is related to sucrose uptake, because similar changes occurred during flower development. In addition, the activity of ATPase was well correlated with membrane fluidity.
It is suggested that sucrose uptake is controlled by ATPase activity, which in turn is modulated by membrane lipid fluidity. The decline in membrane fluidity associated with senescence leads to a corresponding reduction in ATPase activity and sucrose uptake. Further evidence supporting this view comes from experiments in which senescence was enhanced by 1-aminocyclopropane-l-carboxylic acid. It shortened the time to decline in fresh weight, rise in respiration and ethylene production. In parallel, reduction in membrane fluidity, ATPase activity and sucrose uptake were observed.     

Induction of increased calcium uptake in liposomes having membrane proteins of chicken erythrocytes by S-adenosylmethionine

Liposomes having membrane proteins of chicken erythrocytes were prepared and the effect of S-adenosylmethionine on ⁴⁵Ca²⁺ uptake into the liposomes was investigated. S-Adenosylmethionine, a donor of methyl groups in enzymatic methylation, induced an increase of ⁴⁵Ca²⁺ uptake into the proteoliposomes with membrane proteins but not into the liposomes without membrane proteins. Increased release of ⁴⁵Ca²⁺ from the inside of the proteoliposomes was also induced by S-adenosylmethionine. These increases of uptake and release of ⁴⁵Ca²⁺ were inhibited by S-adenosylhomocystein, an inhibitor of enzymatic methylation. Furthermore, membrane proteins from chicken erythrocytes showed protein and phospholipid methyltransferase activities. The uptake of other materials, 3-0-[methyl-³H]glucose, α-[1-¹⁴C]aminoisobutyric acid, ⁴²K⁺ and ⁵⁴Mn²⁺, into the proteoliposomes was not increased by S-adenosylmethionine. These results suggest that enzymatic methylation of membrane components may have an important role in the regulation of calcium transport in the chicken erythrocyte membrane and this regulation is rather specific for calcium.