Effects of membrane-stabilizing agents,cholesterol and cepharanthin,on radiation-induced lipid peroxidation and permeability in liposomes

Effects of two membrane-stabilizing agents, cholesterol and cepharanthin, on radiation-induced lipid peroxidation and membrane permeability were examined. Radiation-induced lipid peroxidation caused an increase in membrane permeability in phosphatidylcholine liposomes. The presence of cholesterol in liposomal membranes caused a decrease in the degree of membrane permeability in spite of an increased lipid peroxidation. On the other hand, cepharanthin suppressed both lipid peroxidation and the changes in permeability induced by radiation. The membrane-stabilizing effect of cholesterol against radiation-induced changes in permeability seemed to depend on the rigidification of membranes, which was estimated by ESR studies. Cepharanthin suppressed the degree of membrane permeability mainly by inhibiting the radiation-induced lipid peroxidation. However, cepharanthin did not exhibit a radical-trapping ability.     

Kinetics of water penetration into unsonicated liposomes effects ofn-alkanols and cholesterol

Summary The rate of swelling of egg lecithin liposomes under osmotic shock has been studied employing a stopped-flow spectrophotometer. Incorporation of cholesterol and simple alcohols into the liposomal structure elicits a biphasic response in swelling rate: at low concentrations these additives increase but at high concentrations they decrease water permeabilty. For simplen-alkanols, the effects can be correlated with structure. Specifically, the concentration of alcohol required to elicit maximal permeability as well as the maximal permeability decreases with increasing length of the alcohol. These effects are accounted for on the basis of modification of the orientation and packing of lecithin molecules in the bilayer membrane of the liposome.     

The effect of temperature on membrane hydraulic conductivity

The objective of this study was to use the temperature dependence of water permeability to suggest the physical mechanisms of water transport across membranes of osmotically slowly responding cells and to demonstrate that insight into water transport mechanisms in these cells may be gained from easily performed experiments using an electronic particle counter. Osmotic responses of V-79W Chinese hamster fibroblast cells were measured in hypertonic solutions at various temperatures and the membrane hydraulic conductivity was determined. The results were fit with the general Arrhenius equation with two free parameters, and also fit with two specific membrane models each having only one free parameter. Data from the literature including that for human bone marrow stem cells, hamster pancreatic islets, and bovine articular cartilage chondrocytes were also examined. The results indicated that the membrane models could be used in conjunction with measured permeability data at different temperatures to investigate the method of water movement across various cell membranes. This approach for slower responding cells challenges the current concept that the presence of aqueous pores is always accompanied by an osmotic water permeability value, P(f)>0.01 cm/s. The possibility of water transport through aqueous pores in lower-permeability cells is proposed.     

Hyperosmotic relaxation lysis of chromaffin granules is caused by interactions between the granular membrane and intragranular vesicles

Bovine chromaffin granules undergo irreversible structural changes during osmotic shrinkage in hypertonic sucrose and salt solutions, such that, on reexposure to isoosmotic conditions they do not regain their original morphology, but undergo lysis ('hyperosmotic relaxation lysis'). Irreversible alterations of granules were induced by hypertonic incubations lasting for as little as 1 min. Fluorescence and EPR membrane labelling experiments showed that hypertonicity did not induce membrane loss for instance by inwardly or outwardly directed pinching off of membrane material. The mean sizes of chromaffin granules as a function of increasing and subsequently decreasing osmotic pressure were measured by photon correlation spectroscopy; there was no significant difference in sizes of hyperosmotically pretreated granules as compared with controls. Freeze-fracture electron micrographs showed the formation of 'twins' and 'triplets' under hypertonic conditions. They also revealed intragranular vesicles of 50-200 nm in diameter in both hypertonically and isotonically suspended granules. 'Twin' and 'triplet' granules were formed by the attachment of intragranular vesicles to the granule membranes. We suggest that hyperosmotic relaxation lysis is caused by the fact that this adhesion partly prevents the granule membrane from reexpanding, thus, leading to its rupture.     

Lipids, curvature stress, and the action of lipid prodrugs: free fatty acids and lysolipid enhancement of drug transport across liposomal membranes

Molecular shape and its impact on bilayer curvature stress are powerful concepts for describing the effects of lipids and fatty acids on fundamental membrane properties, such as passive permeability and derived properties like drug transport across liposomal membranes. We illustrate these relationships by studying the effects of fatty acids and lysolipids on the permeation of a potent anti-cancer drug, doxorubicin, across the bilayer of a liposome in which the drug is encapsulated. Using a simple fluorescence assay, we have systematically studied the passive permeation of doxorubicin across liposomal membranes in different lipid phases: the solid-ordered phase (DPPC bilayers), the liquid-disordered phase (POPC lipid bilayers), and the liquid-ordered phase induced by high levels of cholesterol (DOPC + cholesterol lipid bilayers). The effect of different free fatty acids (FA) and lysolipids (LL), separately and in combination, on permeability was assessed to elucidate the possible mechanism of phospholipase A₂-triggered release in cancer tissue of liposomal doxorubicin formulations. In all cases, FAs applied separately lead to significant enhancement of permeability, most pronounced in liquid-disordered bilayers and less pronounced in solid and solid-ordered bilayers. LLs applied separately had only a marginal effect on permeability. FA and LL applied in combination lead to a synergistic enhancement of permeability in solid bilayers, whereas in liquid-disordered bilayers, the combined effect suppressed the otherwise strong permeability enhancement due to the FAs.     

PIP1 aquaporins,sterols, and osmotic water permeability of plasma membranes from etiolated pea seedlings

Plasma membrane isolated from microsomal membranes of pea seedling root and shoot cells by means of aqueous two-phase polymer system was separated by flotation in discontinuous OptiPrep gradient into “light” (≤1.146 g/cm³) and “heavy” (≥1.146 g/cm³) fractions. Osmotic water permeability of plasma membrane and its two fractions was investigated by inducing transmembrane osmotic gradient on the vesicle membrane and recording the kinetics of vesicle osmotic shrinkage by the stopped-flow method. Rate constants of osmotic shrinkage and coefficients of osmotic water permeability of the membranes were estimated on the basis of the kinetic curve approximation by exponential dependencies and using electron microscope data on vesicles sizes. In plasma membrane and its fractions the content of sterols and PIP1 aquaporins was determined. It was found that in “light” PM fractions from both roots and shoots the content of PIP1 aquaporins and sterols was higher and the osmotic water permeability coefficient was lower than in “heavy” fractions of plasma membrane. The results indicate that plasma membrane of roots and shoots is heterogeneous in osmotic water permeability. This heterogeneity may be related with the presence of microdomains with different content of aquaporins and sterols in the membrane.     

Interaction and transport of poly(L-lysine) dendrigrafts through liposomal and cellular membranes: the role of generation and surface functionalization

Two generations of poly(l-lysine) dendrigrafts (DGLs) were studied with regard to their ability to interact with and translocate through liposomal and cellular membranes. Partial guanidinylation of the surface amino groups of the starting dendrigrafts afforded the guanidinylated derivatives whose membrane translocation properties were also assessed. Mixed liposomes, consisting of dihexadecyl phosphate, phosphatidylcholine, and cholesterol, were employed as model membranes, while A549 human lung carcinoma cells were used for cellular uptake studies. At high surface group/liposomal phosphate molar ratios and depending on the structure of the DGL, the interaction led to aggregation. Dendrigraft liposomal internalization was achieved, however, at low molar ratios. Thus translocation of the second generation dendrigrafts was rather limited at 25 degrees C, which, however, was enhanced when the bilayer was in the liquid-crystalline phase. In contrast, third-generation counterparts exhibited minor translocational ability. Furthermore, the introduction of a guanidinium group to dendrigrafts was found to enhance their transport through liposomal membranes. On the other hand, cellular uptake by A549 cells was monitored up to 3 h incubation time via fluorescence registration employing fluorescein-labeled dendrigrafts. The efficiency of dendrigraft internalization was enhanced by the presence of the guanidinium groups, while DGLs were preferentially localized in the nucleus and nuclear membrane, as revealed by fluorescence microscopy.     

Permeability studies on red cell membranes of dog, cat, and beef

Water permeability coefficients of dog, cat, and beef red cell membranes have been measured under an osmotic pressure gradient. The measurements employed a rapid reaction stop flow apparatus with which cell shrinking was measured under a relative osmotic pressure gradient of 1.25 to 1.64 times the isosmolar concentration. For the dog red cell the osmotic permeability coefficient is 0.36 cm⁴/(sec, osmol). The water permeability coefficient for the dog red cell under a diffusion gradient was also measured (rate constant = 0.10/msec). The ratio between the two permeabilities was used to calculate an equivalent pore radius of 5.9 A. This value agrees welt with an equivalent pore radius of 6.2 A obtained from reflection coefficients of nonelectrolyte water-soluble molecules, and is consistent with data on the permeability of the dog red cell membrane to glucose. These data provide evidence supporting the existence of equivalent pores in single biological membranes.     

Metabolically derived potential on the outer membrane of mitochondria: a computational model

The outer mitochondrial membrane (OMM) is permeable to various small substances because of the presence of a voltage-dependent anion channel (VDAC). The voltage dependence of VDAC's permeability is puzzling, because the existence of membrane potential on the OMM has never been shown. We propose that steady-state metabolically derived potential (MDP) may be generated on the OMM as the result of the difference in its permeability restriction for various charged metabolites. To demonstrate the possibility of MDP generation, two models were considered: a liposomal model and a simplified cell model with a creatine kinase energy channeling system. Quantitative computational analysis of the simplified cell model shows that a MDP of up to -5 mV, in addition to the Donnan potential, may be generated at high workloads, even if the OMM is highly permeable to small inorganic ions, including potassium. Calculations show that MDP and DeltapH, generated on the OMM, depend on the cytoplasmic pH and energy demand rate. Computational modeling suggests that MDP may be important for cell energy metabolism regulation in multiple ways, including VDAC's permeability modulation and the effect of electrodynamic compartmentation. The osmotic pressure difference between the mitochondrial intermembrane space and the cytoplasm, as related to the electrodynamic compartmentation effects, might explain the morphological changes in mitochondria under intense workloads.     

Modifying effect of concanavalin A and diamide on erythrocyte sensitivity to cold shock]

The temperature (0 degrees C and 37 degrees C) and the medium tonicity (0.15-1.20 M NaCl) were shown to affect erythrocyte agglutination by concanavalin A. Treatment of cells with lectin caused no significant decrease in the erythrocyte hemolysis upon cooling. Diamide, unlike concanavalin A used at concentrations above 2.0 M decreases the cell sensitivity to the cold shock. The changes in the erythrocyte susceptibility to cooling within the temperature range of 37-0 degrees C correlate with changes in the electrophoretic spectrum of membrane proteins. The progressive decrease in the spectrin bands intensity with a simultaneous formation of high molecular weight protein aggregates not included in the gel composition was observed after diamide treatment. The diamide effect depends on the medium tonicity, at which the treatment was performed, being especially well pronounced in hypertonic media with 0.8-1.2 M NaCl concentrations, the maximal spectrin aggregation being observed under these conditions. It is suggested that the main factor of the mechanism underlying the erythrocyte hypertonic cold shock is the increase in the association of peripheral cytoskeleton proteins with plasma membrane in osmotically dehydrated cells which limits the ability of lipids to adapt during cooling and results in the stabilization of defects in the membrane structure at low temperatures. Diamide eliminates these unfavourable changes eventually resulting in the dissociation of peripheral proteins from the cytoplasmic surface of the membrane on the protein aggregation.     

Membranotropic effect of phosphocreatine and its structural analogs]

The effects of phosphocreatine (PCr) and its analogues (creatine, phosphocreatinine, phosphoarginine and inorganic phosphate) on liposomal and erythrocyte membranes and on the sarcolemmal membrane of cardiomyocytes were studied. The ESR spectrum of the spin-labeled probe, 5-doxyl-stearate, incorporated into the membrane were recorded for analysis of the structural order of the phospholipid bilayer of these membranes. PCr and its analogues had no effect on the structure of the phospholipid bilayer in liposomes; this effect was temperature-independent. However, in erythrocyte and sarcolemmal membranes the rigidity of the membranes was increased by these compounds (except for creatine) at temperatures above 38-40 degrees C. Analysis of these and literary data revealed that cardiac cell membranes may be the site of protective action of PCr on the ischemic myocardium. The lack of effect on liposomes may suggest that the membrane-stabilizing effect of PCr depends on the presence of membrane proteins. The compounds under study may influence the lipid-protein interactions by increasing the rigidity of membrane phospholipids. These membranotropic effects may be due to the interaction of charged molecules of the compounds with polar heads of phospholipids and/or polar groups of proteins in the membrane interphase which, in turn, may influence the packing of hydrophobic fatty acid chains.     

Hemolysis of human red blood cells by freezing and thawing in solutions containing polyvinylpyrrolidone: relationship with postthypertonic hemolysis and solute movements

An investigation was made into the effects of the presence of polyvinylpyrrolidone (PVP) on changes in human red blood cells suspended in hypertonic solutions, on posthypertonic hemolysis, and on freezing at temperatures down to ?12 °C.PVP is very effective at reducing hemolysis when the red blood cells are frozen at temperatures down to ?12 °C. However, the membranes of the cells recovered on thawing have become very permeable to sodium and potassium ions and there is a much increased hemolysis if the cells are resuspended in an isotonic solution of sodium chloride.The presence of PVP does not affect the dehydration of the cells or the development of a change in membrane permeability when the cells are shrunken in hypertonic solutions at 0 °C. Neither does its presence in the hypertonic solution reduce the extent of posthypertonic hemolysis at 4 °C (as measured by the hemolysis on resuspension in an isotonic solution of sodium chloride), but it is more effective than sucrose at reducing hemolysis when present in the resuspension solution. It is concluded that the PVP is able to prevent swelling and hemolysis of cells which are very permeable to cations by opposing the colloid osmotic pressure due to the hemoglobin. However, this does not explain how PVP is able to protect cells against freezing damage at high cooling rates, and a mechanism by which it might do this is discussed.     

Effect of chilling temperatures on osmotic water permeability and aquaporin activity in the plasma membranes from pea roots

The osmotic water permeability of plasma membrane vesicles was examined after isolation from the roots of 7-day-old etiolated pea ( Pisum sativum, cv. Orlovchanin) seedlings grown at optimal temperature and those exposed to 1-day chilling at 8°C in the end of the growth period. The homogenization medium for obtaining plasma membranes was supplemented with either SH-reagents or protein phosphatase inhibitors. The plasmalemma vesicles were purified from the microsome fraction by means of two-phase polymer system. The osmotic water permeability of membrane vesicles was evaluated from the rate of their osmotically induced shrinkage. The lowering of growth temperature was accompanied by the increase in osmotic water permeability of plasmalemma. These changes occurred without the corresponding increase in aquaporin content or permeability of membrane lipid matrix. The membranes from cooled seedlings were markedly depleted in the content of SH-groups. Furthermore, the treatment of membrane samples with a thiol-reducing agent, tributylphosphine did not raise the SH-group content in membranes from chilled plants, unlike such changes in membranes from warm-grown plants. When the homogenization medium contained dithiothreitol and phenylarsine oxide (an inhibitor of tyrosine protein phosphatases), the osmotic permeability of plasmalemma in preparations from warm-grown seedlings also increased. Based on these results, it is supposed that aquaporin-mediated water permeability of membranes is regulated through different pathways under optimal and adverse conditions for plant growth. Direct action of endogenous SH redox regulators on aquaporin activity is likely under optimal growth conditions, while protein phosphatase might mediate changes in aquaporin activity under unfavorable growth conditions.     

Permeability properties of the membrane of vesicular stomatitis virions

Observations of the light-scattering properties of several enveloped viruses indicate that virions (vesicular stomatitis, SV5 and influenza), in common with other membrane systems, are osmotically active, responding to NaCl gradients by swelling in hypo-osmolar solutions and shrinking in hyperosmolar solutions. The permeability barrier responsible for this osmotic response in vesicular stomatitis virions was modified both by protease treatment to remove the viral glycoprotein and by treatment with the polyene antibiotic filipin, an agent known to interact with cholesterol in liposomes and membranes. Filipin altered the kinetic and equilibrium permeability behavior of virions but the extent of leakage of osmotic shocking agent was less than that in lecithin/cholesterol and lecithin/ergosterol liposomes and in ergosterol-containing ciliary membranes. Negative-staining electron microscopy revealed that filipin treatment caused structural changes in the viral membrane. Intact virions exhibited appreciably larger responses to osmotic change than did protease-treated virus particles. Thus, the osmotic barrier in intact vesicular stomatitis virions may not be exclusively lipid in nature.     

Permeability properties of the membrane of vesicular stomatitis virions.

Observations of the light-scattering properties of several enveloped viruses indicate that virions (vesicular stomatitis, SV5 and influenza), in common with other membrane systems, are osmotically active, responding to NaCl gradients by swelling in hypo-osmolar solutions and shrinking in hyperosmolar solutions. The permeability barrier responsible for this osmotic response in vesicular stomatitis virions was modified both by protease treatment to remove the viral glycoprotein and by treatment with the polyene antibiotic filipin, an agent known to interact with cholesterol in liposomes and membranes. Filipin altered the kinetic and equilibrium permeability behavior of virions but the extent of leakage of osmotic shocking agent was less than that in lecithin/cholesterol and lecithin/ergosterol liposomes and in ergosterol-containing ciliary membranes. Negative-staining electron microscopy revealed that filipin treatment caused structural changes in the viral membrane. Intact virions exhibited appreciably larger responses to osmotic change than did protease-treated virus particles. Thus, the osmotic barrier in intact vesicular stomatitis virions may not be exclusively lipid in nature.     

Localization of DnaK (chaperone 70) from Escherichia coli in an osmotic-shock-sensitive compartment of the cytoplasm.

The chaperone DnaK can be released (up to 40%) by osmotic shock, a procedure which is known to release the periplasmic proteins and a select group of cytoplasmic proteins (including thioredoxin and elongation factor Tu) possibly associated with the inner face of the inner membrane. As distinct from periplasmic proteins, DnaK is retained within spheroplasts prepared with lysozyme and EDTA. The ability to isolate DnaK with a membrane fraction prepared under gentle lysis conditions supports a peripheral association between DnaK and the cytoplasmic membrane. Furthermore, heat shock transiently increases the localization of DnaK in the osmotic-shock-sensitive compartment of the cytoplasm. We conclude that DnaK belongs to the select group of cytoplasmic proteins released by osmotic shock, which are possibly located at Bayer adhesion sites, where the inner and outer membranes are contiguous.     

Osmotic tolerance limits and membrane permeability characteristics of stallion spermatozoa treated with cholesterol

Stallion spermatozoa exhibit osmotic damage during the cryopreservation process. Recent studies have shown that the addition of cholesterol to spermatozoal membranes increases the cryosurvival of bull, ram and stallion spermatozoa, but the exact mechanism by which added cholesterol improves cryosurvival is not understood. The objectives of this study were to determine if adding cholesterol to stallion sperm membranes alters the osmotic tolerance limits and membrane permeability characteristics of the spermatozoa. In experiment one, stallion spermatozoa were treated with cholesterol-loaded cyclodextrin (CLC), subjected to anisotonic solutions and spermatozoal motility analyzed. The spermatozoa were then returned to isotonic conditions and the percentages of motile spermatozoa again determined. CLC treatment increased the osmotic tolerance limit of stallion spermatozoa in anisotonic solutions and when returned to isotonic conditions. The second and third experiments utilized an electronic particle counter to determine the plasma membrane characteristics of stallion spermatozoa. In experiment two, stallion spermatozoa were determined to behave as linear osmometers. In experiment three, spermatozoa were treated with CLC, incubated with different cryoprotectants (glycerol, ethylene glycol or dimethyl formamide) and their volume excursions measured during cryoprotectant removal at 5° and 22 °C. Stallion spermatozoa were less permeable to the cryoprotectants at 5 °C than 22 °C. Glycerol was the least permeable cryoprotectant in control cells. The addition of CLC’s to spermatozoa increased the permeability of stallion spermatozoa to the cryoprotectants. Therefore, adding cholesterol to spermatozoal membranes reduces the amount of osmotic stress endured by stallion spermatozoa during cryopreservation.     

Membrane-damaging action of staphylococcal alpha-toxin on phospholipid-cholesterol liposomes

The mechanism of membrane damage by staphylococcal alpha-toxin was studied using carboxyfluorescein (internal marker)-loaded multilamellar liposomes prepared from various phospholipids and cholesterol. Liposomes composed of phosphatidylcholine or sphingomyelin and cholesterol bound alpha-toxin and released carboxyfluorescein in a dose dependent manner, when they were exposed to alpha-toxin of concentrations higher than 1 or 8 micrograms/ml, respectively. In contrast, the other liposomes composed of phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol or phosphatidylinositol plus cholesterol were not susceptible to the toxin even at high concentrations up to 870 micrograms/ml. The insensitive liposomes containing either phosphatidylserine or phosphatidylglycerol were made sensitive to alpha-toxin by inserting phosphatidylcholine into the liposomal membranes. In addition, phosphorylcholine inhibited the toxin-induced marker release from liposomes. These results indicated that the choline-containing phospholipids are required for the interaction between alpha-toxin and liposomal membranes. Susceptibility of liposomes containing phosphatidylcholine or sphingomyelin increased with the increase in cholesterol contents of the liposomes. Based on these results, we propose that the choline-containing phospholipids are possible membrane components or structures responsible for the toxin-membrane interaction, which leads to damage of membranes. Furthermore, cholesterol may facilitate the interaction between alpha-toxin and membrane as a structural component of the membrane.     

Asymmetric distribution of nitrate reductase subunits in the cytoplasmic membrane of Escherichia coli: evidence derived from surface labeling studies with transglutaminase.

The enzyme transglutaminase has been used to label surface proteins of Escherichia coli cytoplasmic membranes by covalently attaching to them a small fluorescent primary amine, dansyl cadaverine. Spheroplasts lacking outer membrane, osmotically lysed vesicles from the spheroplasts, and vesicles made by breaking cells in a French pressure cell were each labeled with transglutaminase and dansyl cadaverine. When the total cytoplasmic membrane proteins of each were examined on sodium dodecyl sulfate gels, three rather different labeling patterns were obtained. Labeling of the respiratory enzyme, nitrate reductase, in the membranes of each of these preparations was also examined. Membrane-bound nitrate reductase contains three subunits: A, B, and C. Dansyl cadaverine labeling of nitrate reductase in the presence of Triton X-100 indicated that subunits A and C could be labeled. When nitrate reductase was isolated from dansyl cadaverine-labeled spheroplasts, none of the subunits was labeled. When nitrate reductase was isolated from French press vesicles, subunit A was labeled and labeling was enhanced by the presence of nitrate during labeling. When nitrate reductase from osmotic vesicles was examined, subunit A was labeled in the presence of nitrate but no labeled subunits appeared when the vesicles were labeled in the absence of nitrate. It was concluded that (i) nitrate reductase is buried in the membrane with subunit A exposed only on the inner surface of the membrane, (ii) subunit C is sufficiently buried within the membrane so that it is inaccessible to transglutaminase, (iii) subunit B is not labeled under any condition, so its location is not known, and (iv) large osmotic vesicles are probably mosaics in which some protein components have been reoriented.     

Membrane association of the Escherichia coli enterobactin synthase proteins EntB/G, EntE, and EntF

The cytosolic proteins EntE, EntF, and EntB/G, which are Escherichia coli enzymes necessary for the final stage of enterobactin synthesis, are released by osmotic shock. Here, consistent with the idea that cytoplasmic proteins found in shockates have an affinity for membranes, a small fraction of each was found in membrane preparations. Two procedures demonstrated that the enzymes were enriched in a minor membrane fraction of buoyant density intermediate between that of cytoplasmic and outer membranes, providing indirect support for the notion that these proteins have a role in enterobactin excretion as well as synthesis.