Effects of sterol-binding agent nystatin on wheat roots: the changes in membrane permeability, sterols and glycoceramides

Plant sterols are important multifunctional lipids, which are involved in determining membrane properties. Biophysical characteristics of model lipid and isolated animal membranes with altered sterol component have been intensively studied. In plants however, the precise mechanisms of involvement of sterols in membrane functioning remain unclear. In present work the possible interactions between sterols and other membrane lipids in plant cells were studied. A useful experimental approach for elucidating the roles of sterols in membrane activity is to use agents that specifically bind with endogenous sterols, for example the antibiotic nystatin. Membrane characteristics and the composition of membrane lipids in the roots of wheat (Triticum aestivum L.) seedlings treated with nystatin were analyzed. The application of nystatin greatly increased the permeability of the plasma membrane for ions and SH-containing molecules and decreased the total sterol level mainly as a consequence of a reduction in the amount of β-sitosterol and campesterol. Dynamic light-scattering was used to confirm the in vitro formation of stable complexes between nystatin and β-sitosterol or cholesterol. Sterol depletion was accompanied by a significant rise in total glycoceramide (GlCer) content after 2h treatment with nystatin. Analysis of the GlCer composition using mass spectrometry with electrospray ionization demonstrated that nystatin induced changes in the ratio of molecular species of GlCer. Our results suggest that changes in the sphingolipid composition can contribute to the changes in plasma membrane functioning induced by sterol depletion.     

Energy metabolism in wheat root cells under modification of plasma membrane permeability by antibiotic nystatin

This paper reports changes in ion transport and energy metabolism of plant cells during short- and long-term expositions, resp., to antibiotic nystatin, which is known to specifically bind with plasma membrane sterols to form channels. The excised roots of 5 days old wheat seedlings were used as a model system in this research. It has been shown that treatment of excised roots with nystatin leads to activation of energy metabolism expressed as an increase of respiration and heat production by root cells. Furthermore, in the presence of nystatin increased pH of incubation medium, plasma membrane depolarization and a significant loss of potassium ions were observed. Nystatin-induced stimulation of respiration was prevented by malonate, an inhibitor of succinate dehydrogenase, electron acceptor dichlorophenolindophenol, and AgNO3, an inhibitor of H(+)-ATPase. Based on the data obtained it can be suggested that nystatin-induced stimulation of respiration is related to electron transport activation via mitochondrial respiratory chain, and is connected with activation of plasmalemma proton pump. Moreover, nystatin-induced increase of oxygen consumption was prevented by cerulenin, an inhibitor of fatty acid and sterol synthesis. This indicates that additional sterols and phospholipids may be synthesized in root cells to "heal" nystatin-caused damage of plasma membrane. A supposed chain of events of cell response to nystatin action may by as following: formation of nystatin channels-influx of protons--depolarization of plasmalemma-efflux of potassium ions-disturbance of ion homeostasis--activation of H(+)-ATPase work-increase in energy "requests" for H(+)-ATPase function--increase in the rate of oxygen consumption and heat production. The increased energy production under the action of nystatin, may provide the work of proton pump and synthesis of sterols and phospholipids, which are necessary for membrane regeneration.     

Optimisation of plant sterols incorporation in human keratinocyte plasma membrane and modulation of membrane fluidity.

The in vitro effects of plant sterols were investigated with regard to their uptake and membrane lipid fluidity in human keratinocytes. Among the different media tested to transport sterols (liposomes, micelles and organic solvents), the best results in terms of incorporation and viability were obtained by the use of the organic solvents dimethylsulfoxide and ethanol. After 48 h incubation exogenous sterol can account for about 30% of the total cell sterol content. The total sterol amount in plasma membranes increased 2-fold after incubation with cholesterol, whereas it was not altered when phytosterols were incorporated. The incorporation of cholesterol, sitosterol and stigmasterol led to an increase in the percent of unsaturated fatty acid C18:1 in the plasma membrane. The effect of this uptake on membrane fluidity was studied by means of fluorescence polarisation using DPH and TMA-DPH as fluorescent probes. Whereas cholesterol and sitosterol had no significant effect on the DPH fluorescence anisotropy (rs), the presence of stigmasterol induced a 12% decrease of rs reflecting an increase in membrane fluidity. We can conclude from this study that in the presence of sitosterol, the mean fluidity of the membrane is regulated whereas stigmasterol triggers a looseness of molecular packing of phospholipids acyl chains, in accordance with previous results obtained on purely lipid model membranes.     

Influence of sterol structure on yeast plasma membrane properties

Fluorescence anisotropy measurements indicated that physical changes occured in the lipids of plasma membranes of yeast sterol mutants but not in the plasma membrane of an ergosterol wild-type. Parallel experiments with model membrane liposomes verified that the physical changes in lipids observed in the sterol mutants are dependent on the sterol present and not the phospholipid composition. In addition, the physical changes in lipids observed in liposomes derived from wild-type phospholipids were eliminated by addition of ergosterol but persisted in the presence of cholesterol, cholestanol, ergostanol, or sterols from the sterol mutants. No physical changes in lipids were observed, however, in plasma membranes from a sterol auxotroph, even when the auxotroph was grown on cholesterol or cholestanol. The lack of physical changes in lipids in the sterol auxotroph may reflect the ability of the auxotroph to modify its phospholipid composition with respect to its sterol composition. These results indicate that high specificity ‘sparking’ sterol is not required for the regulation of overall bulk lipid properties of the plasma membrane.     

Sterols in hardening winter wheat

The main sterols in winter wheat crowns and roots were sitosterol and campesterol, with significant amounts of stigmasterol and traces of cholesterol. The main groups of sterol-containing lipids were free sterols, steryl glucosides, steryl esters and esterified steryl glucosides. Sterol analysis within each group showed little difference between them. Steryl esters were relatively rich in cholesterol and poor in stigmasterol. Free sterols were rich in stigmasterol. Low temperature caused an increase in sterol content but had little effect on sterol composition and sterol to lipid P ratio. There was some increase in steryl esters and some decrease in free sterols. Cholesterol and stigmasterol decreased in the steryl ester and free sterol fractions, respectively. There was little evidence for involvement of sterols in winter wheat frost hardening.     

Changes in lipid composition and respiratory activity of wheat roots treated with inhibitor of phosphoinositide cycle of lithium ions

A study was made of the effect of lithium ions (5 mM LiCl) on the growth and respiration of roots of 2-5 day old wheat seedlings, composition of phospholipids, free fatty acids and sterols, and superoxide radical generation. Significant growth depression was shown in roots treated with lithium, increasing with age of seedlings. The growth of seedlings on a 5 mM LiCl containing medium influenced the lipid composition in roots. In the roots of these seedlings phospholipid contents increased along with a decrease in free fatty acids, and the ratio of sterol/phospholipids, while superoxide radical production was stimulated. Lithium stimulated oxygen consumption of excised roots of 5 day old seedlings. Due to its physical and chemical properties lithium is supposed to be able to disrupt gradients of Ca2+, H+, K+, and to lead to a partial blockade of signal transduction for triggering proliferation via the phosphoinositide cycle. Changes in the lipid composition, increase in the membrane permeability for ions, and respiration of roots of seedlings grown in LiCl-containing medium are presumably associated with growth depression and juvenility of roots.     

Sterol structure dependence of insulin receptor and insulin-like growth factor 1 receptor activation

The plasma membrane is a dynamic environment with a complex composition of lipids, proteins, and cholesterol. Areas enriched in cholesterol and sphingolipids are believed to form lipid rafts, domains of highly ordered lipids. The unique physical properties of these domains have been proposed to influence many cellular processes. Here, we demonstrate that the activation of insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) depends critically on the structures of membrane sterols. IR and IGF1R autophosphorylation in vivo was inhibited by cholesterol depletion, and autophosphorylation was restored by the replacement with exogenous cholesterol. We next screened a variety of sterols for effects on IR activation. The ability of sterols to support IR autophosphorylation was strongly correlated to the propensity of the sterols to form ordered domains. IR autophosphorylation was fully restored by the incorporation of ergosterol, dihydrocholesterol, 7-dehydrocholesterol, lathosterol, desmosterol, and allocholesterol, partially restored by epicholesterol, and not restored by lanosterol, coprostanol, and 4-cholesten-3-one. These data support the hypothesis that the ability to form ordered domains is sufficient for a sterol to support ligand-induced activation of IR and IGF1R in intact mammalian cells.     

Plasma membrane lipids in the resurrection plant Ramonda serbica following dehydration and rehydration

Plants of Ramonda serbica were dehydrated to 3.6% relative water content (RWC) by withholding water for 3 weeks, afterwards the plants were rehydrated for 1 week to 93.8% RWC. Plasma membranes were isolated from leaves using a two-phase aqueous polymer partition system. Compared with well-hydrated (control) leaves, dehydrated leaves suffered a reduction of about 75% in their plasma membrane lipid content, which returned to the control level following rewatering. Also the lipid to protein ratio decreased after dehydration, almost regaining the initial value after rehydration. Lipids extracted from the plasma membrane of fully-hydrated leaves were characterized by a high level of free sterols and a much lower level of phospholipids. Smaller amounts of cerebrosides, acylated steryl glycosides and steryl glycosides were also detected. The main phospholipids of control leaves were phosphatidylcholine and phosphatidylethanolamine, whereas sitosterol was the free sterol present in the highest amount. Following dehydration, leaf plasma membrane lipids showed a constant level of free sterols and a reduction in phospholipids compared with the well-hydrated leaves. Both phosphatidylcholine and phosphatidylethanolamine decreased following dehydration, their molar ratio remaining unchanged. Among free sterols, the remarkably high cholesterol level present in the control leaves (about 14 mol%) increased 2-fold as a result of dehydration. Dehydration caused a general decrease in the unsaturation level of individual phospholipids and total lipids as well. Upon rehydration the lipid composition of leaf plasma membranes restored very quickly approaching the levels of well-hydrated leaves.     

The influence of plasma membrane ion permeability modulators on superoxide formation and bioelectrical characteristics of wheat root cells

Changes in superoxide radical formation and bioelectrical characteristics of excised wheat root cells under modification of plasma membrane ion permeability were studied. It was shown that a 2 h treatment of excised roots with valinomycin (Val, 20 microM), N, N'-dicyclohexylcarbodimide (DCCD, 100 microM), gramicidin S (Gr, 20 microM), chlorpromazine (CPZ, 100 microM) caused an increased loss of potassium by cells, lowering of membrane potential (MP) and electrical input resistance (Rin) of the cells. The superoxide formation by excised root cells diminished (under DCCD) or remained at the control level (under Val), which was accompanied by a minor decrease of MP and Rin of the cells, a small increase in potassium loss by excised roots, and in no change of pH of incubation medium. Significant depolarization of plasma membrane, dropping of Rin and essential loss of potassium ions by the cells correlated with a rise in the medium alkalinization and superoxide formation by excised roots (in the presence of Gr, CPZ). Ion channel blocker gadolinium (Gd3+, 200 microM) caused an increase of MP and Rin reduction of potassium loss by cells, and a decrease of pH of the incubation medium, and also enhancement of superoxide formation by excised root cells. It is suggested that upon plasma membrane ion permeability modification the activity of superoxide generating systems depends on the specificity and mechanisms of action of modulators, and is determined by their influence on redox state of plasma membrane as well as by peculiarities of ion transport disturbance.     

Cell-free transfer of sterols from dictyosome-like structures to plasma membrane vesicles of guinea pig testes

Summary Transfer of radiolabeled lipids from dictyosome-like structures (DLS) from testis tubules of the guinea pig as donor to unlabeled plasma membrane from testis tubules immobilized on nitrocellulose as acceptor was studied in a completely cell-free system. As a general label for lipids of the donor DLS, isolated testis tubules were incubated with [¹⁴C]acetate. Time- and temperature-dependent transfer of [¹⁴C]acetate labeled constituents was observed in the cellfree system. However, despite the fact that phospholipids and other constituents were highly labeled in the donor fraction, primarily radioactive sterols were transferred to the plasma membrane acceptor vesicles. Transfer at 37°C represented 0.4 to 0.7% of the total radiolabeled cholesterol at 37°C but little or no transfer occurred at 4°C. The sterols transferred exhibited Chromatographic mobilities corresponding to those of cholesterol and lanosterol. Similar results were obtained with [¹⁴C]mevalonic acid. In subsequent experiments, cholesterol transfer from DLS to plasma membrane was demonstrated by incubation of DLS with [³H]squalene which was converted into sterol or with [¹⁴C]cholesterol. Transfer of sterols required ATP, but not cytosol, and was both time- and temperature-dependent. DLS were more effective than either endoplasmic reticulum or plasma membrane as the donor fraction. The results from the cell-free analysis suggest a possible functional role of the DLS in sterol biogenesis and transfer to the plasma membrane during spermatid development.Abbreviations DLS dictyosome-like structure(s) - PBS phosphatebuffered saline - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - BSA bovine serum albumin     

Plasma membrane lipid alterations induced by NaCl in winter wheat roots

A highly enriched plasma membrane traction was isolated by two phase partitioning from wheat roots ( Triticum aestivum L. cv. Vivant) grown with and without 100 m M NaCl. The lipids of the plasma membrane fraction were extracted and characterised. Phosphatidylcholine and phosphatidylethanolamine were the major phospholipids with lesser amounts of phosphandylinositol, phosphatidylglycerol, diphosphalidylglycerol, phosphatidic acid and phosphatidylseriae. NaCl decreased the total phospholipids and the phosphatidylcholine portion of the plasma membranes. Salt treatment had no effect on total sterols and glycolipids. but the relative abundance of the tree sterols was altered: cholesterol, stigma sterol and brassicasterol were significantly increased. Salt treatment resulted in an increase of the more planar/less planar ratio of the free sterols and in introduction of a double bond in the C₂₂ position in the side chain of stigma sterol and brassicasterol. The degree of fatty acid saturation of total phospholipids, phospha-tidylcholine and phosphatidylethanolamine was increased after salt treatment. These lipid changes are discussed in relation to the salt tolerance mechanism.     

Effects of the growth retardant tetcyclacis on the sterol composition of oat (Avena sativa)

The norbornenodiazetine plant growth regulator tetcyclacis, when applied to roots of Avena sativa, caused a substantial increase in the cholesterol content of the shoots. Amounts of the C-24 alkylated sterols campesterol, stigmasterol and sitosterol all declined. A similar alteration in the sterol profile was observed for a plasma membrane preparation from the shoots. Changes in the sterol composition of root tissue were much less pronounced.     

An InCytes from MBC Selection: Sterols Are Mainly in the Cytoplasmic Leaflet of the Plasma Membrane and the Endocytic Recycling Compartment in CHO Cells

The transbilayer distribution of many lipids in the plasma membrane and in endocytic compartments is asymmetric, and this has important consequences for signaling and membrane physical properties. The transbilayer distribution of cholesterol in these membranes is not properly established. Using the fluorescent sterols, dehydroergosterol and cholestatrienol, and a variety of fluorescence quenchers, we studied the transbilayer distribution of sterols in the plasma membrane (PM) and the endocytic recycling compartment (ERC) of a CHO cell line. A membrane impermeant quencher, 2,4,6-trinitrobenzene sulfonic acid, or lipid-based quenchers that are restricted to the exofacial leaflet of the plasma membrane only reduce the fluorescence intensity of these sterols in the plasma membrane by 15–32%. When the same quenchers have access to both leaflets, they quench 70–80% of the sterol fluorescence. Sterol fluorescence in the ERC is also quenched efficiently in the permeabilized cells. In microinjection experiments, delivery of quenchers into the cytosol efficiently quenched the fluorescent sterols associated with the PM and with the ERC. Quantitative analysis indicates that 60–70% of the PM sterol is in the cytoplasmic leaflet. This means that cholesterol constitutes ∼40 mol% of cytoplasmic leaflet lipids, which may have important implications for intracellular cholesterol transport and membrane domain formation.     

Where sterols are required for endocytosis

Sterols are essential membrane components of eukaryotic cells. Interacting closely with sphingolipids, they provide the membrane surrounding required for membrane sorting and trafficking processes. Altering the amount and/or structure of free sterols leads to defects in endocytic pathways in mammalian cells and yeast. Plasma membrane structures functioning in the internalization step in mammalian cells, caveolae and clathrin-coated pits, are affected by cholesterol depletion. Accumulation of improper plasma membrane sterols prevents hyperphosphorylation of a plasma membrane receptor in yeast. Once internalized, sterols still interact with sphingolipids and are recycled to the plasma membrane to keep an intracellular sterol gradient with the highest amount of free sterols at the cell periphery. Interestingly, cells from patients suffering from sphingolipid storage diseases show high intracellular amounts of free cholesterol. We propose that the balanced interaction of sterols and sphingolipids is responsible for protein recruitment to specialized membrane domains and their functionality in the endocytic pathway.     

Role of sterol photoconversion products in the regulation of cholesterol biosynthesis in rat skin

In order to clarify the effect of products of photochemical conversion of sterols on cholesterol biosynthesis, rat skin samples were incubated with 2-(14)C-acetate in the presence of the antirachitic agent Dk and 7beta-hydroxycholesterol. The synthesis of sterols from acetate was activated in the presence of Dk. A correlation between the activation of sterol synthesis and the concentration of the antirachitic agent was found. An addition of 7beta-hydroxycholesterol to the incubation medium inhibited acetate incorporation into the sterols. The level of synthesis inhibition increased with an elevation of the 7beta-hydroxysterol concentration in the incubation medium. This indicates that both products of sterol photoconversion can be involved in the control of cholesterol biosynthesis.     

Relationship between membrane sterol composition and responsiveness to 12-O-tetradecanoylphorbol 13-acetate in HL-60 human promyelocytic leukaemia cell lines.

We have examined the sterol composition and metabolism of promyelocytic leukaemia cell lines (HL-60) after treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). A variant cell line (Blast II cells) which is resistant to TPA was used as control. Analysis of the sterols of TPA-sensitive cells radiolabelled with [3H]leucine, [14C]acetate or [14C]pyruvate showed a high incorporation into cholesterol and a low incorporation in lanosterol + dihydrolanosterol. The inverse relationship was observed in TPA-resistant cells. Experiments with other cellular variants representing TPA-sensitive and TPA-resistant classes gave similar results. Analysis of the cellular sterol composition by gas chromatography confirmed that TPA-resistant cells are particularly rich in lanosterol/dihydrolanosterol. TPA treatment enhanced the incorporation of [14C]pyruvate into the sterol fraction of both cell types. This was accompanied by an alteration of incorporation into several lipids, particularly phospholipids. Pulse-chase studies with [14C]acetate revealed that TPA induced the release of radioactive lipids into the medium from HL-60 and Blast II cells. However this treatment released phospholipids from the TPA-sensitive cells and sterols and fatty acids from the TPA-resistant cells. We conclude that the sterol composition can regulate specific biochemical processes in the membrane and can be considered as a factor that plays a role in the responsiveness of HL-60 cells to TPA.     

Lipid acyl chain-dependent effects of sterols in Acholeplasma laidlawii membranes.

Acholeplasma laidlawii was grown with different fatty acids for membrane lipid synthesis (saturated straight- and branched-chain acids and mono- and di-unsaturated acids). The ability of 12 different sterols to affect cell growth, lipid head group composition, the order parameter of the acyl chains, and the phase equilibria of in vivo lipid mixtures was studied. The following two effects were observed with respect to cell growth: with a given acyl chain composition of the membrane lipids, growth was stimulated, unaffected, reduced, or completely inhibited (lysis), depending on the sterol structure; and the effect of a certain sterol depended on the acyl chain composition (most striking for epicoprostanol, cholest-4-en-3-one, and cholest-5-en-3-one, which stimulated growth with saturated acyl chains but caused lysis with unsaturated chains). The three lytic sterols were the only sterols that caused a marked decrease in the ratio between the major lipids monoglucosyldiglyceride and diglucosyldiglyceride and hence a decrease in bilayer stability when the membranes were enriched in saturated (palmitoyl) chains. With these chains correlations were found for several sterols between the glucolipid ratio and the order parameter of the acyl chains, as well as the lamellar-reversed hexagonal phase transition, in model systems. A shaft experiment revealed a marked decrease in the ratio of monoglucosyldiglyceride to diglucosyldiglyceride with the lytic sterols in unsaturated (oleoyl) membranes. The two cholestenes induced nonlamellar phases in in vivo mixtures of oleoyl A. laidlawii lipids. The order parameters of the oleoyl chains were almost unaffected by the sterols. Generally, the observed effects cannot be explained by an influence of the sterols on the gel-to-liquid crystalline phase transition.     

Study of the resistance of Candida guilliermondii to polyene antibiotics

250 Candida guilliermondii strains resistant to the polyene antibiotics nystatin, levorin and amphotericin B were obtained using UV irradiation. When the mutant strains became resistant to one of the polyene antibiotics, their resistance to the other ones changed. Phenotypic analysis showed that the resistance of the strains to polyene antibiotics did not make them susceptible to a rise in osmotic pressure and to a change of the temperature of incubation. Some of the polyene-resistant strains were stained in a medium with methylene blue. Analysis of the sterol composition in the mutants by UV spectroscopy showed that the resistance to polyene antibiotics sometimes involved changes in the sterol composition. Two new UV spectrum types were recorded for the sterols of the mutant strains; they differed from the UV spectrum for the sterols of the parent sensitive strain.     

Effect of NaCl and Polyamines on Plasma Membrane Lipids of Wheat Roots

Caryopses of a salt sensitive wheat cultivar (Triticum aestivum L. cv. Giza 163) were presoaked in 2.5 mM putrescine (Put), 5 mM spermidine (Spd) or 2.5 mM spermine (Spm) for 24 h and then subjected to 150 mM NaCl added to the growth medium for 15 d. Effects of NaCl and polyamines (PAs) on plasma membrane (PM) lipids, phospholipids, fatty acids, and free sterols were determined. NaCl treatment caused a decrease in total phospholipids, increase in saturated fatty acids and altered distribution of sterols and phospholipids. NaCl also induced increase in sterol/phospholipid ratio. PAs treatments (particularly Put and Spd) counterbalanced the NaCl deleterious effects on PM lipids.     

Stress tolerance in the marsh plant Spartina patens: Impact of NaCl on growth and root plasma membrane lipid composition

The C‐4 salt marsh grass, Spartina patens , thrives in the upper portion of the marsh where soil salinities may be equal to coastal seawater. Spartina patens was grown in hydroponic culture in a greenhouse at 0, 340, and 510 m M NaCl, and measured for growth, tissue cation content, and root plasma membrane (PM) lipid composition. From 0 to 340 and 510 m M , the shoot growth decreased, but root growth was not affected. The Na⁺ content increased in both shoots and roots when plants were grown in salt, while the shoots had a decreased K⁺ content and the roots had a decreased Ca²⁺ content. Spartina patens root plasma membrane was isolated with an aqueous polymer two‐phase system. The purity of the plasma membrane was verified with cytochemical tests on membrane enzyme markers. Plasma membrane lipids were stable relative to the membrane protein content. Molar percentages of sterols (including free sterols) and phospholipid decreased with increasing salinity. However, glycolipid showed a statistically significant increase in the total lipid as salinity in the medium was increased from 0 to 510 m M . Even at a salinity of 510 m M , the plasma membrane sterol/phospholipid ratio was unaffected by NaCl. When the plants were grown in NaCl media, the plasma membrane had a decreased phosphatidylcholine (PC) and phosphatidylethanolamine (PE) content, but the PC/PE ratios were not affected. The plasma membrane molar percentage of sitosterol in total free sterol increased when plants were grown in salt media. The predominant membrane fatty acids were C11 and C14, and the major unsaturated one was C14:1. An increase in growth medium salinity resulted in a decreased root plasma membrane fluidity.