Lipid Bilayer Of Plasma Membrane Research Articles

Differences in the Transbilayer and Lateral Motions of Fluorescent Analogs of Phosphatidylcholine and Phosphatidylethanolamine in the Apical Plasma Membrane of Bovine Aortic Endothelial Cells

In the plasma membrane of various eucaryotic cell types, in particular blood platelets and erythrocytes, it is known that phospholipids are asymmetrically distributed between the two leaflets of the lipid bilayer and that this transverse asymmetry is controlled by an aminophospholipid translocase activity. In this respect, it was of interest to check whether there are differential transbilayer movements between amino- and neutral phospholipids in the apical plasma membrane of vascular endothelial cells which form the inner nonthrombogenic lining of the large blood vessel. In the first step we compared the transbilayer localization and also the rate of lateral motion of two fluorescent analogs of phosphatidylcholine and phosphatidylethanolamine, namely C6-NBD-PC and C6-NBD-PE, inserted into the apical plasma membrane of bovine aortic endothelial cells, in vitro. By the use of back-exchange experiments we have found that C6-NBD-PC could be removed from the cell membrane toward the culture medium regardless of the incubation conditions used, i.e., just after cell labeling at 0°C or even after further cell incubation for 1 h at 0 or 20°C. In contrast, C6-NBD-PE could be removed only when the cells were maintained at 0°C. After incubation for 1 h at 20°C, 85% of the probe molecules remained nonexchangeable, indicating probe translocation from the outer to the inner leaflet of the lipid bilayer. This "flip" process, which occurred at 20°C, was abolished when the endothelial cells were preincubated with N-ethylmaleimide, diamide, vanadate (VO 3- 4) and vanadyl (VO 2+) ions, a set of substances which inhibit aminophospholipid translocase activity in various systems, and with a combination of sodium azide and 2-deoxyglucose which led to nearly complete ATP depletion in the cells. Fluorescence recovery after photobleaching experiments were also carried out to specify more precisely the localization and dynamics of the probes in the two leaflets of the plasma membrane lipid bilayer. They produced lateral diffusion coefficients D of 1.2 ± 0.05 × 10 -9 cm 2/s for C6-NBD-PC and 2.8 ± 0.3 × 10 -9 cm 2/s for C6-NBD-PE, when the two probes were located in the outer leaflet of the plasma membrane, just after cell labeling at 0°C. After cell incubation for one hour at 20°C, i.e., when C6-NBD-PC was still in the outer leaflet whereas C6-NBD-PE was translocated in the inner leaflet, D was observed to slightly increase for C6-NBD-PC ( D = 1.9 ± 0.06 × 10 -9 cm 2/s) and to greatly increase by at least a factor of 3 for C6-NBD-PE ( D = 9.1 ± 0.9 × 10 -9 cm 2/s). These results show that the plasma membrane of bovine aortic endothelial cells is equipped with a protein-dependent and energy-mediated phosphatidylethanolamine translocase activity and that the lateral diffusion rate of this phospholipid is much faster in the inner than in the outer leaflet of the lipid bilayer, thus indicating large differences in the fluidity of the two halves of this membrane.

Synthetic peptides corresponding to the calmodulin-binding domains of skeletal muscle myosin light chain kinase and human erythrocyte Ca2+ pump interact with and permeabilize liposomes and cell membranes.

Synthetic calmodulin-binding (CaM-binding) peptides (CBPs) representing CaM-binding domains of Ca2+/CaM-dependent enzymes have been reported to interfere with the activity of the melanocyte-stimulating hormone (MSH) receptor function in melanoma cells [Gerst, J. E., & Salomon, Y. (1988) J. Biol. Chem. 263, 7073-7078]. We postulated that membrane lipids may play an important role in the mode of action of CBPs on cells. We therefore tested the ability of CBPs to interact with membrane bilayers. Using artificial phospholipid vesicles, or M2R melanoma cells and cell membranes derived therefrom, as models, we report here that synthetic peptides representing the CaM-binding domains of skeletal muscle myosin light chain kinase (M5) and the human erythrocyte calcium pump (C28W), as well as other CBPs, interact with lipid bilayers and cell membranes. Significant interactions of CBPs with the lipid bilayer were detected in both model systems. M5 and C28W were found to partition into the lipid bilayer of melanoma cell membranes and soybean lecithin vesicles, and surface partition constants obtained (for the liposome model) were in the range 10(3)-10(4) M-1. In addition, C28W and its N-modified NBD derivative were found to inhibit [125I]iodo-[Nle4,D-Phe7]alpha MSH binding to cultured M2R melanoma cells. These and other CBPs were also found to induce the release of cations and calcein from liposomes, suggesting that the interaction of CBPs with the lipid bilayer increases membrane permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

X-ray studies on phospholipid bilayers. XII. Interactions of pentachlorophenol with myelin.

Pentachlorophenol (PCP) is a widely used pesticide, particularly for the preservation of wood. Given its high toxicity and resistance to degradation it has become a dangerous environmental pollulant. Due to its high lipophilicity, PCP is able to partition into the lipid bilayer of cell membranes disrupting several vital functions. The present research was concerned with the effects that the chronic administration of PCP could produce in vivo to the sciatic nerve of rats. X-ray diffraction patterns obtained from freshly dissected and dried sciatic nerves of PCP treated rats did not show significant differences in their reflections with respect to those present in the patterns from untreated animals. However, morphological studies performed by optical and electron microscopy showed degenerative changes in about 10% of the A and B type of nerve fibers.

Neuronal protein NP185 is developmentally regulated, initially expressed during synaptogenesis, and localized in synaptic terminals.

Evidence is presented here that demonstrates the presence of NP185 (AP3) in neuronal cells, specifically within syn-aptic terminals of the central nervous system and in the peripheral nervous system, particularly in the neuro-muscular junction of adult chicken muscle. Biochemical results obtained in our laboratories indicate that NP185 is associated with brain synaptic vesicles, with clathrin-coated vesicles, and with the synaptosomal plasma membrane. Also, NP185 binds to tubulin and clathrin light chains and the binding is regulated by phosphorylation (Su et al., 1991). Based on these properties and the data reported here, we advance the postulate that NP185 fulfills multiple functions in synaptic terminals. One function is that of a plasma membrane docking or channel protein, another of a signaling molecule for brain vesicles to reach the synaptic terminal region, and a third is that of a recycling molecule by binding to protein components on the lipid bilayer of the synaptic plasma membrane during the process of endocytosis. In support of these premises, a thorough study of NP185 using the developing chick brain, adult mouse brain, and chicken straited muscle was begun by temporally and spatially mapping the expression and localization of NP185 in evolving and mature nerve endings. To achieve these objectives, monoclonal antibodies to NP185 were used for immunocytochemistry in tissue sections of chicken and mouse cerebella. The distribution of NP185 was compared with those of other cytoskeletal and cytoplasmic proteins of axons and synapses, namely synaptophysin, vimentin, neurofilament NF68, and the intermediate filaments of glial cells (GFAP). The data indicate that expression of NP185 temporally coincides with synaptogenesis, and that the distribution of this protein is specific for synaptic terminal buttons of the CNS and the PNS.

Changes in erythrocyte membrane lipid composition affect the transient decrease in membrane order which accompanies insulin receptor down-regulation

We have recently demonstrated, using electron paramagnetic resonance (EPR) spectroscopy, that insulin receptor internalization in response to insulin incubation (down-regulation) in human erythrocytes is accompanied by a transient decrease in membrane order, as measured by the 2T' parallel order parameter. Since membrane lipids play such an important role in receptor internalization, we investigated the possible effects that an alteration of the normally-occurring lipid profile might have on down-regulation and the concomitant transient decrease in membrane order. Consequently, human erythrocytes enriched with cholesterol and erythrocytes from cirrhotic patients were examined, because both of these groups of cells have a higher cholesterol/phospholipid molar ratio (CH/PL) than controls. The 5-nitroxystearate spin label, which inserts into the lipid bilayer of cell membranes, was used to monitor changes in 2T' parallel for a 3-h period at 37 degrees C. We report here that both cholesterol-enriched and cirrhotic erythrocytes do not down-regulate, as demonstrated by binding assays, and that they do not show the typical transient decrease in membrane order observed in controls. The results seem to indicate that a more ordered membrane inhibits internalization of the insulin receptor in erythrocytes, and that an increase in membrane disorder is necessary for insulin receptor down-regulation.

Oxidant and angiotensin II-induced subcellular translocation of protein kinase C in pulmonary artery endothelial cells.

We recently reported that nitrogen dioxide (NO2), an environmental oxidant, alters the dynamics of the plasma membrane lipid bilayer structure, resulting in increased phosphatidylserine content and angiotensin II (Ang II) receptor binding. Angiotensin II is known to elicit receptor-mediated stimulation of diacylglycerol (DAG) production in pulmonary artery endothelial cells. Because protein kinase C (PKC) is a phosphatidylserine-dependent enzyme and is activated by DAG, we examined whether NO2 resulted in activation and/or translocation of PKC from predominantly cytosolic to membrane fractions of these cells. We also evaluated whether NO2 exposure resulted in increased production of DAG in pulmonary artery endothelial cells. Exposure to 5 ppm NO2 for 1-24 hr resulted in significant increases in PKC activity in the cytosolic and membrane fractions (p less than 0.05 for both fractions) compared to activities in control fractions. Exposure to Ang II resulted in translocation of PKC activity from cytosol to membrane fractions of both control and NO2-exposed cells. This translocation of PKC from cytosolic to membrane fraction was prevented by the specific receptor antagonist [Sar1 Ile8] Ang II. Exposure of 5 ppm NO2 for 1-24 hr provoked rapid increases in [3H]glycerol labeling of DAG in pulmonary artery endothelial cells. These results demonstrate that exposure to NO2 increases the production of second messenger DAG and activates PKC in both the cytosolic and membrane fractions, whereas Ang II stimulates the redistribution of PKC from cytosolic to membrane fractions of pulmonary artery endothelial cells.

‘Pharmacokinetic’ and ‘pharmacodynamic’ design of lipophilic drugs based on a structural model for drug interactions with biological membranes

AbstractThe pharmacokinetic profile a drug exhibits will ultimately depend on how the drug molecule interacts with the different cell membranes it encounters en route to its target site of action. Even if the drug molecule targets a soluble protein as its final site of action, there is a reasonable probability that it will interact with some cell membrane on its pathway to this biological target. In some cases, the final protein, lipid or sugar moiety target could be an integral part of the cell membrane architecture whereby the drug's interaction with the cell membrane could influence its pharmacodynamic profile as well, especially if the final site of action is an integral membrane‐bound receptor protein. In either case, drug transport across a cell membrane or within the cell membrane lipid bilayer compartment can dictate partial or dominant control of the drug's bioavailability. A molecular understanding of the equilibrium membrane partition coefficient, the rates of drug partitioning into and out of a cell membrane and the equilibrium location of the drug within the cell membrane can provide critical chemical design strategies for altering overall drug bioavailability and optimal pharmacokinetics.

The relationship of chemical modification of membrane proteins and plasma lipoproteins to reduced membrane fluidity of erythrocytes from diabetic subjects.

The significance of the two most common hallmarks of the diabetic state, hyperglycaemia and hyperlipidaemia, was investigated in terms of disorders of cell membrane dynamics. In order to examine whether the alterations in cell membrane lipid bilayer dynamics are somehow related to protein chemical modifications in plasma low-(LDL) and high-density lipoproteins (HDL) and blood cell membranes, we compared 19 poorly controlled diabetic subjects with 19 age- and sex-matched controls. The extent of (non-enzymatic) glycation, lipid peroxidation and the cholesterol/phospholipid ratio were increased in plasma low density lipoproteins and high density lipoproteins from diabetic patients. The mean steady-state fluorescence polarization values in 1,6-diphenyl-1,3,5-hexatriene-labelled isolated erythrocyte membranes from diabetic subjects were significantly greater than from control subjects (0.186 +/- 0.008 vs 0.173 +/- 0.006, p < 0.001); the fluorescence polarization values in erythrocyte membranes from diabetic and control subjects positively correlated with the extent of membrane protein glycation, lipid peroxidation and the cholesterol content. The cholesterol to phospholipid molar ratios in low density lipoproteins and high-density lipoproteins from diabetic and control subjects correlated significantly with the fluorescence polarization values in erythrocyte membranes from these subjects. Furthermore, the extent of glycation of low density lipoproteins appears to be strongly correlated with the extent of lipoprotein lipid peroxidation (r = 0.789, p < 0.001). The atherosclerotic potential of plasma lipoproteins in diabetes mellitus was discussed in terms of membrane and plasma protein chemical modifications.

Antagonism of platelet‐activating factor in isolated rat colon: Possible mechanism

The contractions of three different regions of rat colon in response to platelet-activating factor (PAF) were compared. The ascending colon was found to be the most responsive. The slow contraction of the ascending colon induced by PAF was dependent on external Ca2+. CV-3988, a structural analog of PAF, slowly induced irreversible inhibition of PAF-induced contraction, whereas FR-900452, which is structurally unrelated to PAF, caused rapid reversible inhibition of PAF-induced contraction. No inhibitory effects of CV-3988 were observed when the strip was washed with Tyrode's solution containing 1% bovine serum albumin (BSA). The results suggest that PAF and CV-3988 penetrate slowly into the outer half of the lipid bilayer of plasma membranes of cells in isolated rat colon, and then rapidly diffuse laterally to associate firmly with specific binding sites.

Molecular architecture and dynamics of the plasma membrane lipid bilayer: The red blood cell as a model

The structural backbone of the erythrocyte membrane, as well as that of any other plasma membrane, is provided by a lipid bilayer which is composed of more than 100 molecular species. The individual lipid classes are distributed over both halves of the bilayer in a highly asymmetric fashion: all glycolipids are exclusively confined to the outer membrane leaflet where we also find the majority of the two choline-containing phospholipids, sphingomyelin and phosphatidylcholine. The two amino-phospholipids are predominantly (phosphatidylethanolamine) or even exclusively (phosphatidylserine) localized in the cytoplasmic half of the bilayer. Glycolipids and sphingomyelin are not subject to transbilayer movements, a property that (under normal conditions) is shared by phosphatidylserine. Phosphatidylcholine exhibits a relatively slow transbilayer movement, revealing half-time values from 3 to 27 h, whereas phosphatidylethanolamine is subject to an ATP-dependent "flippase"-catalyzed inward movement with a half-time of approximately 30 min. Probably much faster than that of any other lipid, is the transbilayer movement of cholesterol, revealing a half-time value in the order of seconds.

Strategies of Nutrient Transport by Ruminal Bacteria

The survival of bacteria in natural environments like the rumen depends on the ability of the bacteria to scavenge nutrients. It is now evident that ruminal bacteria use a variety of transport mechanisms. Hydrophobic substances, such as ammonia and acetate, are permeable to the lipid bilayers of cell membranes and can be taken up by passive diffusion. Hydrophilic compounds (e.g., sugars, amino acids, peptides) do not easily pass through lipid bilayers and must be transported across cell membranes on carrier proteins. Facilitated diffusion can display saturable kinetics but does not result in accumulation of solute. Active transport can establish extremely high concentration gradients, and this work may be driven by the hydrolysis of chemical bonds (e.g., ATP) or ion gradients, which are coupled to solute symport. Many solute symports involve protons, but sodium systems also are common in ruminal bacteria. The phosphotransferase system chemically modifies sugars as they pass across the cell membrane, and several ruminal bacteria have this method of group translocation. Many feed additives have either a direct or indirect effect on rumen bacterial transport. For instance, ionophores can inhibit transport by destroying (sometimes even reversing) ion gradients, lowering intracellular pH, or causing excessive ATP hydrolysis.

Human erythrocyte insulin receptor down-regulation is accompanied by a transient decrease in membrane order

Insulin receptor internalization in response to insulin incubation (down-regulation) has been shown to occur in human erythrocytes as well as in human erythrocyte ghosts. It is also known that changes in cell membrane events can be detected with electron paramagnetic resonance (EPR) spectroscopy using spin labels. In the present study, changes in erythrocyte membrane order during down regulation as measured by the 2 T′ II parameter were investigated using EPR. The spin label, 5-nitroxystearate, which inserts into the lipid bilayer of cell membranes, was used. Changes in 2 T′ II at 37° C were followed over a 3 h time period. A transient decrease in erythrocyte membrane order began within 30 min of the start of insulin incubation and reached a minimum level of 52.5 Gauss (G) within 90 min. This represented nearly a 2 G decrease from the zero incubation time value. Membrane order returned to the initial value by 2.5 h. These time-related changes in membrane order corresponded well with the insulin receptor internalization process as followed by surface binding assays. Surface insulin binding began to decrease within 30 min of the start of insulin incubation, and was reduced to 30% of control values within 2 h. Similar correlations between membrane order and receptor internalization were observed at 23° C. Erythrocytes incubated with denatured insulin, and ATP-depleted erythrocytes incubated with native insulin, did not down-regulate their insulin receptors. Under these conditions, these erythrocytes also did not exhibit the transient decreases in membrane order. These findings are consistent with the hypothesis that an increase in membrane disorder is part of the mechanism of insulin receptor down-regulation.

Antimutagens from Momordica charantia

The antimutagenic principle of the green fruits of Momordica charantia was shown by the micronucleus test to be an intractable mixture of novel acylglucosylsterols. The antimutagens were extracted from the green fruits with ethanol and isolated from the bioactive petroleum ether and carbon tetrachloride extracts by repeated and sequential flash column chromatography. The major component of the mixture is 3-O-[6′-O- palmitoyl-β- d- glucosyl- stigmasta-5,25(27)- dien and the minor component is the stearyl derivative (Guevara, 1989). At a dosage range in mice of 50–12.5 μg extract/g, the mixture reduced by about 80% the number of micronucleated polychromatic erythrocytes induced by the well-known mutagen mitomycin C. Structure-activity correlation studies suggested that the antimutagenic activity may reside in the peculiar lipid-like structure of the acylglucosylsterols. Ingestion of these compounds may result in their absorption in the plasma membrane lipid bilayer which could adversely affect the membrane permeability towards mitomycin C and disrupt the cellular activity of the latter.

Interferon-α Modulates the Plasma Membrane–Cytoskeletal Complex of Human Lymphoblastoid Cells Sensitive to the Antiproliferative Action of Interferon-α

Interferon (IFN)-induced structural changes in the plasma membrane-cytoskeletal complex were investigated in human lymphoblastoid cell lines that differed in sensitivity to the antiproliferative action of IFN-alpha. These cell lines have structurally indistinguishable high-affinity IFN-alpha receptors. By using electron spin resonance techniques, we determined the effect of IFN-alpha on fluidity of the plasma membrane lipid bilayer. By using the ability of cells to redistribute fluorescent anti-immunoglobulin (Ig) into caps at the cell surface, we investigated the effect of IFN-alpha on the mobility of the cell surface. IFN-alpha decreases the fluidity of the plasma membrane lipid bilayer of lymphoblastoid cells sensitive to the antiproliferative action of IFN-alpha. Similarly, IFN-alpha inhibits the anti-Ig-induced redistribution of surface immunoglobulins (capping) of IFN-sensitive cells. These effects of IFN-alpha occur within the first few hours after IFN-alpha addition to cells. However, IFN-alpha had no effect on membrane fluidity or anti-Ig-induced capping of lymphoblastoid cells resistant to the antiproliferative action of IFN-alpha.

Effect of Vitamin E Deficiency on Oxidative Metabolism and Antioxidant Enzyme Activity of Macrophages

In AUG rats, deprived of vitamin E for 90 days, we noted a 3-fold increase of kinetic parameters of luminol-dependent chemiluminescence of macrophages, stimulated with opsonized zymosan, superoxide dismutase activity decrease and increment of plasma membrane lipid bilayer microviscosity, which was estimated by fluorescent probe pyrene eximerization method. Vitamin E deficiency did not affect glutathione peroxidase and glutathione reductase activities of macrophages.

Stable cell membrane labelling.

Binding fluorescent or radioactive reporter molecules to the lipid bilayer of cell membranes allows cell growth and trafficking to be monitored in vivo.

Accumulation of lipid peroxidation products in human cataracts.

The role of free-radical-induced lipid peroxidation (LPO) in the development of human lens opacity was investigated. 130 human crystalline lenses impaired with cataract of various etiology (senile, traumatic, complicated cataracts) at different stages of cataract development as well as 18 transparent human lenses were studied. Primary products of LPO (diene conjugates), secondary products (ketodienes) and the end products (Schiff bases) were distinctly accumulated in cataractous lenses. Initial stages of cataract were characterized by the accumulation of primary LPO products, while in the latest stages there was a prevalence of fluorescent end products of LPO. It was concluded that the accumulation of polar hydroxy groups in the lipid bilayer of plasma membranes of lenticular fibers is one of the possible causes of their injury in cataracts.

Morphological changes in human spermatozoa as examined under scanning electron microscope after [formula omitted] exposure to saponins isolated from [formula omitted

Saponins isolated from Sapindus mukorossi have potent spermicidal activity. Morphological changes in human ejaculated spermatozoa after exposure to these saponins were evaluated under Scanning Electron Microscope. The minimum effective concentration (0.05% in spot test) did not affect the surface topography after exposure for 1 minute. However, incubation of spermatozoa for 10 minutes resulted in extensive vesiculation and disruption of plasma membrane in the head region. Higher concentrations (0.1%, 1.25%, 2.5% and 5.0%) caused more or less similar changes which included vesiculation, vacuolation, disruption or erosion of membranes in the head region. These findings suggest that the morphological changes observed are due to alterations in the glycoproteins associated with the lipid bilayer of plasma membrane of spermatozoa.

Interaction of amphiphilic polymers with model membranes

AbstractThe lipid bilayers of cell membranes are usually connected to two types of biopolymers: the cytoskeleton and the glycocalix. These structures are not only responsible for the stabilization of the lipid bilayer, they also alter the surface properties of the cells. Among the various attempts to mimick membrane properties using polymeric systems(1), one method is the fixation of amphiphilic polymers via hydrophobic anchor groups to lipid bilayers. In the experiments reported here the influence of amphiphilic polymers on the morphology of giant unilamellar liposomes has been investigated. Therefore a series of amphiphilic polyvinylpyrrolidone and dextran derivatives has been synthesized. The fixation of these polymers to the liposomal membranes could be shown with a fluorescence‐labelled derivative by using epifluorescence microscopy. The polymer‐membrane interaction caused by the insertion of the anchor groups leads to drastic morphological changes in this model membrane system. The exclusive interaction with the outer membrane of multilamellar liposomes could be demonstrated by freeze fracture electron microscopy. In addition, the anchoring of a natural capsular polysaccharide in liposomes has been proven by immunofluorescence. In these experiments, also a clustering (patching) of the antigen caused by the binding of the antibody could be observed.

The mechanisms of action of nonsteroidal antiinflammatory drugs.

In mammalian species and in the oldest of multicellular animal forms, NSAIDs inhibit cell activation, apparently in the absence of effects on PG biosynthesis. Thus, an alternative hypothesis can be proposed to account for the antiinflammatory effects of these drugs. Clearly, at low doses aspirin and most of the newer NSAIDs inhibit the biosynthesis of PGs from arachidonic acid, and stable PGs have been shown to mediate fever, hyperalgesia, vasodilation (edema), and several interleukin-1-dependent responses. At high doses, however, aspirin, sodium salicylate, and the newer NSAIDs (at antiinflammatory doses) inhibit non-PG-dependent processes, such as the activity of a variety of enzymes, proteoglycan synthesis by chondrocytes, transmembrane ion fluxes, and chemoattractant binding. These effects are most likely due to the capacity of aspirin-like drugs to insert into the lipid bilayer of plasma membranes, where they disrupt normal signaling events and protein-protein interactions. The ability of NSAIDs to thereby inhibit the activation of inflammatory cells such as the neutrophil may contribute to the antiinflammatory properties of this class of drugs.