Influence of lipid composition on physical properties and peg-mediated fusion of curved and uncurved model membrane vesicles: "nature's own" fusogenic lipid bilayer

Poly(ethylene glycol) (PEG)-mediated fusion of phosphatidylcholine model membranes has been shown to mimic the protein-mediated biomembrane process [Lee, J., and Lentz, B. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9274-9279]. Unlike the simple model membranes used in this earlier study, the lipid composition of fusogenic biomembranes is quite complex. The purpose of this paper was to examine PEG-mediated fusion of highly curved (SUV) and largely uncurved (LUV) membrane vesicles composed of different lipids in order to identify lipid compositions that produce highly fusogenic membranes. Starting with liposomes composed of five lipids with different physical properties, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylserine (DOPS), bovine brain sphingomyelin (SM), and cholesterol (CH), we systematically varied the composition and tested for the extent of PEG-mediated fusion after 5 min of treatment. We found that a vesicle system composed of four lipids, DOPC/DOPE/SM/CH, fused optimally at a 35/30/15/20 molar ratio. Each lipid seemed to play a part in optimizing the membrane for fusion. PE disrupted outer leaflet packing as demonstrated with TMA-DPH lifetime, C(6)-NBD-PC partitioning, and DPH anisotropy measurements, and thus significantly enhanced fusion and rupture, without significantly altering interbilayer approach (X-ray diffraction). An optimal ratio of PC/PE (35/30) produced a balance between fusion and rupture. CH and SM, when present at an optimal ratio of 3/4 in vesicles containing the optimal PC/PE ratio, reduced rupture without significantly reducing fusion. This optimal CH/SM ratio also enhanced outer leaflet packing, suggesting that fusion is dependent not only on outer leaflet packing but also on the properties of the inner leaflet. Addition of CH without SM enhanced rupture relative to fusion, while SM alone reduced both rupture and fusion. The optimal lipid composition is very close to the natural synaptic vesicle composition, suggesting that the synaptic vesicle composition is optimized with respect to fusogenicity.     

Cholesterol and lipid/protein ratio control the oligomerization of a sphingomyelin-specific toxin, lysenin

Lysenin is a pore-forming toxin that specifically binds sphingomyelin (SM). The binding of the toxin to the membrane is accompanied by the oligomerization of the protein, leading to pore formation. The interaction of lysenin with SM is affected by the presence of other lipids found in the plasma membrane. Although a previous study showed that SM/cholesterol liposomes were 10,000 times more effective than SM liposomes in inhibiting lysenin-induced hemolysis (Yamaji, A., Sekizawa, Y., Emoto, K., Sakuraba, H., Inoue, K., Kobayashi, H., and Umeda, M. (1998) J. Biol. Chem. 273, 5300-5306), the role of cholesterol is not precisely clarified. In the present study, we examined the effects of the presence of cholesterol in the SM membrane on the inhibition of hemolysis, the binding of lysenin to SM, and the oligomerization of lysenin. The addition of cholesterol to SM liposomes dramatically inhibited lysenin-induced hemolysis as described previously. However, the presence of cholesterol did not affect the binding of lysenin to SM liposomes. The oligomerization of lysenin was facilitated by the presence of cholesterol in SM liposomes. The oligomerization of lysenin was also dependent on the SM/lysenin ratio, that is, the amount of lysenin oligomer was increased with the decrease in the SM/lysenin ratio. When the SM/lysenin molar ratio was high, lysenin associated with the membrane as a monomer, which was able to transfer to the erythrocyte membrane. Our results indicate that both cholesterol and the SM/lysenin ratio control the amount of lysenin monomer via altering the state of protein oligomerization, thus affecting hemolysis.     

Effects of changed lipid composition on responses of liposomes to various odorants: possible mechanism of odor discrimination

In a previous paper [Nomura, T., & Kurihara, K. (1987) Biochemistry (preceding paper in this issue)], we showed that azolectin liposomes are depolarized by various odorants and there is a good correlation between the responses in the liposomes and the frog or porcine olfactory responses. In this study, we examined effects of changed lipid composition on responses of liposomes to various odorants. The membrane potential changes in response to odorants were monitored with the fluorescent dye 3,3'-dipropylthiocarbocyanine iodide [diS-C3(5)]. Egg phosphatidylcholine (PC) liposomes showed depolarizing responses to nine odorants among ten odorants tested. The magnitudes of depolarization by alcohols were similar to those in azolectin liposomes, but those by other odorants were much less than those in azolectin liposomes. Addition of sphingomyelin (SM) to PC led to an increase in the magnitude of depolarization by most odorants. Addition of phosphatidylethanolamine (PE) to PC (PE/PC = 0.25) led to depolarizing responses to four odorants among six odorants tested, and a further increase in PE content (PE/PC = 0.54) led to depolarizing responses only to two odorants. Addition of SM to the lipids of this composition of PC and PE [SM/(PC + PE) = 0.22] led to depolarizing responses to four odorants again. Liposomes made of a mixture of SM, PE, and PC exhibited depolarizing responses to four odorants tested, and addition of cholesterol to the lipids [cholesterol/(PC + PE + SM) = 0.05 and 0.11] led to depolarizing responses only to two and one odorant, respectively. Thus, changes in lipid composition of liposomes led to great changes in specificity of the responses to odorants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of lipids in age-related changes in the properties of muscarinic receptors in cultured rat heart myocytes

The effects of the culture's age and of liposome treatments on the properties of muscarinic receptors in cultured rat heart myocytes prepared from the hearts of newborn (1-3 days old) rats were investigated. In these studies we investigated the binding characteristics of antagonists and agonists to the myocyte muscarinic receptors in young (5 days after plating) vs. older (14 days after plating) cultures. Our findings demonstrate that the aging of the cells in culture is accompanied by a reduction in the muscarinic binding capacity and by alterations in the proportion of high- and low-affinity states toward muscarinic agonists, as well as by striking changes in the mode of coupling of the receptors with guanine nucleotide binding protein(s) [G protein(s)]. The above effects of the culture's age occur concomitantly with alterations in the lipid composition of the cultured myocytes (in 14-day old cultures, the phosphatidylcholine/sphingomyelin ratio is reduced, and the cholesterol level is elevated). In order to explore whether the lipid composition is involved in the mechanism that alters the properties and coupling of the muscarinic receptors, we treated aging cultures with liposomes containing egg phosphatidylcholine. This treatment resulted in 14 day old cultures with a lipid composition similar to that of young cultures, and the treated myocytes demonstrated muscarinic receptor properties similar to those of young myocyte cultures. The implications for the role of membrane lipid composition and organization in determining the properties of the muscarinic receptors and their coupling with G proteins are discussed.     

Electron spin resonance characterization of liquid ordered phase of detergent-resistant membranes from RBL-2H3 cells.

The dynamic structure of detergent-resistant membranes (DRMs) isolated from RBL-2H3 cells was characterized using two different acyl chain spin-labeled phospholipids (5PC and 16PC), a headgroup labeled sphingomyelin (SM) analog (SD-Tempo) and a spin-labeled cholestane (CSL). It was shown, by comparison to dispersions of SM, dipalmitoylphosphatidylcholine (DPPC), and DPPC/cholesterol of molar ratio 1, that DRM contains a substantial amount of liquid ordered phase: 1) The rotational diffusion rates (R( perpendicular)) of 16PC in DRM between -5 degrees C and 45 degrees C are nearly the same as those in molar ratio DPPC/Chol = 1 dispersions, and they are substantially greater than R( perpendicular) in pure DPPC dispersions in the gel phase studied above 20 degrees C; 2) The order parameters (S) of 16PC in DRM at temperatures above 4 degrees C are comparable to those in DPPC/Chol = 1 dispersions, but are greater than those in DPPC dispersions in both the gel and liquid crystalline phases. 3) Similarly, R( perpendicular) for 5PC and CSL in DRM is greater than in pure SM dispersions in the gel phase, and S for these labels in DRM is greater than in the SM dispersions in both the gel and liquid crystalline phases. 4) R( perpendicular) of SD-Tempo in DRM is greater than in dispersions of SM in both gel and liquid phases, consistent with the liquid-like mobility in the acyl chain region in DRM. However, S of SD-Tempo in DRM is substantially less than that of this spin label in SM in gel and liquid crystalline phases (in absolute values), indicating that the headgroup region in DRMs is less ordered than in pure SM. These results support the hypothesis that plasma membranes contain DRM domains with a liquid ordered phase that may coexist with a liquid crystalline phase. There also appears to be a coexisting region in DRMs in which the chain labels 16PC and 5PC are found to cluster. We suggest that other biological membranes containing high concentrations of cholesterol also contain a liquid ordered phase.     

A mutual inhibitory effect on absorption of sphingomyelin and cholesterol

Several studies have shown that there is a strong physical interaction between cholesterol and sphingomyelin (SM). The critical factor is thought to be the high degree of saturation in the very long acyl chains of SM. In this study we examined the effects of SM on cholesterol absorption in the rat and compared them with those of phosphatidylcholine (PC). Cholesterol absorption was studied by use of the dual-isotope plasma ratio method. We also studied the effect of sterols on the fecal excretion of undigested SM and its metabolites after a single oral meal of (3)H-dihydrosphingosine-labeled SM. When cholesterol was given dissolved in soybean oil, without addition of SM or other phospholipids, absorption was 68 +/- 12% in the rat intestine. As a general feature the absorption was less efficient from the cholesterol/phospholipid dispersions. In dispersions with cholesterol and SM, the lowest cholesterol absorption (9 +/- 2%) was seen with a cholesterol:SM molar ratio of 1:1. With dispersions of cholesterol and different PC substrates the absorption of cholesterol was lower with saturated PC (16 +/- 8%) than with soybean-PC (22 +/- 4%) or dioleoyl PC (23 +/- 8%). Uptake of SM in the rat intestine was reduced by sterols. For example, percentage recovery of (3)H radioactivity in fecal lipids was 38 +/- 8% when SM was given with cholesterol and 16 +/- 3% without any sterol. One third of the radioactivity in feces was present as ceramide. Sitostanol had the same effect on uptake of SM as cholesterol. This study shows that when rats are fed mixtures of SM and cholesterol the intestinal uptake of both lipids is decreased. By feeding mixtures of SM and sterols the exposure of the colon to ceramide can be increased.     

Cholesterol reverts Triton X-100 preferential solubilization of sphingomyelin over phosphatidylcholine: A P-NMR study

The distribution of phosphatidylcholine (PC) and sphingomyelin (SM) between the solubilized (micellar) and non-solubilized (lamellar) fractions arising from bilayers composed of PC and SM, with or without cholesterol (Chol) has been measured under conditions of partial, incomplete solubilization by Triton X-100. Quantitation is achieved by ³¹P-NMR determination of the composition of mixed micelles in the range of bilayer-micelle coexistence. We find that the solubilized fraction of bilayers consisting of binary mixtures of PC and SM is rich in SM, as expected from previous data on solubilization of pure PC and pure SM liposomes. In contrast, after partial solubilization of ternary mixtures of PC, SM and Chol, the solubilized fraction becomes SM-poor, as observed in the partial solubilization of biomembranes.     

参麦注射液对肠缺血/再灌注肺损伤大鼠肺组织p38MAPK及凋亡相关基因表达的影响

目的:观察参麦注射液(SM)对肠缺血/再灌注(I/R)肺损伤大鼠肺组织p38MAPK和凋亡相关基因Bax、Bcl-2蛋白表达的影响,探讨其保护机制。方法:采用夹闭肠系膜上动脉(SMA)方法建立大鼠肠I/R损伤模型。24只SD大鼠随机分为对照组(Control组)、肠缺血/再灌注组(I/R组)、参麦注射液组(SM+I/R组),每组8只。比较各组大鼠肺湿/干比(W/D)、肺表面活性物质主要成分卵磷脂(PC)及总磷脂(TPL)含量的变化;同时免疫组织化学法检测各组大鼠肺组织中p38MAPK、Bax及Bcl-2蛋白的表达水平。结果:与对照组比较,I/R组肺组织W/D明显升高,而PC和TPL的含量显著降低,肺组织p38MAPK、Bcl-2和Bax蛋白表达明显增强(P均＜0.01),其中Bax的增强比Bcl-2的增强更为明显,Bcl-2/Bax比值降低(P＜0.01);与I/R组比较,SM+I/R组大鼠肺组织W/D明显降低,PC和TPL的含量增加,肺组织p38MAPK和Bax蛋白表达下降(P均＜0.01),Bcl-2的表达增强,Bcl-2/Bax比值明显升高(P＜0.01)。相关分析显示,肠I/R时肺组织p38MAPK蛋白表达水平与肺表面活性物质主要功能成分PC含量及凋亡基因Bcl-2/Bax比值呈负相关(r分别为-0.787,-0.731,P均＜0.01)。结论:SM可能通过抑制p38MAPK信号通路的激活,提高Bcl-2/Bax比值来阻抑细胞凋亡,从而减轻肠I/R时的肺损伤。     

ABCB4 exports phosphatidylcholine in a sphingomyelin-dependent manner

ABCB4, which is specifically expressed on the canalicular membrane of hepatocytes, exports phosphatidylcholine (PC) into bile. Because SM depletion increases cellular PC content and stimulates PC and cholesterol efflux by ABCA1, a key transporter involved in generation of HDL, we predicted that SM depletion also stimulates PC efflux through ABCB4. To test this prediction, we compared the lipid efflux activity of ABCB4 and ABCA1 under SM depletion induced by two different types of inhibitors for SM synthesis, myriocin and (1R,3S)-N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide, in human embryonic kidney 293 and baby hamster kidney cells. Unexpectedly, SM depletion exerted opposite effects on ABCB4 and ABCA1, suppressing PC efflux through ABCB4 while stimulating efflux through ABCA1. Both ABCB4 and ABCA1 were recovered from Triton-X-100-soluble membranes, but ABCB4 was mainly recovered from CHAPS-insoluble SM-rich membranes, whereas ABCA1 was recovered from CHAPS-soluble membranes. These results suggest that a SM-rich membrane environment is required for ABCB4 to function. ABCB4 must have evolved to exert its maximum activity in the SM-rich membrane environment of the canalicular membrane, where it transports PC as the physiological substrate.     

Aging of rat heart myocytes disrupts muscarinic receptor coupling that leads to inhibition of cAMP accumulation and alters the pathway of muscarinic-stimulated phosphoinositide hydrolysis

The biochemical responses to muscarinic stimulation (inhibition of isoproterenol-stimulated cAMP accumulation and stimulation of phosphoinositide turnover) were investigated in intact myocyte cultures prepared from the hearts of newborn rats. The studies employed young (5 days after plating) and aged (14 days old) myocyte cultures. Aging of the myocyte cultures was accompanied by marked alterations in both the inhibition of cAMP accumulation and the stimulation of the phosphoinositide metabolism via the muscarinic receptors. However, the effects on the two muscarinic responses were different. The first response was disrupted at the level of the coupling of the muscarinic receptors with adenylate cyclase through Gi. On the other hand, muscarinic stimulation of phosphoinositide hydrolysis still occurred in the aged myocyte cultures; however, the inositol trisphosphate generated was not converted to inositol 1-phosphate as in young cultures or as in aged cultures stimulated by norepinephrine. This raises the possibility that muscarinic activation of aged myocyte cultures shifts the metabolic state of the cells and alters the pathway of phosphoinositide hydrolysis. Treatment of aging cultures with phosphatidylcholine liposomes under conditions that yielded aged myocyte cultures with a lipid composition resembling that of young ones restored the muscarinic effect on cAMP accumulation, where the impairment in aged cultures was at the coupling stage (which takes place in the plasma membrane). This treatment had no effect on the response of the phosphoinositide metabolism to muscarinic stimulation.     

Modifications in the activities of membrane-bound enzymes during in vivo ageing of human and rabbit erythrocytes

Erythrocyte plasma membranes were isolated from a homogeneous population of human or rabbit erythrocytes fractionated into classes representing young, middle-age and old age in vivo. Lipid analyses of human erythrocyte plasma membranes reveal a decrease of the cholesterol to phospholipid molar ratio, followed by a marked decrease in the activities of the membrane-bound enzymes (Na+,K+)-stimulated ATPase, acetylcholinesterase and NAD+ase from young to old age. Such changes were not observed between young and middle-age rabbit erythrocytes. Incubation of rabbit young erythrocytes with phosphatidylcholine vesicles (liposomes) to obtain partial depletion of their membrane cholesterol, indicated that cholesterol depletion causes a statistically significant decrease of the (Na+,K+)-stimulated ATPase and acetylcholinesterase activities, but the NAD+ase activity remained almost unchanged. The biological significance of these data are discussed in terms of the differences and modifications in the interaction of membrane-bound enzymes with membrane lipids during in vivo ageing of erythrocytes.     

Phosphatidylcholine-rich acceptors, but not native HDL or its apolipoproteins, mobilize cholesterol from cholesterol-rich insoluble components of human atherosclerotic plaques

To examine the potential of high density lipoproteins (HDL) to ameliorate atherosclerotic plaques in vivo, we examined the ability of native HDL, lipid-free HDL apolipoproteins (apo HDL), cholesterol-free discoidal reconstituted HDL (R-HDL) comprised of apo HDL and phosphatidylcholine (PC) and PC liposomes to release cholesterol from cholesterol-rich insoluble components of plaques (ICP) isolated from atherosclerotic human aorta. Isolated ICP had a free cholesterol (FC) to phospholipid (PL) mass ratio (0.8-3.1) and a sphingomyelin (SPM) to PC mass ratio (1.2-4.2) that exceeded those of plasma membranes of cultured cells. Surprisingly, native HDL and its apolipoproteins were not able to release cholesterol from ICP. However, R-HDL and PC liposomes were effectively released cholesterol from ICP. The release of ICP cholesterol by R-HDL was dose-dependent and accompanied by the transfer of > 8 x more PC in the reverse direction (i.e., from R-HDL to ICP), resulting in a marked enrichment of ICP with PC. Compared to R-HDL, PC liposomes were significantly less effective in releasing cholesterol from ICP but were somewhat more effective in enriching ICP with PC. Native HDL was minimally effective in enriching ICP with PC, but became effective after prior in vitro enrichment of HDL with PC from multilamellar PC liposomes. The enrichment of ICP with PC resulted in the dissolution of cholesterol crystals on ICP and allowed the removal of ICP cholesterol by apo HDL and plasma. Our study revealed that the removal of cholesterol from ICP in vivo will be possible through a change in the level, composition, and physical state of ICP lipids mediated by PC-enriched HDL.     

Protection of membrane cholesterol by sphingomyelin against free radical-mediated oxidation

Although the free radical-mediated oxidation of free cholesterol (FC) is critical in the generation of regulatory sterols and in atherogenesis, the physiological regulation of this process is poorly understood. We tested the hypothesis that sphingomyelin (SM), a major phospholipid of cell membranes, which is closely associated with FC, protects FC against oxidation, because of its unique structure, and affinity to the sterol. We employed phosphatidylcholine (PC) liposomes containing varying amounts of SM, and either radioactive FC or a fluorescent analog, dehydroergosterol (DHE), and determined the oxidative decay of the sterol in presence of 2,2′-azo-bis(2-amidinopropane hydrochloride) (AAPH). Incorporation of 25 mol% of SM in the liposomes inhibited the oxidation of FC or DHE by up to 50%. This inhibition was specific for SM among phospholipids, and was abolished by sphingomyelinase treatment. SM was not degraded during the oxidation reaction, and its effect was not dependent on the nature of the oxidizing agent, because it also inhibited sterol oxidation by FeSO₄/ascorbate, and by cholesterol oxidase. These studies show that SM plays a physiological role in the regulation of cholesterol oxidation by free radicals.     

Small and large unilamellar vesicle membranes as model system for bile acid diffusion in hepatocytes.

Uptake of bile acids into the liver cell occurs via active transport or passive diffusion. In a model system, passive diffusion was studied in liposomes using pyranine fluorescence. Rate constants for the diffusion of diverse more polar or more apolar bile acids were examined. Hydrophobic lithocholic acid (LCA) revealed a maximal rate constant of 0.057 s(-1); with the polar ursodeoxycholic acid (UDCA), the value was 0.019 s(-1). UDCA (3 mol%) effectively decreased the rate constant of 0.1 mM chenodeoxycholic acid (CDCA), whereas cholesterol reached a similar decrease only between 5 and 10 mol%. At higher concentrations of CDCA (above 1 mM) or LCA (0.3-0.4 mM), breaking up of liposomal structure was confirmed by light-scattering decrease and increase of carboxyfluorescein fluorescence. Changes in lipid composition of phosphatidylcholine (PC)- small unilamellar vesicles (SUVs) or large unilamellar vesicles (LUVs) also caused decreasing rate constants. For a cardiolipin (CL):PC ratio of 1:20 the CDCA (0.1 mM) rate constant was 71% lower (0.015 s(-1)) and for a sphingomyelin (SM):PC ratio of 2:1 the rate constant was 50% lower (0.026 s(-1)). Changes in membrane fluidity were detected using membrane anisotropy measurements with the 1,6-diphenyl-1,3, 5-hexatriene (DPH) method. Membrane fluidity was reduced with cholesterol- but not with CL- or SM-containing SUVs (ratio: cholesterol, CL, SM:PC of 1:5). This model system is currently used for the analysis of more complex lipid vesicles resembling the plasma/hepatocyte membrane, which is either stabilized or destabilized by appropriate conditions. The results should become clinically relevant.     

Effects of enrichment of fibroblasts with unesterified cholesterol on the efflux of cellular lipids to apolipoprotein A-I.

This study elucidates the factors underlying the enhancement in efflux of human fibroblast unesterified cholesterol and phospholipid (PL) by lipid-free apolipoprotein (apo) A-I that is induced by cholesterol enrichment of the cells. Doubling the unesterified cholesterol content of the plasma membrane by incubation for 24 h with low density lipoprotein and lipid/cholesterol dispersions increases the pools of PL and cholesterol available for removal by apoA-I from about 0.8-5%; the initial rates of mass release of cholesterol and PL are both increased about 6-fold. Expression of the ATP binding cassette transporter A1 (ABCA1) is critical for this increased efflux of lipids, and cholesterol loading of the fibroblasts over 24 h increases ABCA1 mRNA about 12-fold. The presence of more ABCA1 and cholesterol in the plasma membrane results in a 2-fold increase in the level of specific binding of apoA-I to the cells with no change in binding affinity. Characterization of the species released from either control or cholesterol-enriched cells indicates that the plasma membrane domains from which lipids are removed are cholesterol-enriched with respect to the average plasma membrane composition. Cholesterol enrichment of fibroblasts also affects PL synthesis, and this leads to enhanced release of phosphatidylcholine (PC) relative to sphingomyelin (SM); the ratios of PC to SM solubilized from control and cholesterol-enriched fibroblasts are approximately 2/1 and 5/1, respectively. Biosynthesis of PC is critical for this preferential release of PC and the enhanced cholesterol efflux because inhibition of PC synthesis by choline depletion reduces cholesterol efflux from cholesterol-enriched cells. Overall, it is clear that enrichment of fibroblasts with unesterified cholesterol enhances efflux of cholesterol and PL to apoA-I because of three effects, 1) increased PC biosynthesis, 2) increased PC transport via ABCA1, and 3) increased cholesterol in the plasma membrane.     

Regulation of the activity and fatty acid specificity of lecithin-cholesterol acyltransferase by sphingomyelin and its metabolites, ceramide and ceramide phosphate

Sphingomyelin (SM), the second most abundant phospholipid in plasma lipoproteins, was previously shown to be a physiological inhibitor of the lecithin-cholesterol acyltransferase (LCAT) reaction. In this study, we investigated the effects of its metabolites, ceramide and ceramide phosphate, on the activity and fatty acid specificity of LCAT in vitro. Treatment of SM-containing substrate with SMase C, which hydrolyzes SM to ceramide, abolished the inhibitory effect of SM, whereas treatment with SMase D, which hydrolyzes it to ceramide phosphate, increased the level of inhibition. Although incorporation of ceramide into the substrate in the absence of SM activated the LCAT reaction only modestly, its co-incorporation with SM neutralized the inhibitory effect of SM. Ceramide phosphate, on the other hand, inhibited the LCAT reaction more strongly than SM. The effects of the sphingolipids on the phospholipase A and cholesterol esterification reactions of the enzyme were similar, indicating that they regulate the binding of phosphatidylcholine (PC) to the active site, rather than the esterification step. Incorporation of ceramide into the substrate stimulated the synthesis of unsaturated cholesteryl esters at the expense of saturated esters. However, these effects on fatty acid specificity disappeared when the PC substrates were incorporated into an inert diether PC matrix, suggesting that ceramide increases the availability of polyunsaturated PCs to the enzyme by altering the macromolecular structure of the substrate particle. Since the plasma ceramide levels are increased during inflammation, these results indicate that the activity and fatty acid specificity of LCAT may be altered during the inflammatory response.     

Asymmetric distribution of phosphatidylcholine and sphingomyelin between micellar and vesicular phases. Potential implications for canalicular bile formation.

Both phosphatidylcholine (PC) and sphingomyelin (SM) are the major phospholipids in the outer leaflet of the hepatocyte canalicular membrane. Yet, the phospholipids secreted into bile consist principally (>95%) of PC. In order to understand the physical;-chemical basis for preferential biliary PC secretion, we compared interactions with bile salts (taurocholate) and cholesterol of egg yolk (EY)SM (mainly 16:0 acyl chains, similar to trace SM in bile), buttermilk (BM)SM (mainly saturated long (>20 C-atoms) acyl chains, similar to canalicular membrane SM) and egg yolk (EY)PC (mainly unsaturated acyl chains at sn-2 position, similar to bile PC). Main gel to liquid-crystalline transition temperatures were 33. 6 degrees C for BMSM and 36.6 degrees C for EYSM. There were no significant effects of varying phospholipid species on micellar sizes or intermixed-micellar/vesicular bile salt concentrations in taurocholate-phospholipid mixtures (3 g/dL, 37 degrees C, PL/BS + PL = 0.2 or 0.4). Various phases were separated from model systems containing both EYPC and (EY or BM)SM, taurocholate, and variable amounts of cholesterol, by ultracentrifugation with ultrafiltration and dialysis of the supernatant. At increasing cholesterol content, there was preferential distribution of lipids and enrichment with SM containing long saturated acyl chains in the detergent-insoluble pelletable fraction consisting of aggregated vesicles. In contrast, both micelles and small unilamellar vesicles in the supernatant were progressively enriched in PC. Although SM containing vesicles without cholesterol were very sensitive to micellar solubilization upon taurocholate addition, incorporation of the sterol rendered SM-containing vesicles highly resistant against the detergent effects of the bile salt. These findings may have important implications for canalicular bile formation.     

The organ distribution of liposome-encapsulated and free cobalt in rats. Liposomes decrease the cardiac uptake of the metal

Rats were administered intravenously liposome-encapsulated or free cobalt, and the organ distribution of the metal was explored using Co57 tracer. Two hours after administration, the cobalt level in the heart was about 40% of the control when given in sphingomyelin (SM)/cholesterol (CH) (1:1 mole ratio) liposomes. These vesicles also tended to decrease the uptake of cobalt in the kidney and the carcass, and to increase it in the spleen and the bones. Liposomes prepared from soybean phosphatidylcholine (SPC)/CH (1:1) had no effect on the uptake of cobalt in the heart, whereas increased its level in the spleen, liver and lung. The time-course of cobalt deposition in the organs displayed substantial variation with the different preparations. Most importantly, no buildup of cobalt level was observed in the heart when the metal was administered in SM/CH vesicles. While confirming known effects of liposomes on the organ-distribution of entrapped drugs, our findings suggest that administration of cobalt in SM/CH liposome-encapsulated form may result in decreased cardiotoxicity and thus increased safety of cobalt-treatment in some anemias.     

Metabolism of cholesterol and phospholipids in cultured human vascular smooth muscle cells: differences between artery and vein-derived cells and the effect of oxygen partial pressure

Human smooth muscle (SM) cells derived from vena saphena magna, aorta abdominalis and arteria mamaria were grown in culture under 40 or 145 mmHg oxygen partial pressure (pO2) and their lipid metabolism studied. Esterification of the cellular [3H]cholesterol was higher by 2.5-fold in artery derived than in vein-derived cells and was slightly higher in cultures exposed to 145 mmHg than to 40 mmHg pO2. Cholesterol efflux in the presence of high density lipoprotein (HDL) in the incubation medium was higher in artery-derived than vein-derived cells. Apolipoprotein (apo) AI also supported cholesterol efflux to a higher extent in artery than in vein-derived cells. Cholesterol efflux in the presence of apo AI was accompanied by a decrease of 50% in cellular [3H]cholesteryl ester in both cell types. SM cultures exposed to [3H]choline incorporated about 90% of the radioactivity to phosphatidylcholine (PC) and 10% to sphingomyelin (SPM). During 5 days exposure to [3H]choline, 10 to 15% and 20 to 30% of the newly synthesized PC and SPM, respectively, were released by vein-derived cells into the incubation medium. The relative amount of SPM of the total radioactive phospholipids released by vein-derived cultures was significantly higher in cultures growing under 40 mmHg than 145 mmHg pO2 reaching a value of up to 33% of the radioactive phospholipids in the incubation medium. HDL was shown to serve as an acceptor for phospholipids released by both vein and artery-derived SM cells, while free apo AI supported phospholipid efflux in artery but not in vein-derived SM cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stability of small unilamellar liposomes in serum and clearance from the circulation: The effect of the phospholipid and cholesterol components

Small unilamellar liposomes containing carboxyfluorescein (CF) and composed of various unsaturated and saturated phospholipids with or without cholesterol were incubated in the presence of mouse serum at 37°C. Liposomes composed of egg L-α-phosphatidylcholine (PC), L-α-dioleoylphosphatidylcholine (DOPC) or sphingomyelin (SM) became rapidly permeable to entrapped CF but incorporation of cholesterol into such liposomes reduced CF leakage. Under similar conditions, CF leakage from cholesterol-free liposomes composed of saturated phospholipids of increasing fatty acid chain length was dependant on the liquid-crystalline phase transition temperature (Tc) of the phospholipid component. Thus, L-α-dilaureoylphos-phatidylcholine (DLPC), L-α-dimyristoyl phosphatidylcholine (DMPC) and L-α-dipalmitoylphosphatidylcholine (DPPC) with Tc's below or near the temperature of the incubation (37°C) released CF rapidly whereas L-α-diheptedecanoyl phosphatidylcholine (DHPC), L-α-distearoylphosphatidylcholine (DSPC) and hydrogenated egg PC (HPC) liposomes with Tc's above 37°C retained the dye quantitatively. After incorporation of cholesterol into liposomes composed of saturated phospholipids, CF release was reduced for DLPC and DMPC and increased for DPPC, DSPC, DHPC and HPC vesicles. Liposomes with or without cholesterol exhibiting greatest stability (in terms of CF retention) in the presence of serum were injected intravenously into mice and rates of clearance of quenched CF from the circulation measured. Observed clearance rates were linear and, when liposomes contained tritiated phospholipid, identical to those of the radiolabel suggesting retention of liposomal integrity in the intravascular space. However, half-lifes of liposomes ranging from 0.1 to 16 h did not correlate with the physical characteristics of their phospholipid component. After intraperitoneal injection, there was quantitative entry of quenched CF (stable liposomes) into the blood from which it was eliminated at rates corresponding to those observed after intravenous injection. These results suggest that solute retention by liposomes and their half-life in the circulation can be controlled by the appropriate manipulation of liposomal membrane fluidity and composition.