Lysosome lipid storage disorder in NCTR-BALB/c mice: spleen and lung lysosomes store unesterified cholesterol but differ in their phospholipid composition

A strain derived from a colony of BALB/c mice at the National Center for Toxicological Research, Jefferson, AR, USA (NCTR-BALB/c) suffers from an autosomal recessive disorder characterized by proliferation of secondary lysosomes with accumulation ofunesterified cholesterol in several tissues. The unesterified cholesterol content of spleens and lungs from the affected mice were elevated 8- and 3-fold respectively over age- and sex-matched controls. Postnuclear supernatants of tissue homogenates were fractionated by sucrose density gradient centrifugation and the fractions were analyzed for unesterified cholesterol, protein and marker enzyme activities for lysosomes (N-acetyl-beta-D-glucosaminidase, beta-D-glucuronidase), plasma membrane (alkaline phosphodiesterase I), endoplasmic reticulum (glucose-6-phosphatase) and mitochondria (cytochrome oxidase). The enzyme distribution profile showed that lysosomes of affected tissues floated at low density regions (density 1.05-1.08) of the gradient and contained substantial amount of tissue unesterified cholesterol. These low density lysosomes were purified about 17-fold (58% yield) from spleen and about 6-fold (32% yield) from lungs with minimal contamination by other organelles They were mostly intact as judged by high latency for N-acetyl-beta-D-glucosaminidase activity (70-100%). Lysosomes of control tissues were not found at the low density regions. The distribution profiles for other organelles were similar between affected and control tissues. Phospholipid composition of low density lysosomes were distinctly different from their respective tissue homogenates. Spleen and lung lysosomes were enriched in sphingomyelin and phosphatidylcholine respectively. The results suggest that these lysosomes acquire their low densities due to accumulation of unesterified cholesterol, the retention of which may be aided by sphingomyelin and phosphatidylcholine content of the lysosomes.     

A genetic storage disorder in BALB/C mice with a metabolic block in esterification of exogenous cholesterol

Cholesterol metabolism has been investigated in a strain of BALB/C mice that carry an autosomal recessive mutation associated with decreased sphingomyelinase and glucocerebrosidase activity and storage of sphingomyelin and glucocerebroside as well as cholesterol in lysosomes (Pentchev, P. G., Gal, A. E., Boothe, A. D., Omodeo-Sale, F., Fouks, J., Neumeyer, B. A., Quirk, J. M., Dawson, G., and Brady, R. O. (1980) Biochim. Biophys. Acta 619, 669-679). When affected animals are placed on a diet high in cholesterol, they develop hepatomegaly associated with an extensive accumulation of unesterified cholesterol in the liver. Cultured skin fibroblasts derived from these mice also manifest a defect in cholesterol esterification although the uptake and intracellular location of exogenous cholesterol is comparable to that of controls. Microsomal fatty acyl-CoA:cholesterol acyltransferase activity was normal or elevated in extracts of tissues from the affected animals. Furthermore, the subcellular distribution and membrane orientation of acyl-CoA:cholesterol acyltransferase appeared normal in microsomal preparations isolated from affected mice. The blockage of esterification of exogenous cholesterol in the presence of normal transferase activity is suggestive of a defect in a component involved in the intracellular disposition of this sterol. The attenuation in tissue levels of sphingomyelinase and glucocerebrosidase and the accumulation of sphingolipids may reflect alterations in lysosomal function resulting from an imbalance of unesterified cholesterol in these organelles.     

Lysosomal lipolytic enzymes,lipid peroxidation,and injury

Summary We have used highly purified lysosomes to investigate three models of hydrolytic injury by lysosomal phospholipases. Lysosomes, enriched up to 70-fold in marker enzyme activities, can be isolated from homogenized hepatic tissue by differential centrifugation and subsequent free flow electrophoresis. These organelles remain latent and can also be utilized to obtain lysosol, the soluble fraction of the lysosomes tissue containing acid active phospholipases. The first model investigated the effect of lysosol on non-lysosomal membranes. When this soluble fraction was incubated with plasmalemma (sarcolemma) from cardiac cells, selective hydrolysis of the phospholipids was observed: phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin were the preferred substrates, and only lysophosphatidylcholine and lysophosphatidylethanolamine accumulated in significant amounts. Hydrolysis of sphingomyelin was enhanced significantly by Triton-X-100. In the second model, when intact lysosomes were incubated at acid pH, hydrolysis of phospholipids by the endogenous lipases was observed. Once again this lipolysis was specific for phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin: significant amounts of lysophospholipids also accumulated in this model. Concurrent with these lipid changes, an increase in lysosomal permeability also occurred and pH 5.0 was optimal for this lipolytic activity. However, no phospholipase activity was detected when lysosomes were incubated at pH ranges found in acidotic tissue (pH 6.0 or higher). In the third model, lysosomes were incubated at pH 6.0 in the presence of exogenously generated free radicals (dihydroxyfumarate-FeADP). A rapid loss of membrane phospholipids was observed, and most of this loss could be contributed to peroxidation of membrane phospholipids; the production of malondialdehyde preceded loss of N-acetylglucosaminidase from the lysosome. However, significant accumulation of lysophospholipids, from 2% at control time to 6.6 and 8.7% at 10 and 20 minutes, suggested that lysosomal phospholipase were hydrolyzing lysosomal phospholipids. Thus, we hypothesize that this free radical-induced lipolysis is a result of peroxidized phospholipids serving as preferred substrate for phospholipases at pH 6.0.     

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.     

Dual pathways for the secretion of lysosomal cholesterol into a medium from cultured macrophages

The removal of cholesterol from macrophages is important for reversing foam cell formation. In a previous study, we demonstrated that mouse peritoneal macrophages in culture secrete significant amounts of unesterified cholesterol from the lysosomes into the medium during endocytosis and subsequent metabolism of cholesterol-containing liposomes [Furuchi, T., Aikawa, K., Arai, H., and Inoue, K. (1993) J. Biol. Chem. 268, 27345-27348]. In this study, we found that at least two distinct mechanisms are involved in this process. The efflux of unesterified cholesterol into the medium was greatly suppressed by pregnenolone, an inhibitor of lysosomal cholesterol transport, but an appreciable proportion of the unesterified cholesterol was still released into the medium. Analysis of the medium containing the secreted cholesterol by NaBr density gradient ultracentrifugation revealed that the unesterified cholesterol was distributed in two different density peaks (bottom and d =/ approximately 1.1). The d =/ approximately 1.1 peak material formed high-density lipoprotein (HDL)-like particles that were produced and secreted by the macrophages. The lipid components of these particles were phosphatidylcholine and sphingomyelin, while the sole protein component was apolipoprotein E (apo E). Treatment with pregnenolone completely abolished the production of these HDL-like particles but had little effect on the bottom fractions. These data indicate that macrophages release lysosomal cholesterol via both pregnenolone-sensitive and -insensitive pathways, and that only the cholesterol secreted through the pregnenolone-sensitive pathway is associated with endogenously synthesized apo E-containing HDL-like particles. Moreover, we found that the pregnenolone-sensitive pathway operated independently of the presence or absence of exogenous HDL, whereas secretion via the pregnenolone-insensitive pathway was greatly stimulated by exogenously added HDL.     

High density lipoprotein phospholipid composition is a major determinant of the bi-directional flux and net movement of cellular free cholesterol mediated by scavenger receptor BI

The role of high density lipoprotein (HDL) phospholipid in scavenger receptor BI (SR-BI)-mediated free cholesterol flux was examined by manipulating HDL(3) phosphatidylcholine and sphingomyelin content. Both phosphatidylcholine and sphingomyelin enrichment of HDL enhanced the net efflux of cholesterol from SR-BI-expressing COS-7 cells but by two different mechanisms. Phosphatidylcholine enrichment of HDL increased efflux, whereas sphingomyelin enrichment decreased influx of HDL cholesterol. Although similar trends were observed in control (vector-transfected) COS-7 cells, SR-BI overexpression amplified the effects of phosphatidylcholine and sphingomyelin enrichment of HDL 25- and 2.8-fold, respectively. By using both phosphatidylcholine-enriched and phospholipase A(2)-treated HDL to obtain HDL with a graded phosphatidylcholine content, we showed that SR-BI-mediated cholesterol efflux was highly correlated (r(2) = 0.985) with HDL phosphatidylcholine content. The effects of varying HDL phospholipid composition on SR-BI-mediated free cholesterol flux were not correlated with changes in either the K(d) or B(max) values for high affinity binding to SR-BI. We conclude that SR-BI-mediated free cholesterol flux is highly sensitive to HDL phospholipid composition. Thus, factors that regulate cellular SR-BI expression and the local modification of HDL phospholipid composition will have a large impact on reverse cholesterol transport.     

Labeling of cholesterol with filipin in cellular membranes of parenchymatous organs

Summary Filipin is used for ultrastructural cytochemical localization of cholesterol in biological membranes. It binds to unesterified 3-hydroxy-sterols forming 25 nm complexes which are readily recognized in freeze-fracture replicas. Since most investigations with filipin have been performed in isolated cells (tissue culture, cell suspensions etc.) we have investigated the conditions for reproducible labeling of cholesterol in membranes of parenchymatous organs. Vibratome sections of rat kidney fixed by glutaraldehyde perfusion were incubated in filipin and freeze-fracture replicas were prepared using standard techniques. The concentration of filipin, the thickness of vibratome sections and the incubation time and temperature were varied over a wide range. Optimal results were obtained with 50 m thick tissue slices incubated in 400 g/ml of filipin for 46 h at room temperature. Under these conditions lysosomes were consistently labeled while mitochondria and the endoplasmatic reticulum were negative. Peroxisomes showed a little or no labeling at all while the nuclear envelope was heavily labeled in some cells being negative in others. The method described here should be useful in investigation of the role of cholesterol in function of biological membranes in parenchymatous organs and compact tissues.     

Lipid profile of rat myelin subfractions

Myelin from adult rat brains was separated on a discontinuous sucrose gradient into three subfractions. Analysis of light, heavy and membrane fraction lipid classes was performed by HPTLC and densitometry while fatty acid composition was determinated by GLC. The more interesting results observed are: i) the membrane fraction resembles in its lipid and fatty acid composition other cell membranes (particulary oligodentrocytes); ii) light and heavy myelin are quite similar between them but the former has a higher content of sphingomyelin, a lower hydroxy/nohhydroxy cerebrosides ratio and a lower content of monoenoic fatty acids than the heavy subfraction. The results obtained could explain the different structures observed in each myelin subfraction since fatty acid composition, hydroxy fatty acids, sphingomyelin and cholesterol play a key role in the stability and structure of membranes.     

Localization of lysosomal antigens in activated T-lymphocytes

Summary The lysosomal compartment has been examined in activated T-lymphocytes by immunogold electron microscopy and subcellular fractionation. Immunoprecipitation and sodium dodecyl sulphate-polyacrylamide gel, electrophoresis (SDS-PAGE) of radiolabelled extracts of the T-cells showed that they contained three antigens which are fundamental to normal lysosomal function: a representative lysosomal enzyme -glucuronidase, a lysosomal associated membrane protein (LAMP-1), and the cation-independent mannose 6-phosphate lysosomal enzyme targeting receptor (MPR). Immunogold labelling showed that -glucuronidase was present in the rough endoplasmic reticulum, the Golgi complex and Golgi-associated vesicles. The enzyme was also found to accumulate in distinct, non-Golgi organelles in which LAMP-1 was co-localized, probably lysosomes. LAMP-1 was also found in tubular elements of the golgi and in a complex of vesicles clustered near the nucleus where MPR was also present at high density.Fractionation of homogenates from lymphocytes on Percoll gradients revealed that -glucuronidase was distributed throughout the low density region containing rough endoplasmic reticulum, Golgi and plasma membrane components, and the high density region which contained only lysosomal activity. Multiple immunogold electron microscopy of the latter fraction showed the presence of homogenous vesicles which had large amounts of -glucuronidase within the lumen, LAMP-1 at the periphery and no MPR. These vesicles were probably mature lysosomes, arising from pre-lysosomal organelles enriched for LAMP-1 and MPR.     

Interaction of cholesterol with sphingomyelin in mixed membranes containing phosphatidylcholine, studied by spin-label ESR and IR spectroscopies. A possible stabilization of gel-phase sphingolipid domains by cholesterol

The ESR spectra from different positional isomers of sphingomyelin and phosphatidylcholine spin-labeled in their acyl chain have been studied in sphingomyelin(cerebroside)-phosphatidylcholine mixed membranes that contain cholesterol. The aim was to investigate mechanisms by which cholesterol could stabilize possible domain formation in sphingolipid-glycerolipid membranes. The outer hyperfine splittings in the ESR spectra of sphingomyelin and phosphatidylcholine spin-labeled on the 5 C atom of the acyl chain were consistent with mixing of the components, but the perturbations on adding cholesterol were greater in the membranes containing sphingomyelin than in those containing phosphatidylcholine. Infrared spectra of the amide I band of egg sphingomyelin were shifted and broadened in the presence of cholesterol to a greater extent than the carbonyl band of phosphatidylcholine, which was affected very little by cholesterol. Two-component ESR spectra were observed from lipids spin-labeled on the 14 C atom of the acyl chain in cholesterol-containing membranes composed of sphingolipids, with or without glycerolipids (sphingomyelin/cerebroside and sphingomyelin/cerebroside/phosphatidylcholine mixtures). These results indicate the existence of gel-phase domains in otherwise liquid-ordered membranes that contain cholesterol. In the gel phase of egg sphingomyelin, the outer hyperfine splittings of sphingomyelin spin-labeled on the 14-C atom of the acyl chain are smaller than those for the corresponding spin-labeled phosphatidylcholine. In the presence of cholesterol, this situation is reversed; the outer splitting of 14-C spin-labeled sphingomyelin is then greater than that of 14-C spin-labeled phosphatidylcholine. This result provides some support for the suggestion that transbilayer interdigitation induced by cholesterol stabilizes the coexistence of gel-phase and "liquid-ordered" domains in membranes containing sphingolipids.     

Relationships between membrane lipid composition and biological properties of rat myocytes. Effects of aging and manipulation of lipid composition

Cultures of newborn rat heart myocytes undergo major age-related alterations as demonstrated by comparing 5-6-day-old cells ("young cells") and 14-15-day-old cells ("old cells"). This includes: changes from spherical to elongated shape; sphingomyelin and cholesterol level/cell increase by 100% and 50%, respectively, while the phosphatidylcholine is reduced by 15-20% with almost no change in content of total phospholipids. There is a 50% increase in total protein content/cell while DNA content remain constant. The specific activity of seven marker enzymes representing most subcellular organelles is increased. Beating rate is reduced from 160 +/- 20 to 20 +/- 20 beats min-1. All the above age-dependent alterations are affected by modification of cellular polar lipid composition. Small unilamellar vesicles of egg phosphatidylcholine added to the growth medium of old cells serve as donor of egg phosphatidylcholine to the cells and as acceptor of cellular sphingomyelin and cholesterol. Sphingomyelin-phospholipid exchange can be separated from cholesterol depletion either by using vesicles of egg phosphatidylcholine/cholesterol mixtures which serve only in the phospholipid exchange process, or by small unilamellar vesicles of sphingomyelin which act only as efficient cholesterol acceptors. Such experiments indicated that the major response of old cells is to alteration in the phosphatidylcholine to sphingomyelin mole ratio, while changes in the cholesterol level induce smaller effects. Thus, reversal of phosphatidylcholine to sphingomyelin mole ratio to the values shown by young cells reverse cellular functions and features which were altered by cell aging to levels found in young cells. This includes: increase in the beating rate back to 160 +/- 20, reduction in the total protein level and in the specific activity per DNA content of seven marker enzymes and reappearance of spherical cell shape. These results suggest that membrane lipid composition has major influence on cellular properties which as described in the accompanying paper (Yechiel, E., Barenholz, Y., and Henis, Y. I. (1985) J. Biol. Chem. 260, 9132-9136), may be mediated through the organization and dynamics of the cell membranes.     

Characterization of rabbit lung lysosomes and their role in surfactant dipalmitoylphosphatidylcholine catabolism

Although alveolar surfactant is rapidly catabolized in adult rabbit lungs, the pathways have not been characterized. Pathways of surfactant secretion and recycling involve lamellar bodies and multivesicular bodies, organelles shown to be related to lysosomes by cytochemistry and autoradiography. Since lysosomes are central to intracellular catabolic events, it is possible that lysosomes are involved in intrapulmonary surfactant catabolism. Lysosomes relatively free of contaminating organelles (as determined morphologically and by marker enzymes for mitochondria, endoplasmic reticulum, peroxisomes, and plasma membranes) were obtained from post-lavage lung homogenates of 1-kg rabbits by differential centrifugation in buffered sucrose and gradient separation in percoll (density, 1.075-1.165). The role of lung lysosomes in catabolism of dipalmitoylphosphatidylcholine (DPC) was then studied in rabbits killed 4, 12, and 24 h following intratracheal injection of [3H]DPC and [14C] dihexadecyl phosphatidylcholine (DPC-ether). While equal amounts of label were in the lamellar body containing fractions at 4 h, nearly 6-fold more DPC-ether label than DPC label was recovered in the lysosomal fractions. By 24 h, there was 15-fold more DPC-ether in the lysosomes. This is the first report of successful isolation of lysosomes relatively free of other organelles from rabbit lungs. The tracer studies indicate DPC and DPC-ether follow similar intracellular processing after alveolar uptake. The subsequent accumulation of the ether analog in the lysosomal fractions supports a role for these organelles in surfactant DPC catabolism.     

Enhanced apoA-I-dependent cholesterol efflux by ABCA1 from sphingomyelin-deficient Chinese hamster ovary cells

ATP binding cassette protein A1 (ABCA1) plays a major role in cholesterol homeostasis and high density lipoprotein (HDL) metabolism. It is proposed that ABCA1 reorganizes the plasma membrane and generates more loosely packed domains that facilitate apoA-I-dependent cholesterol efflux. In this study, we examined the effects of the cellular sphingomyelin level on HDL formation by ABCA1 by using a Chinese hamster ovary-K1 mutant cell line, LY-A, which has a missense mutation in the ceramide transfer protein CERT. When LY-A cells were cultured in Nutridoma-BO medium and sphingomyelin content was reduced, apoA-I-dependent cholesterol efflux by ABCA1 from LY-A cells increased 1.65-fold compared with that from LY-A/CERT cells stably transfected with human CERT cDNA. Exogenously added sphingomyelin significantly reduced the apoA-I-dependent efflux of cholesterol from LY-A cells, confirming that the decrease in sphingomyelin content in the plasma membrane stimulates cholesterol efflux by ABCA1. The amount of cholesterol available to cold methyl-beta-cyclodextrin (MbetaCD) extraction from LY-A cells was increased by 40% by the expression of ABCA1 and was 1.6-fold higher than that from LY-A/CERT cells. This step in ABCA1 function, making cholesterol available to cold MbetaCD, was independent of apoA-I. These results suggest that the function of ABCA1 could be divided into two steps: (i) a flopping step to move phosphatidylcholine and cholesterol from the inner to outer leaflet of the plasma membrane, where cholesterol becomes available to cold MbetaCD extraction, and (ii) a loading step to load phosphatidylcholine and cholesterol onto apoA-I to generate HDL.     

Rapid turn-over of plasma membrane sphingomyelin and cholesterol in baby hamster kidney cells after exposure to sphingomyelinase

Plasma membrane sphingomyelin in baby hamster kidney (BHK-21) cells was hydrolyzed with sphingomyelinase (Staphylococcus aureus) and the effects on membrane cholesterol translocation and the properties of membrane bound adenylate cyclase and Na+/K(+)-ATPase were determined. Exposure of confluent BHK-21 cells to 0.1 U/ml of sphingomyelinase led to the degradation (at 37 degrees C) of about 60% of cell sphingomyelin. No simultaneous hydrolysis of phosphatidylcholine occurred. The hydrolysis of sphingomyelin subsequently led to the translocation (within 40 min) of about 50-60% of cell [3H]cholesterol from a cholesterol oxidase susceptible pool to an oxidase resistant compartment. The translocation of [3H]cholesterol from the cell surface to intracellular membranes was accompanied by a paralleled increase in [3H]cholesterol ester formation. When cells were first exposed to sphingomyelinase (to degrade sphingomyelin) and then incubated without the enzyme in serum-free media, the mass of cell sphingomyelin decreased initially (by 60%), but then began to increase and reached control levels within 3-4 h. The rapid re-synthesis of sphingomyelin was accompanied by an equally rapid normalization of cell [3H]cholesterol distribution. The re-formation of cell sphingomyelin also led to a decreased content of cellular [3H]cholesterol esters, indicating that unesterified [3H]cholesterol was pulled out of the cholesterol ester cycle and transported to the cell surface. Exposure of BHK-21 cells to sphingomyelinase further led to a dramatically decreased activity of ouabain-sensitive Na+/K(+)-ATPase, whereas forskolin-stimulated adenylate cyclase activity was not affected. The activity of Na+/K(+)-ATPase returned to normal in parallel with the normalization of cell sphingomyelin mass and cholesterol distribution. We conclude that sphingomyelin has profound effects on the steady-state distribution of cell cholesterol, and that manipulations of cell sphingomyelin levels directly and reversibly affects the apparent distribution of cholesterol. Changes in the lipid composition of the plasma membrane also appears to selectively affect important metabolic reactions in that compartment.     

Effect of LCAT on HDL-mediated cholesterol efflux from loaded EA.hy 926 cells

• 1.1. Human endothelial cells (EA.hy 926 line) were loaded with cholesterol, using cationized LDL, and the effect of lecithin:cholesterol acyltransferase (LCAT) on cellular cholesterol efflux mediated by high density lipoproteins (HDL) was measured subsequently.
• 2.2. In plasma, lecithin:cholesterol acyltransferase (LCAT) converts unesterified HDL cholesterol into cholesteryl esters, thereby maintaining the low UC/PL ratio of HDL. It was tested if further decrease in UC/PL ratio of HDL by LCAT influences cellular cholesterol efflux in vitro.
• 3.3. Efflux was measured as the decrease of cellular cholesterol after 24 hr of incubation with various concentrations of HDL in the presence and absence of LCAT. LCAT from human plasma (about 3000-fold purified) was added to the cell culture, resulting in activity levels in the culture media of 60–70% of human serum.
• 4.4. Although LCAT had a profound effect on HDL structure (UC/TC and UC/PL ratio's decreased), the enzyme did not enhance efflux of cellular cholesterol, using a wide range of HDL concentrations (0.05–2.00 mg HDL protein/ml).
• 5.5. The data indicate that the extremely low unesterified cholesterol content of HDL, induced by LCAT, does not enhance efflux of cholesterol from loaded EA.hy 926 cells. It is concluded that the HDL composition (as isolated from plasma by ultracentrifugation) is optimal for uptake of cellular cholesterol.

     

Phospholipid/cholesteryl ester microemulsions containing unesterified cholesterol: model systems for low density lipoproteins

As models for the effects of unesterified cholesterol (UC) on the lipid organization of low density lipoprotein (LDL), microemulsions containing either egg yolk phosphatidylcholine (EYPC) or dimyristoyl phosphatidylcholine (DMPC) as the surface component, cholesteryl oleate (CO) as the core component, and varying amounts of unesterified cholesterol were prepared by sonication. Gel filtration chromatography showed coelution of each of the lipid components, demonstrating the formation of well-defined microemulsion populations. Unesterified cholesterol incorporation into the microemulsions was proportional to the composition of the original mixture at low unesterified cholesterol compositions, but reached saturation at compositions of approximately 15 and 10 mol% unesterified cholesterol for EYPC/CO and DMPC/CO microemulsions, respectively. The Stokes' radius of the microemulsions was constant and similar to native LDL for initial compositions less than 15 mol% unesterified cholesterol, but increased at compositions above 15 mol%. In both EYPC/CO/UC and DMPC/CO/UC microemulsions, no significant changes were observed for the calorimetric or Van't Hoff enthalpy for the thermal transition of the core cholesteryl ester; however, increases in the transition temperature as a function of increasing unesterified cholesterol composition suggests that unesterified cholesterol has a stabilizing effect on the core transition. In DMPC/CO/UC microemulsions, the effect of unesterified cholesterol on the surface-located DMPC could be clearly observed as a broadening of the thermal transition of the acyl chains. These results demonstrate that unesterified cholesterol is located primarily in the surface of these protein-free lipid model systems for LDL.     

The interaction of prostaglandins with human serum lipoproteins

Using high density and low density lipoproteins (HDL and LDL) labeled with fluorescent analogues of phosphatidylcholine or sphingomyelin it was found that low amounts (10^–12 M) of prostaglandins E₁ and F₂ induced different structural rearrangements of the lipoprotein surface, whereas prostaglandins E₂ and F₁ had no effect. The effects of prostaglandin E₁ on HDL were largely paralled by those of this prostaglandin on synthetic recombinants prepared from pure apolipoprotein A₁, phospholipids and cholesterol and were demonstrated to be caused by prostaglandin-apolipoprotein interaction. The interaction resembled that of a ligand with a specific receptor protein because it was specific, reversible, concentration and temperature dependent and saturable. However the retaining capacity of HDL or LDL for prostaglandin E₁ as determined by equilibrium dialysis was very low and a single prostaglandin E₁ molecule was able to induce structural changes in large numbers of discrete lipoprotein particles. To explain this remarkable fact a non-equilibrium model of ligand-receptor interaction is proposed. According to that model in open systems characterized by weak ligand-receptor binding, high diffusion rate of the ligand and long relaxation times which exceed the interval between two successive receptor occupations, the ligand-induced changes will accumulate, resulting in transformation of the system into a new state which may be far away from equilibrium. It is emphasized that the low mobility of lipids constituting the environment of the receptor protein plays a critcal role in this type of signal amplification.It was further demonstrated that the PGE₁-induced changes of the lipoprotein surface resulted in an enhancement of LDL-to-HDL transfer of cholesterol esters and phosphatidylcholine especially in the presence of serum lipid transfer proteins. The acceleration of the interlipoprotein transfer caused by prostaglandin E₁ in turn increases the rate of cholesterol esterification in serum. It is suggested that in such a way prostaglandin E₁ may influence the homeostasis of cholesterol.Abbreviations LDL low density lioproteins - HDL high density lipoproteins - PG prostaglandin - ASM anthrylvinyl-labeled sphingomyelin (N-12-(9-anthryl)-11-trans-dodecanoylsphingosin-1-phosphocholine - APC anthrylvinylphosphatidylcholine (1-radyl-2-[(9-anthryl)-11-transdodecanoyl)-sn-glycerophosphocholine - NAP-SM nitroazidophenyl labeled sphingomyelin (N-[N-(2-nitro-4azidophenyl)-12-aminododecanoyl]-sphingosin-1-phosphocholine) - NAP-PC adizophenyl labeled phosphatidylcholine (1-radyl-2-[N-(2-nitro-4azidophenyl)-12-aminododecanoyl]-sn-glycero-3-phosphocholine - DPPC dipalmitoylphosphatidylcholine - P fluorescence polarization - E parameter of tryptophanyl to ASM resonance energy transfer - LEP lipid-exchange protein     

Changes of gangliosides and other lipids in skeletal muscle from rabbits with experimental dystrophy

Comparison of the skeletal muscles from vitamin E-deficient and control rabbits showed that the muscles from the deficient animals had lower contents of protein and glycogen but more water and lipid. Increases of individual lipids per unit weight of muscle from deficient animals compared with those from control animals were 2.2-fold for gangliosides, 2.18-fold for cholesterol, 1.74-fold for sulfatides, and 1.45-fold for neutral glycosylceramides. Total phospholipids did not change; this was the result of an increase in sphingomyelin (1.47-fold) and a decrease of phosphatidylcholine to 83% of the control, while the other fractions remained unchanged. When the measurements were referred to total muscle, the contents of cholesterol, gangliosides, sulfatides, neutral glycosylceramides, and sphingomyelin in muscle from vitamin E-deficient rabbits were also above those of the control rabbits, and only the phosphatidylcholine content was decreased. It was not possible to determine whether the alteration of lipid content preceded or followed the onset of signs of muscular dystrophy.     

Formation of unesterified choline by rat brain

Two preparations of rat brain (ischemic intact brain and homogenized whole brain) formed large amounts of unesterified (free) choline when incubated at 37 degrees C. The accumulation of choline was inhibited by microwave irradiation of brain, or by heating of brain to 50 degrees C, and was maximal at 37 degrees C at pH 7.4-8.5. Choline formation was only observed in subcellular fractions of brain that contained membranes. In homogenates of brain, choline accumulated at a rate exceeding 10 nmol/mg protein per h. There was a significant decrease in brain phosphatidylcholine concentration (of 50 nmol/mg protein) during incubation for 1 h at 37 degrees C. Concentrations of phosphocholine rose (by 2.3 nmol/mg protein), and concentrations of glycerophosphocholine and sphingomyelin did not change during this period. We used radiolabeled phospholipids to trace the fate of phosphatidylcholine and sphingomyelin during incubations of homogenates of brain. Phosphatidylcholine was degraded to form phosphocholine, glycerophosphocholine and free choline. No lysophosphatidylcholine accumulated. Sphingomyelin was degraded to form phosphocholine and a small amount of free choline. Magnesium ions stimulated choline production, while zinc ions were a potent inhibitor. Other divalent cations (calcium, manganese) had little effect on choline accumulation. ATP concentrations in brain homogenates were less than 5 nmol/mg protein (rapidly microwaved brain contained 27 nmol/mg protein). Addition of ATP or ADP to brain homogenates increased ATP concentrations and significantly inhibited choline accumulation. ATP diminished the formation of choline from added phosphatidylcholine, lysophosphatidylcholine, phosphocholine and glycerophosphocholine. The effects of ATP, zinc ion, or magnesium ion upon choline accumulation were not mediated by changes in the rates of utilization of choline for formation of phosphocholine or phosphatidylcholine. In summary, we showed that there was enhanced formation of choline when ATP concentrations within brain were low. This choline was derived, in part, from the degradation of phosphatidylcholine, and we suggest that phospholipase A activity was the primary initiator of choline release from this phospholipid.