Fluid shear stress increases membrane fluidity in endothelial cells: a study with DCVJ fluorescence

Fluid shear stress (FSS) has been shown to be an ubiquitous stimulator of mammalian cell metabolism. Although many of the intracellular signal transduction pathways have been characterized, the primary mechanoreceptor for FSS remains unknown. One hypothesis is that the cytoplasmic membrane acts as the receptor for FSS, leading to increased membrane fluidity, which in turn leads to the activation of heterotrimetric G proteins (13). 9-(Dicyanovinyl)-julolidine (DCVJ) is a fluorescent probe that integrates into the cell membrane and changes its quantum yield with the viscosity of the environment. In a parallel-plate flow chamber, confluent layers of DCVJ-labeled human endothelial cells were exposed to different levels of FSS. With increased FSS, a reduced fluorescence intensity was observed, indicating an increase of membrane fluidity. Step changes of FSS caused an approximately linear drop of fluorescence within 5 s, showing fast and almost full recovery after shear cessation. A linear dose-response relationship between shear stress and membrane fluidity changes was observed. The average fluidity increase over the entire cell monolayer was 22% at 26 dyn/cm(2). This study provides evidence for a link between FSS and membrane fluidity, and suggests that the membrane is an important flow mechanosensor of the cell.     

Effect of 8-alkylberberine homologues on erythrocyte membrane

8-alkylberberine homologues (Ber-C8-n, where n indicates carbon atom number of gaseous normal alkyl at 8 position, n = 0, 2, 4, 6, 8, 10, 12, or 16) were synthesized and their effects on the hemolysis of rabbit erythrocyte, the fluidity of membrane and the fluorescence of membrane protein were investigated by fluorescence analysis technique. Ber-C8-n with mediate length alkyl (4 < n < 10) exhibited obvious hemolysis effect on rabbit erythrocyte when their concentration exceed 1.25 x10(-4) mol/L, and Ber-C8-8 displayed the highest hemolysis effect among all tested homologues. All of Ber-C8-n influenced the fluidity of erythrocyte membrane to different extents, which exhibited an obvious dose-effect relationship. The effect of Ber-C8-n on fluidity increased as the length of alkyl chain was elongated and decreased gradually when the alkyl carbon atoms exceeded 8. The fluorescence of erythrocyte membrane protein was quenched by Ber-C8-n, which showed a similar changing tendency on membrane fluidity. Experiments in vitro suggested that disturbing effects of Ber-C8-n on the conformation and function of membrane protein leaded to the changes of membrane fluidity and stability, and then the membrane was broken down.     

Direct effects of ethanol on bone resorption and formation in vitro

In vitro studies indicate that low concentrations of ethanol can have direct effects on bone formation and resorption. Bone resorption was increased when embryonic chick tibiae were exposed to ethanol at 0.03-0.3% (v/v), and bone formation was inhibited when tibiae were exposed to 0.2% ethanol in the presence of NaF or parathyroid hormone (P less than 0.01 for each). Ethanol also had direct effects on isolated bone cells in vitro, increasing both cAMP and PGE2 production (P less than 0.001 for each), and affecting cell proliferation in a biphasic, time- and dose-dependent manner. After 24 h of exposure, 0.03% ethanol increased bone cell proliferation (P less than 0.001), but 0.3% ethanol was inhibitory (P less than 0.01). Paradoxically, mitogenic doses of ethanol prevented the effects of two other mitogens, NaF and human skeletal growth factor, to increase bone cell proliferation (P less than 0.001). But how were these effects produced? Several observations suggest that these direct effects of ethanol on skeletal tissues in vitro were mediated by changes in bone cell membrane fluidity. (a) Dimethyl sulfoxide, ethylene glycol, and lecithin, which act, like ethanol, to increase membrane fluidity, mimicked the effects of ethanol on bone cell proliferation. Dimethyl sulfoxide also mimicked the effect of ethanol to increase cAMP (P less than 0.001). (b) Cholesterol, which decreases cell membrane fluidity, acted oppositely to ethanol and enhanced the mitogenic response to human skeletal growth factor (P less than 0.001). (c) Preincubation of calvarial cells with ethanol or with cholesterol altered the in situ reaction kinetics of the membrane-bound enzyme, alkaline phosphatase. Together, these data demonstrate that ethanol has direct effects on skeletal tissue in vitro, and suggest that those effects may be secondary to changes in bone cell membrane fluidity.     

The role of the plasma membrane fluidity on the shear sensitivity of hybridomas grown under hydrodynamic stress

The role of the plasma membrane fluidity (PMF) on the shear sensitivity of HB-32 hybridomas to laminar fluid shear was investigated. Steady-state fluorescence anisotropy (r(s)) of the cationic fluorescent probe 1-[4-(trimethylamino) phenyl]-6-phenylhexa-1,3,5-triene, was used to evaluate the PMF of whole hybridoma cells. The PMF was manipulated by the addition of the anesthetic benzyl alcohol, by temperature changes and by cholesterol enrichment. The effect of these PMF modifying procedures on the shear sensitivity of HB-32 was assessed by exposing the cells to defined levels of laminar shear stress in a Couette flow device. Conditions that resulted in lower r(s) values (indicating higher PMF) yielded a more fragile cell. Batch cultivations supplemented with the shear protective agent Pluronic(R) F-68 showed higher values of r(s) compared to control experiments during various growth phases, suggesting that the protective mechanism of Pluronic F-68 relies on its ability to decrease the PMF through direct interaction with the plasma membrane. The protective mechanism of serum against turbulent fluid shear is also discussed from analysis of growth and death kinetics of agitated and static cultures at increasing serum levels. The results of this study show that the fluid state of the plasma membrane is important in determining the integrity of hybridomas when exposed to lethal shear levels. It is concluded that increasing membrane fluidity correlates with increasing shear sensitivity.     

Characterization of changes in the viscosity of lipid membranes with the molecular rotor FCVJ

Membrane viscosity is a key parameter in cell physiology, cell function, and cell signaling. The most common methods to measure changes in membrane viscosity are fluorescence recovery after photobleaching (FRAP) and fluorescence anisotropy. Recent interest in a group of viscosity sensitive fluorophores, termed molecular rotors, led to the development of the highly membrane-compatible (2-carboxy-2-cyanovinyl)-julolidine farnesyl ester (FCVJ). The purpose of this study is to examine the fluorescent behavior of FCVJ in model membranes exposed to various agents of known influence on membrane viscosity, such as alcohols, dimethyl sulfoxide (DMSO), cyclohexane, cholesterol, and nimesulide. The influence of key agents (propanol and cholesterol) was also examined using FRAP, and backcalculated viscosity change from FCVJ and FRAP was correlated. A decrease of FCVJ emission was found with alcohol treatment (with a strong dependency on the chain length and concentration), DMSO, and cyclohexane, whereas cholesterol and nimesulide led to increased FCVJ emission. With the exception of nimesulide, FCVJ intensity changes were consistent with expected changes in membrane viscosity. A comparison of viscosity changes computed from FRAP and FCVJ led to a very good correlation between the two experimental methods. Since molecular rotors, including FCVJ, allow for extremely easy experimental methods, fast response time, and high spatial resolution, this study indicates that FCVJ may be used to quantitatively determine viscosity changes in phospholipid bilayers.     

The production of nitric oxide and prostaglandin E(2) by primary bone cells is shear stress dependent

Loading-induced flow of interstitial fluid through the lacuno-canalicular network is a likely signal for bone cell adaptive responses. However, the nature of the stimulus that activates the cell is debated. Candidate stimuli include wall shear stress, streaming potentials, and chemotransport. We have addressed the nature of the flow-derived cell stimulus by comparing variations in fluid transport with variations in wall shear stress, using nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production as a parameter of bone cell activation. Adult mouse long bone cell cultures were treated for 15min with or without pulsating fluid flow using the following regimes: Low PFF, mean flow rate 0.20 cm(3)/s, 3 Hz, shear stress 0.4+/-0.12 Pa; Medium PFF, 0.33 cm(3)/s, 5 Hz, 0.6+/-0.27 Pa; and High PFF, 0.63 cm(3)/s, 9Hz, 1.2+/-0.37 Pa. In some Low PFF experiments, 2.8% neutral dextran (mol. wt. 4.98x10(4)) was added to the flow medium to increase the viscosity, thereby increasing the wall shear stress 3-fold to a level similar of the High PFF stimulus, but without affecting streaming potentials or chemotransport. NO and PGE(2) production were stimulated by Low, Medium, and High PFF in a dose-dependent manner. Application of Low PFF using dextran-supplemented medium, enhanced both the NO and PGE(2) response by 3-fold, to a level mimicking the response to High PFF at normal viscosity. These results show that the production of NO and PGE(2) by bone cells can be enhanced in a dose-dependent manner by fluid flow of increasing wall shear stress. Therefore, the stimulus leading to NO and PGE(2) production is the flow-derived shear stress, and not streaming potentials or chemotransport.     

Effects of solvent polarity and solvent viscosity on the fluorescent properties of molecular rotors and related probes

Fluorescent molecular rotors belong to a group of twisted intramolecular charge transfer complexes (TICT) whose photophysical characteristics depend on their environment. In this study, the influence of solvent polarity and viscosity on several representative TICT compounds (three Coumarin derivatives, 4,4-dimethylaminobenzonitrile DMABN, 9-(dicyanovinyl)-julolidine DCVJ), was examined. While solvent polarity caused a bathochromic shift of peak emission in all compounds, this shift was lowest in the case of molecular rotors. Peak intensity was influenced strongly by solvent viscosity in DMABN and the molecular rotors, but polarity and viscosity influences cannot be separated with DMABN. Coumarins, on the other hand, did not show viscosity sensitivity. This study shows the unique suitability of molecular rotors as fluorescent viscosity sensors.     

Comparison of 9-aminoacridine and atebrine induced changes in optical, electrical and mechanical characteristics of lipid bilayers.

The effects of fluorescent probes 9-aminoacridine (9AA) and atebrine (AT) on physical properties of liposomes and planar bilayer lipid membranes (BLM) were studied. The method of fluorescence spectroscopy and the electrostriction method based on measurement of higher current harmonics were used. At low concentrations (10(-5)-5 x 10(-5) mol/l), 9AA increased fluorescence intensity, while in liposomes from soybean phosphatidylcholine fluorescence quenching occurred at higher probe concentration. Fluorescence quenching occurred over the entire concentration range tested (10(-5)-10(-4) mol/l) in liposomes made from a mixture of egg phosphatidylcholine and cardiolipin. In contrast to 9AA, AT, thanks to its hydrophobic chain, penetrates deeper into the hydrophobic membrane moiety; thus, immobilization of the molecule and an increase in fluorescence intensity was always observed. Probes adsorbed to membranes, leaving their electric capacitance effectively unchanged. Adsorption of charged dye particles induced small changes in transmembrane potential. In the presence of 10(-5) mol/l AT, the modulus of elasticity E perpendicular increased somewhat for soft membranes (E perpendicular approximately 2.5 x 10(7) Pa), whereas it decreased for hard membranes (E perpendicular approximately 5 x 10(7) Pa). pH gradient present on the membrane affected the ability of the dyes to incorporate into the membranes. Our results provide evidence against the proposed model of the quenching mechanism introduced by Rottenberg and Lee (1975).     

Characterization of changes in the viscosity of lipid membranes with the molecular rotor FCVJ

Membrane viscosity is a key parameter in cell physiology, cell function, and cell signaling. The most common methods to measure changes in membrane viscosity are fluorescence recovery after photobleaching (FRAP) and fluorescence anisotropy. Recent interest in a group of viscosity sensitive fluorophores, termed molecular rotors, led to the development of the highly membrane-compatible (2-carboxy-2-cyanovinyl)-julolidine farnesyl ester (FCVJ). The purpose of this study is to examine the fluorescent behavior of FCVJ in model membranes exposed to various agents of known influence on membrane viscosity, such as alcohols, dimethyl sulfoxide (DMSO), cyclohexane, cholesterol, and nimesulide. The influence of key agents (propanol and cholesterol) was also examined using FRAP, and backcalculated viscosity change from FCVJ and FRAP was correlated. A decrease of FCVJ emission was found with alcohol treatment (with a strong dependency on the chain length and concentration), DMSO, and cyclohexane, whereas cholesterol and nimesulide led to increased FCVJ emission. With the exception of nimesulide, FCVJ intensity changes were consistent with expected changes in membrane viscosity. A comparison of viscosity changes computed from FRAP and FCVJ led to a very good correlation between the two experimental methods. Since molecular rotors, including FCVJ, allow for extremely easy experimental methods, fast response time, and high spatial resolution, this study indicates that FCVJ may be used to quantitatively determine viscosity changes in phospholipid bilayers.     

膜融合剂对脂质体及血影膜膜流动性的影响

本文用荧光探针ANS,DPH与A研究了几种膜融合剂对脂质体与血影膜流动性的影响.蔗糖使PS脂质体的脂双层流动性降低,探针越是在极性区流动性越小,说明蔗糖主要作用于脂双层的极性区;蔗糖也使血影膜流动性降低,此作用是可逆的.油酸甘油脂(GMO)使PS脂质体的流动性增加,且越是在疏水区内部,流动性增加得越大,说明GMO主要是作用于脂双层的非极性区:GMO也使血影膜流动性增加,此作用是不可逆的.二甲亚砜(DMSO)对血影膜的作用,两种不同荧光探针不一样,对DPH的作用出现双相让,低浓度与高浓度的作用结果分别与蔗糖和GMO的作用一致.     

Cell suspensions from porcine olfactory mucosa. Changes in membrane potential and membrane fluidity in response to various odorants

A suspension of olfactory epithelial cells was prepared from porcine olfactory mucosa and the physiological functions of the suspension were examined. The membrane potential of the cell suspension, which was monitored by measuring the fluorescence changes of rhodamine 6G, was depolarized by an increase in the K+ concentration in the external medium. Various odorants depolarized the cell suspension in a dose-dependent fashion. The magnitude of depolarization by odorants was either unchanged or slightly increased by a reduction of the concentration of Na+, Ca2+, and Cl- in the external medium, which suggests that changes in the permeabilities of specific ions are not involved in depolarization by odorants. The application of various odorants to the cell suspension induced changes in the membrane fluidity at different sites of the membrane that were monitored with various fluorescent dyes [8-anilino-1-naphthalene sulfonate, n-(9-anthroyloxy) stearic acids, 12-(9-anthroyloxy) oleic acid, and (1,6-diphenyl-1,3,5-hexatriene)], which suggests that the odorants having different odors are adsorbed on different sites in the membrane. On the basis of these results, a possible mechanism of odor discrimination is discussed.     

Optimizing the rotor design for controlled-shear affinity filtration using computational fluid dynamics

Controlled shear affinity filtration (CSAF) is a novel integrated processing technology that positions a rotor directly above an affinity membrane chromatography column to permit protein capture and purification directly from cell culture. The conical rotor is intended to provide a uniform and tunable shear stress at the membrane surface that inhibits membrane fouling and cell cake formation by providing a hydrodynamic force away from and a drag force parallel to the membrane surface. Computational fluid dynamics (CFD) simulations are used to show that the rotor in the original CSAF device (Vogel et al., 2002) does not provide uniform shear stress at the membrane surface. This results in the need to operate the system at unnecessarily high rotor speeds to reach a required shear stress of at least 0.17 Pa at every radial position of the membrane surface, compromising the scale-up of the technology. Results from CFD simulations are compared with particle image velocimetry (PIV) experiments and a numerical solution for low Reynolds number conditions to confirm that our CFD model accurately describes the hydrodynamics in the rotor chamber of the CSAF device over a range of rotor velocities, filtrate fluxes, and (both laminar and turbulent) retentate flows. CFD simulations were then carried out in combination with a root-finding method to optimize the shape of the CSAF rotor. The optimized rotor geometry produces a nearly constant shear stress of 0.17 Pa at a rotational velocity of 250 rpm, 60% lower than the original CSAF design. This permits the optimized CSAF device to be scaled up to a maximum rotor diameter 2.5 times larger than is permissible in the original device, thereby providing more than a sixfold increase in volumetric throughput.     

Liposomes as a model for olfactory cells: changes in membrane potential in response to various odorants

Various odorants were found to depolarize azolectin liposomes. The results obtained are as follows. (1) Changes in the membrane potential of azolectin liposomes in response to various odorants were monitored by measuring changes in the fluorescence intensity of 3,3'-dipropylthiocarbocyanine iodide [disS-C3(5)]. Ten odorants examined increased the fluorescence intensity of the liposome-dye suspensions in a dose-dependent manner, which indicates that odorants depolarize the liposomes. Concentrations of odorants that depolarized the liposomes greatly varied among the odorants. There existed a good correlation between the minimum concentrations of odorants to depolarize the liposomes and the thresholds of respective odorants in the frog or porcine olfactory responses. (2) Addition of sphingomyelin (SM) to azolectin led to a large enhancement of depolarizations by nonanol, citral, and n-amyl acetate. The results indicate that lipid composition of liposomes is one of the factors that control the sensitivity to odorants. (3) Odorants changed the membrane fluidity of the liposomes, which was monitored by changes in the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH). The membrane fluidity was changed in concentration ranges of odorants similar to those where the membrane potential changes occurred, which suggests that changes in the membrane fluidity are related to generation of the membrane potential changes. (4) Changes in the membrane potential in response to odorants were electrically measured with the planar lipid bilayer made of an azolectin-SM (2:1 w/w) mixture. It was shown that odorants (nonanol, citral, and n-amyl acetate) depolarized the planar lipid bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of nitric oxide and bcl-2 expression by shear stress in human osteoarthritic chondrocytes in vitro

Onset and progression of cartilage degeneration is associated with shear stress occurring in diarthrodial joints subjected to inappropriate loading. This study tested the hypothesis that shear stress induced nitric oxide is associated with altered expression of regulatory onco-proteins, bcl-2, and Fas (APO-1/CD95) and apoptosis in primary human osteoarthritic chondrocyte cultures. Shear stress induced membrane phosphatidylserine and nucleosomal degradation were taken as evidence of chondrocyte apoptosis. Application of shear stress upregulated nitric oxide in a dose-dependent manner and was associated with increases in membrane phosphatidylserine and nucleosomal degradation. Increasing levels of shear stress decreased expression of the anti-apoptotic factor, bcl-2, from 44 to 10 U/ml. Addition of the nitric oxide antagonists, L-N(5)-(1-iminoethyl) ornithine and Nomega-nitro-L-arginine methyl ester (L-NAME), reduced shear stress induced nucleosomal degradation by 62% and 74%, respectively. Inhibition of shear stress induced nitric oxide release by L-NAME coincided with a 2.7-fold increase of bcl-2, when compared to chondrocytes exposed to shear stress in the absence of L-NAME. These data suggest that shear stress induced nitric oxide is associated with changes in apoptotic regulatory factors that alter chondrocyte metabolism and may contribute to joint degeneration.     

Changes in membrane properties of erythrocytes and polymorphonuclear cells in psoriasis

Using fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene and its cationic derivative, 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene, we evaluated membrane fluidity in living polymorphonuclear leukocytes and in erythrocytes of psoriatic patients. Our results have shown that erythrocyte membranes of psoriatic patients exhibit a decrease of fluidity. These changes were not associated with any relevant modifications of the cholesterol to phospholipid molar ratio. Moreover, we observed a decrease in polymorphonuclear leukocytes membrane fluidity associated with changes in chemotactic migration. Our results indicate changes of membrane fluidity involving membranes different from the epidermal cells and suggest the hypothesis of a defective membrane-cytoskeleton interaction in psoriasis.     

Osteoblasts respond to pulsatile fluid flow with short-term increases in PGE(2) but no change in mineralization.

Although there is no consensus as to the precise nature of the mechanostimulatory signals imparted to the bone cells during remodeling, it has been postulated that deformation-induced fluid flow plays a role in the mechanotransduction pathway. In vitro, osteoblasts respond to fluid shear stress with an increase in PGE(2) production; however, the long-term effects of fluid shear stress on cell proliferation and differentiation have not been examined. The goal of this study was to apply continuous pulsatile fluid shear stresses to osteoblasts and determine whether the initial production of PGE(2) is associated with long-term biochemical changes. The acute response of bone cells to a pulsatile fluid shear stress (0.6 +/- 0.5 Pa, 3.0 Hz) was characterized by a transient fourfold increase in PGE(2) production. After 7 days of static culture (0 dyn/cm(2)) or low (0.06 +/- 0.05 Pa, 0.3 Hz) or high (0.6 +/- 0.5 Pa, 3.0 Hz) levels of pulsatile fluid shear stress, the bone cells responded with an 83% average increase in cell number, but no statistical difference (P > 0.53) between the groups was observed. Alkaline phosphatase activity per cell decreased in the static cultures but not in the low- or high-flow groups. Mineralization was also unaffected by the different levels of applied shear stress. Our results indicate that short-term changes in PGE(2) levels caused by pulsatile fluid flow are not associated with long-term changes in proliferation or mineralization of bone cells.     

Correction by 1-25-dihydroxycholecalciferol of the abnormal fluidity and lipid composition of enterocyte brush border membranes in vitamin D-deprived rats

Weanling male Wistar rats were deprived of dietary and light sources of vitamin D for 11-18 weeks along with age-matched diet vitamin D-repleted controls to evaluate the role of lipid fluidity in the stimulatory effect of calcitriol on Ca transport. The "static" component of fluidity of proximal small intestine brush border membrane, as assessed by steady-state fluorescence techniques using the fluorophore 1,6-diphenyl-1,3,5-hexatriene, was similar between these two groups. In contrast, the "dynamic" component of fluidity, as assessed by DL-2-(9-anthroyl)-stearic acid and DL-12-(9-anthroyl)-stearic acid, was decreased in membranes of D-deprived animals. Lipid composition was analyzed to evaluate the potential mechanism mediating these fluidity changes. In vitamin D-deprived rats, linoleic (18:2) and arachidonic (20:4) acids of the phosphatidylcholine and phosphatidylethanolamine fractions of the membrane were decreased, whereas palmitic (16:0) and stearic (18:0) acids were increased in the phosphatidylethanolamine fraction of the membrane. These associated fatty acyl alterations could explain, at least in part, the differences in membrane fluidity between D-repleted and D-deprived rats. Membrane fluidity, lipid composition, and duodenal Ca transport were also analyzed 1, 2, and 5 h after the acute administration of 1-25-dihydroxycholecalciferol to D-deprived animals. In D-deprived rats, within 1-2 h, this hormone restored to levels of vitamin D-repleted controls the dynamic component of fluidity and concentrations of the same membrane phospholipid fatty acids. Since these changes temporally precede detectable increases in Ca absorption (demonstrable only during the 5th h), these data support the hypothesis that alterations in membrane fluidity and lipid composition may play an important role in the stimulation of intestinal calcium transport by calcitriol.     

A study of the Interaction Between Cetirizine and Plasma Membrane of Eosinophils, Neutrophils, Platelets and Lymphocytes using A fluorescence Technique

The effect of cetirizine on plasma membrane fluidity and heterogeneity of human eosinophils, neutrophils, platelets and lymphocytes was investigated using a fluorescence technique. Membrane fluidity and heterogeneity were studied by measuring the steady-state fluorescence anisotropy and fluorescence decay of 1-(4- trimethylammonium-phenyl)-6-phenyl-1, 3, 5-hexatriene (TMA-DPH) incorporated in the membrane. The results demonstrate that cetirizine (1 mug/ml) induced a significant increase in the Hpid order in the exterior part of the membrane and a decrease in membrane heterogeneity in eosinophils, neutrophils and platelets. Moreover, cetirizine blocked the PAF induced changes in membrane fluidity in these cells. Cetirizine did not influence significantly the plasma membrane of lymphocytes. These data may partially explain the effect ofcetirizine on inflammatory cell activities.     

Stabilizing effects of cholesterol on changes in membrane permeability and potential induced in red blood cells by lysolecithin.

Effects of cholesterol on permeability of K+ ion and on change in membrane potential induced by lysolecithin were studied. Cholesterol inhibited K+ release from rabbit red blood cells treated with lysolecithin (1.25 micrograms/ml), 3.3 X 10(-6) M of cholesterol being the optimum concentration for blocking K+ release. Changes in membrane potential, monitored by changes in intensity of fluorescence of cyanine dye, were induced by lysolecithin and inhibited by cholesterol. The inhibitory action on both K+ permeability and membrane potential varied with the cholesterol concentration. The observed effects are thought to be due to membrane-stabilizing activities such as decreasing membrane fluidity and hardening the membrane at the fluid-phase transition temperature. These properties of cholesterol may have significance in relation to transformed cells (tumor cells, lymphomed cells).     

Composition,biophysical properties,and morphometry of plasma membranes in pulmonary interstitial edema

We evaluated the changes in plasma membrane composition, biophysical properties, and morphology of pulmonary endothelial cells in anesthetized rabbits receiving 0.5 ml. kg(-1). min(-1) saline infusion for 180 min, causing mild interstitial edema. Plasma membrane fractions were obtained from lung homogenates with gradient centrifugation, allowing a sixfold enrichment in caveolin-1. In edematous lungs, cholesterol content and phospholipidic phosphorus increased by 15 and 40%, respectively. These data correlated with morphometric analysis of lungs fixed in situ by vascular perfusion with 2.5% glutaraldehyde, suggesting a relative increase in surface of luminal to interstitial front of the capillary endothelial cells, due to a convoluted luminal profile. In edematous lungs, the fraction of double-bound fatty acids increased in membrane lipids; moreover, the phosphatidylcholine/phosphatidylethanolamine and the cholesterol/phospholipid ratios decreased. These changes were consistent with the increase in fluorescence anisotropy of plasma membrane, indicating an increase in its fluidity. Data suggest that mechanical stimuli elicited by a modest (approximately 4%) increase in extravascular water cause marked changes in plasma membranes that may be of relevance in signal transduction and endothelial cell activation.