Role of protein kinase C δ in apoptotic signaling of oxidized phospholipids in RAW 264.7 macrophages.

The oxidized phospholipids (oxPl) 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) are cytotoxic components of oxidized LDL (oxLDL). Sustained exposure to oxLDL or isolated oxPl induces apoptotic signaling in vascular cells, which is a hallmark of the late phase of atherosclerosis. Activation of sphingomyelinase, the coordinate formation of ceramide and activation of caspase 3/7 as well as the activation of stress-associated kinases are causally involved in this process. Here, we provide evidence for a role of PKCδ in oxPl cytotoxicity. Silencing of the enzyme by siRNA significantly reduced caspase 3/7 activation in RAW 264.7 macrophages under the influence of oxPl. Concomitantly, PKCδ was phosphorylated as a consequence of cell exposure to PGPC or POVPC. Single molecule fluorescence microscopy provided direct evidence for oxPl-protein interaction. Both oxPl recruited an RFP-tagged PKCδ to the plasma membrane in a concentration-dependent manner. In addition, two color cross-correlation number and brightness (ccN&B) analysis of the molecular motions revealed that fluorescently labeled PGPC or POVPC analogs co-diffuse and are associated with the fluorescent protein kinase in live cells. The underlying lipid-protein interactions may be due to chemical bonding (imine formation between the phospholipid aldehyde POVPC with protein amino groups) and physical association (with POVPC or PGPC). In summary, our data supports the assumption that PKCδ acts as a proapototic kinase in oxPl-included apoptosis of RAW 264.7 macrophages. The direct association of the bioactive lipids with this enzyme seems to be an important step in the early phase of apoptotic signaling.

Uptake and protein targeting of fluorescent oxidized phospholipids in cultured RAW 264.7 macrophages.

The truncated phospholipids 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) are oxidation products of 1-palmitoyl-2-arachidonoyl phosphatidylcholine. Depending on concentration and the extent of modification, these compounds induce growth and death, differentiation and inflammation of vascular cells thus playing a role in the development of atherosclerosis. Here we describe the import of fluorescent POVPC and PGPC analogs into cultured RAW 264.7 macrophages and the identification of their primary protein targets. We found that the fluorescent oxidized phospholipids were rapidly taken up by the cells. The cellular target sites depended on the chemical reactivity of these compounds but not on the donor (aqueous lipid suspension, albumin or LDL). The great differences in cellular uptake of PGPC and POVPC are a direct consequence of the subtle structural differences between both molecules. The former compound (carboxyl lipid) can only physically interact with the molecules in its immediate vicinity. In contrast, the aldehydo-lipid covalently reacts with free amino groups of proteins by forming covalent Schiff bases, and thus becomes trapped in the cell surface. Despite covalent binding, POVPC is exchangeable between (lipo)proteins and cells, since imines are subject to proton-catalyzed base exchange. Protein targeting by POVPC is a selective process since only a limited subfraction of the total proteome was labeled by the fluorescent aldehydo-phospholipid. Chemically stabilized lipid-protein conjugates were identified by MS/MS. The respective proteins are involved in apoptosis, stress response, lipid metabolism and transport. The identified target proteins may be considered primary signaling platforms of the oxidized phospholipid.

Interaction between ether glycerophospholipid vesicles and serum proteins in vitro.

The effect of blood serum on the stability of small unilamellar vesicles consisting of 1-O-(1'-alkenyl)-2-acyl-sn-glycerophosphocholine (choline plasmalogen) or of the alkylacyl-, dialkyl- and diacyl analogs was evaluated by measuring either release of entrapped calcein or transfer of phospholipids from vesicles to serum high-density lipoproteins. The following order of stability was found: alkenyloleoylGPC greater than dioleoylGPC greater than di-O-octadecenylGPC greater than acyloleoylGPC = egg phosphatidylcholine = alkyloleoylGPC. AlkyloleoylGPC and acyloleoylGPC had aliphatic chain compositions similar to that of alkenyloleoylGPC. From the results obtained it is concluded that stability of vesicles in the presence of serum depends on vesicle size (larger vesicles are more stable) and on the type of bond (ether or ester) in position 2 of glycerol. Dioctadecenyl vesicles are about the same size as alkylacylGPC vesicles, but are significantly more stable in the presence of serum. Thus, it appears that an ester bond in position 2 of glycerol (which is replaced by an ether bond in dioctadecenylglycerol) favors the interaction of phospholipids with serum high-density lipoproteins or lipid-exchange proteins. The addition of cholesterol greatly enhances vesicle stability; among the vesicles used in this study those composed of alkenylacylGPC plus 30 mol% cholesterol were most resistant to disruption by serum. Experiments with sn-1 and sn-3 enantiomers of alkylacylGPC and diacylGPC have shown that interaction of vesicle membranes with serum components is independent of the steric configuration of vesicle phospholipids.

Structure and dynamics of plasmalogen model membranes containing cholesterol: a deuterium NMR study.

Deuterium nuclear magnetic resonance (2H-NMR) was used to investigate the structure and dynamics of the sn-2 hydrocarbon chain of semi-synthetical choline and ethanolamine plasmalogen in bilayers containing 0, 30, and 50 mol% cholesterol. The deuterium NMR spectra of the choline plasmalogen yielded well-resolved quadrupolar splittings which could be assigned to the corresponding hydrocarbon chain deuterons. The sn-2 acyl chain was found to adopt a similar conformation as observed in the corresponding diacyl phospholipid, however, the flexibility at the level of the C-2 methylene segment of the plasmalogen was increased. Deuterium NMR spectra of bilayers composed of the ethanolamine plasmalogen yielded quadrupolar splittings of the C-2 segment much larger than those of the corresponding diacyl lipids, suggesting that the sn-2 chain is oriented perpendicular to the membrane surface at all segments. Cholesterol increased the ordering of the choline plasmalogen acyl chain to the same extent as in diacyl lipid bilayers. T1 relaxation time measurements demonstrated only minor dynamical differences between choline plasmalogen and diacyl lipids in model membranes.

Transfer of pyrene-labelled diacyl-, alkylacyl-, and alkenylacyl-glycerophospholipids from vesicles to human blood platelets.

The present study was aimed at investigating the spontaneous transfer of fluorescently labelled serine- and choline-glycerophospholipids from unilamellar vesicles to resting human blood platelets. The most effectively transferred phospholipids were pyrene-phosphatidylserine (PS) and the ether analogues of choline-glycerophospholipids, e.g., pyrene-alkylacyl- and pyrene-1'-alkenylacyl-glycerophosphocholines (plasmalogens). Transfer of pyrene-diacyl-glycerophosphocholine and pyrene-phosphatidic acid was almost not detectable under the same experimental conditions. The fast intermembrane PS-transfer could be explained by the very high degree of adsorption of PS donor vesicles to the platelet plasma membrane. The short halftime of transfer rate (12-14 min) and the high incorporation (1.08-2.16% of total platelet glycerophosphocholines) observed for ether choline-phospholipids in contrast to pyrene-PS (20 min, 0.8% of total platelet PS), could be interpreted in terms of their bulk membrane properties.

Protein-catalyzed transport of ether phospholipids.

The protein-catalyzed transfer of alkenylacyl-, alkylacyl-, or diacyl-glycerophosphocholines, carrying a pyrenedecanoyl residue as a fluorogenic acyl chain, was studied using unilamellar bilayer vesicles as donor and acceptor membranes in a fluorescence assay. Specific phospholipid transfer proteins, such as phosphatidylinositol transfer protein from yeast and phosphatidylcholine transfer protein from bovine liver showed higher transfer rates with ether lipid substrates. Transfer rates for alkylacyl- and alkenylacyl-glycerophosphocholine as compared to the diacyl analog were rather similar in the presence of non-specific lipid transfer proteins from maize or from bovine liver, respectively. When vesicles of fluorogenic compounds were titrated with the yeast phosphatidylinositol transfer protein, only a 15-20% higher binding affinity for alkenylacyl- and alkylacyl-glycerophosphocholine than for diacyl-glycerophosphocholine was observed. Thus the marked differences of transfer rates measured with this transfer protein cannot be attributed to different binding affinities for the respective phospholipid subclasses. A possible explanation for differences in transfer rates could be differences in the organization of the phospholipid subclasses at the hydrophobic/hydrophilic interface of bilayer membranes.

A study of protein dynamics from anisotropy decays obtained by variable frequency phase-modulation fluorometry: internal motions of N-methylanthraniloyl melittin.

Internal motions of melittin and its lipid complexes were studied by anisotropy decays determined by frequency-domain fluorometry. A covalent anthraniloyl probe was attached, probably to lysine-21. The emission spectra indicate that the anthraniloyl moiety is exposed to solvent in both monomeric and tetrameric forms and is present at the lipid-water interfacial region in the lipid complexes. The fluorescence intensity decay of melittin in solution and its lipid complexes was characterized by three lifetimes. The lifetimes were near 1-2 ns, 6-7 ns and 10 ns. At increased temperatures there was an increase in the amplitude of the intermediate lifetime and a decrease in that of the longer lifetime. For all the melittin systems, at least three correlation times were required to fit the anisotropy data. Of the three correlation times, the shortest correlation time represents the local motions of the probe, while the longest represents global motions of the whole molecule. The intermediate correlation time probably represents the dynamics of domains/helices within the molecule. The melittin monomer is highly flexible, with greater than 90% of its anisotropy being lost by the local motions. Even though it is well organized (greater than 75% helical), the tetramer is still a highly flexible molecule, with 70% of its anisotropy being lost by the local motions. The internal motions of melittin decrease upon binding to lipids and are sensitive to the phase state of the lipid complexes.

Membrane properties modulate the activity of a phosphatidylinositol transfer protein from the yeast, Saccharomyces cerevisiae.

A phospholipid transfer protein from yeast (Daum, G. and Paltauf, F. (1984) Biochim. Biophys. Acta 794, 385-391) was 2800-fold enriched by an improved procedure. The specificity of this transfer protein and the influence of membrane properties of acceptor vesicles (lipid composition, charge, fluidity) on the transfer activity were determined in vitro using pyrene-labeled phospholipids. The yeast transfer protein forms a complex with phosphatidylinositol or phosphatidylcholine, respectively, and transfers these two phospholipids between biological and/or artificial membranes. The transfer rate for phosphatidylinositol is 19-fold higher than for phosphatidylcholine as determined with 1:8 mixtures of phosphatidylinositol and phosphatidylcholine in donor and acceptor membrane vesicles. If acceptor membranes consist only of non-transferable phospholipids, e.g., phosphatidylethanolamine, a moderate but significant net transfer of phosphatidylcholine occurs. Phosphatidylcholine transfer is inhibited to a variable extent by negatively charged phospholipids and by fatty acids. Differences in the accessibility of the charged groups of lipids to the transfer protein might account for the different inhibitory effects, which occur in the order phosphatidylserine which is greater than phosphatidylglycerol which is greater than phosphatidylinositol which is greater than cardiolipin which is greater than phosphatidic acid which is greater than fatty acids. Although mitochondrial membranes contain high amounts of negatively charged phospholipids, they serve effectively as acceptor membranes, whereas transfer to vesicles prepared from total mitochondrial lipids is essentially zero. Ergosterol reduces the transfer rate, probably by decreasing membrane fluidity. This notion is supported by data obtained with dipalmitoyl phosphatidylcholine as acceptor vesicle component; in this case the transfer rate is significantly reduced below the phase transition temperature of the phospholipid.

Lipid topology and physical properties of the outer mitochondrial membrane of the yeast, Saccharomyces cerevisiae.

The outer membrane of yeast mitochondria was studied with respect to its lipid composition, phospholipid topology and membrane fluidity. This membrane is characterized by a high phospholipid to protein ratio (1.20). Like other yeast cellular membranes the outer mitochondrial membrane contains predominantly phosphatidylcholine (44% of total phospholipids), phosphatidylethanolamine (34%) and phosphatidylinositol (14%). Cardiolipin, the characteristic phospholipid of the inner mitochondrial membrane (13% of total phospholipids) is present in the outer membrane only to a moderate extent (5%). The ergosterol to phospholipid ratio is higher in the inner (7.0 wt%) as compared to the outer membrane (2.1 wt.%). Attempts to study phospholipid asymmetry by selective degradation of phospholipids of the outer leaflet of the outer mitochondrial membrane failed, because isolated right-side-out vesicles of this membrane became leaky upon treatment with phospholipases. Selective removal of phospholipids of the outer leaflet with the aid of phospholipid transfer proteins and chemical modification with trinitrobenzenesulfonic acid on the other hand, gave satisfactory results. Phosphatidylcholine and phosphatidylinositol are more or less evenly distributed between the two sides of the outer mitochondrial membrane, whereas the majority of phosphatidylethanolamine is oriented towards the intermembrane space. The fluidity of mitochondrial membranes was determined by measuring fluorescence anisotropy using diphenylhexatriene (DPH) as a probe. The lower anisotropy of DPH in the outer as compared to the inner membrane, which is an indication for an increased lipid mobility in the outer membrane, was attributed to the higher phospholipid to protein and the lower ergosterol to phospholipid ratio. The data presented here show, that the outer mitochondrial membrane, in spite of its close contact to the inner membrane, is distinct not only with respect to its protein pattern, but also with respect to its lipid composition and physical membrane properties.

Uptake of fluorescent plasmalogen analogs by cultured human skin fibroblasts deficient in plasmalogen.

One of the consequences of hereditary peroxisomal dysfunction in the cerebro-hepato-renal (Zellweger) syndrome (CHRS) is a dramatic decrease in the biosynthesis and cellular content of ether lipids. In the present study effects of reduced cellular plasmalogen levels on membrane-membrane interactions were investigated. Cultured CHRS fibroblasts were incubated with unilamellar phospholipid vesicles consisting of 1-O-alkenyl-2-acyl- or 1,2-diacyl-sn-glycerophosphocholines and ethanolamines, carrying either the trans-parinaroyl or the 1,6-diphenyl-1,3,5-hexatriene propionyl group in position 2. Transfer of the fluorogenic phospholipids from vesicles to cells was followed by measuring the concomitant increase in fluorescence intensity. Transfer of phospholipids from cells to vesicles was monitored by incubating cells, prelabeled with [3H]oleic acid, in the presence of phospholipid vesicles. Fibroblasts from healthy donors or CHRS fibroblasts supplemented with the plasmalogen precursor 1-O-hexadecylglycerol served as controls. Plasmalogen-deficient cells exhibited a significantly increased tendency to take up exogenous choline or ethanolamine plasmalogens. Cellular plasmalogens were transferred from control cells to vesicles at a higher rate if the acceptor vesicles consisted of plasmalogens as compared to diacylglycerophosphocholine. Thus, it appears as if mechanisms existed which preserve cellular plasmalogen levels during interaction with exogenous phospholipid pools. Preliminary experimental evidence suggests that the observed exchange of phospholipids between cultured fibroblasts and vesicles occurs by a protein-catalyzed process.

Inhibition of microbial lipases with stereoisomeric triradylglycerol analog phosphonates.

1,2(2,3)-Diradylglycero O-(p-nitrophenyl) n-hexylphosphonates were synthesized, with the diradylglycerol moiety being di-O-octylglycerol, 1-O-hexadecyl-2-O-pyrenedecanylglycerol, or 1-O-octyl-2-oleoyl-glycerol, and tested for their ability to inactivate lipases from Chromobacterium viscosum (CVL) and Rhizopus oryzae (ROL). The experimental data indicate the formation of stable, covalent 1:1 enzyme-inhibitor adducts with the di-O-alkylglycero phosphonates. The differences in reactivity of diastereomeric phosphonates with opposite configuration at the glycerol backbone was less expressed with both enzymes tested as compared to the influence of the stereochemistry at the phosphorus. Both lipases exhibited the same preference for the chirality at the phosphorus that was independent from the absolute configuration at the glycerol backbone. However, with CVL and ROL the inhibitors with the active site serine-directed phosphonate linked at position sn-1 of the glycerol moiety reacted significantly faster than the corresponding sn-3 analogs, reflecting the sn-1 stereopreference of the enzymes towards triacylglycerol analogs with a sn-2 O-alkyl substituent. In contrast, the phosphonates based on the 1-O-octyl-2-oleoylglycerol did not significantly inactivate CVL. Unexpectedly, these substances were hydrolyzed in the presence of lipase.

Stabilization of non-bilayer structures by the etherlipid ethanolamine plasmalogen.

The thermotropic phase behavior of mixtures between diradylphosphatidylethanolamines and diacylphosphatidylcholine was studied using polarized light microscopy, 31P-NMR spectroscopy and synchrotron X-ray diffraction. Multilamellar liposomes composed of alkenylacylphosphatidylethanolamine (ethanolamine plasmalogen) undergo a phase transition from a lamellar to an inverse hexagonal lipid structure at 30 degrees C, which is about 20 degrees C and 30 degrees C lower as compared to its alkylacyl- and diacyl-analog, respectively. These results indicate a higher affinity to non-bilayer structures for the ether lipids. In the presence of the bilayer stabilizing phospholipid, palmitoyloleoylphosphatidylcholine, the transition is shifted to higher temperature without any significant changes in the overall structural parameters as revealed by X-ray diffraction experiments. Again, ethanolamine plasmalogen stabilizes the inverted hexagonal phase to the highest extent, i.e. even in the presence of 40 mol% palmitoyloleoylphosphatidylcholine a pure inverse hexagonal phase is formed at 60 degrees C. Such a result was not reported so far for a diacylphosphatidylethanolamine. This property of ethanolamine plasmalogen might be predominantly explained by an optimized packing of the hydrocarbon chains in the corners and interface region of the hexagonal tubes, owing to a different conformation of the sn-2 chain, which was deduced from 2H-NMR experiments (Malthaner, M., Hermetter, A., Paltauf, F. and Seelig, J. (1987) Biochim. Biophys. Acta 900, 191-197). Data obtained by time resolved X-ray diffraction show a coexistence of lamellar and inverse hexagonal structures in the phase transition region, but do not indicate the existence of non-lamellar intermediates or disorder within the sensitivity limits of the method.

Influence of plasmalogen deficiency on membrane fluidity of human skin fibroblasts: a fluorescence anisotropy study.

The influence of plasmalogen deficiency on membrane lipid mobility was determined by measuring fluorescence anisotropy of trimethylammoniumdiphenylhexatriene (TMA-DPH) and diphenylhexatrienylpropanoylhydrazylstachyose (glyco-DPH) inserted in the plasma membranes of human skin fibroblasts deficient in plasmalogens. The cells used were from patients affected with cerebrohepatorenal (Zellweger) syndrome (CHRS) or rhizomelic chondrodysplasia punctata. Their plasmalogen content (0-5% of total phospholipid) is significantly reduced compared with that of control cells from healthy donors (13-15% of total phospholipid) or of CHRS fibroblasts supplemented with the plasmalogen precursor, hexadecylglycerol. Plasmalogen-deficient cells consistently showed lower fluorescence anisotropies of membrane-bound DPH fluorophores corresponding to higher membrane lipid mobilities as compared to controls. However, very similar lipid mobilities were found for sonicated aqueous dispersions of phospholipids extracted either from CHRS or control cells. Therefore, the differences observed with living cells are not due to differences in the overall physical properties of the membrane lipid constituents. Other phenomena such as lipid asymmetry and/or plasmalogen-protein interactions may be responsible for the effects observed in the biomembranes.

Effects of culture and incubation conditions on membrane fluidity in monolayers of cultured cells measured as fluorescence anisotropy using trimethylammoniumdiphenylhexatriene (TMA-DPH).

Membrane fluidity of coverslip attached living cells was measured as fluorescence anisotropy using 5 microM trimethylammoniumdiphenylhexatriene (TMA-DPH) as fluorescent probe. Fluorescence anisotropy is inversely related to membrane fluidity. Cells were grown on glass coverslips that were inserted and directly incubated in quarz cuvettes. The coverslips were fixed with special holders at an angle of 30 degrees in respect to the incident light. Effects of incubation temperature, of cell growth and densities and of the ionic and nonionic composition of the incubation medium on membrane fluorescence anisotropy were measured. Membranes of growing cells were more fluid than those of stationary cells, while cell densities had no effect except at very low cell numbers. Calcium concentrations increasing from 0 to 8 mmol/l in the incubation medium proportionally decreased membrane fluidity. Hypotonicity of the incubation media increased membrane fluidity while hypertonicity compared to normotonicity had no effect. Differentiated human fibroblasts from different origins exhibited similar membrane fluidities. They were, however, different from those of rat cells. Membrane fluidity of rat brain tumor cells increased with age in culture while membrane fluidity of primary differentiating rat brain cells decreased in with age in culture. Measurement of fluorescence anisotropy in living cells attached to glass coverslips is a convenient tool to study effects of culture--as well as of environmental--conditions on membrane fluidity.

Reversible reduction of phospholipid bound arachidonic acid after low density lipoprotein apheresis. Evidence for rapid incorporation of plasmalogen phosphatidylethanolamine into the red blood cell membrane.

In order to evaluate whether acute changes in fatty acids bound to phospholipids in plasma are transmitted into red blood cell membrane (RBCM) phospholipids, molecular species of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were analyzed after reduction of apo B containing lipoproteins through low density lipoprotein (LDL) apheresis in patients with severe hypercholesterolemia. As compared to the control, increases and decreases in molecular species with arachidonic acid (20:4) and with linoleic acid (18:2), respectively, at sn-2 of plasma diacyl-PC were seen in the patients before the apheresis. Directly after the procedure, the sum of species of plasma and RBCM PC plus PE with 20:4 were reduced. Two days after apheresis major species of plasma diacyl-PC reapproached preapheresis values while, in contrast, the composition of plasma alkenylacyl(plasmalogen)-PE was distinctly altered. In plasmalogen-PE of RBCM similar modifications were induced by the apheresis as in the same subgroup in plasma. In vitro experiments using vesicles with plasmalogen-PE labeled at sn-2 with either [14C]20:4 or a fluorescent pyrenedecanoyl residue indicated fast incorporation of the subgroup into the RBCM. In contrast, diacyl-PE was not taken up by the RBCM. In conclusion, apo B containing lipoproteins are partially responsible for the supply of phospholipids with arachidonic acid to RBCM, in particular by means of the fast incorporation of plasmalogen-PE. The transmission of changes induced by apheresis in plasma into those of the RBCM suggest that erythrocytes play an important role in the homeostasis of fatty acids bound to plasma phospholipids in vivo.

Modified low density lipoprotein delivers substrate for ceramide formation and stimulates the sphingomyelin-ceramide pathway in human macrophages.

Exposure of human blood monocytes derived macrophages to modified (oxidized or acetylated) LDL induced a approximately 40% elevation (60 pmol/10(6) cells) of the endogenous level of the sphingolipid ceramide. A rise of both neutral and acidic SMase activity was found after treatment with oxidized LDL (250 and 80%), while addition of acLDL stimulated only the neutral enzyme (280%). Sphingo(phospho)lipids from LDL were transferred to the cell membrane and distributed into intracellular compartments as observed with acLDL containing BODIPY-FL-C5-SM. Quantitation of ceramide after the addition of [3H-N-acetyl]- or BODIPY-FL-C5-SM-labeled modified LDL (27 microg/ml) to the cell culture medium indicated that approximately 210 pmol CA/10(6) cells was generated from exogenous (ox/acLDL) SM. These results demonstrate a stimulation of the sphingomyelin-ceramide pathway by modified LDL utilizing primarily exogenous (LDL-derived) substrate and suggest that the effects of modified LDL are at least partially due to an increased level of the messenger ceramide.

Effects of dietary fish oil supplementation on platelet aggregability and platelet membrane fluidity in normolipemic subjects with and without high plasma Lp(a) concentrations.

The purpose of this study was to compare the relative effect of n-3 fatty acids on plasma lipids and platelet function in normolipemic subjects (n = 8) with plasma Lp(a) levels greater than 30 mg/dl and normolipemic subjects (n = 7) without detectable plasma Lp(a) concentrations. Six weeks of dietary supplementation (3.8 g EPA and 2.9 g DHA/d) significantly reduced (P less than 0.005) plasma TGs in both groups whereas no changes of plasma TC, LDL-C, HDL-C, and Lp(a), respectively, were found. Collagen- or thrombin-stimulated platelet aggregation and collagen- or thrombin-induced TXB2 generation from platelets decreased by approx. 45% in Lp(a)-negative and Lp(a)-positive platelet donors after a 6 week dietary intake. Four more weeks without n-3 supplementation restored the pretreatment values of TGs, platelet aggregability and TXB2 release. The biophysical properties of platelets from normolipemics with and without high plasma Lp(a) concentrations revealed a similar structural order of platelets at 37 degrees C using DPH, TMA-DPH, or 6-AS as fluorescent probes. Also similar temperature-dependent changes in platelet fluidity from 37 degrees C to 17 degrees C were observed in platelet preparations from Lp(a)-positive and Lp(a)-negative subjects. However, no subtle changes in the structural order of platelets due to nutrient intakes were found in all subjects (n = 15, 19-28 yrs) using fluorescence polarization technique. The present data suggest a similar in vitro platelet behaviour from normolipemic subjects with and without high plasma levels of Lp(a) (which is considered a risk for premature atherosclerosis) in contrast to platelet aggregability and platelet fluidity in certain hyperlipidemic stages.

Novel reversible, irreversible and fluorescent inhibitors of platelet-activating factor acetylhydrolase as mechanistic probes.

Phosphatidylcholines (1-O-alcoxy-2-amino-2-desoxy-phosphocholines and 1-pyrene-labeled analogs) were synthesized and used to examine interactions with recombinant human PAF-acetylhydrolase (PAF-AH), an enzyme purified from plasma, and with macrophage-like U937 cells. Novel phosphatidylcholines containing a sn-2-carbamoylester group such as 1-O-hexadecyl-2-desoxy-2-amino-methylcarbamoyl-2-methyl-rac-glycer o-3-phosphocholine 11 were found to act as site-specific irreversible enzyme inhibitors with Ki-values up to 83 (K(irev)) and 177 (Ki(inact)) microm. The compounds exhibit only marginal inhibition of Ca2+-dependent phospholipases. Kinetic data show that phosphocholines carrying a terminal sn-1-pyrene moiety inhibit PAF-AH activity with an effectivity similar to analogs with an aliphatic chain. 1-O-Decyloxy-[10-(4-pyrenyl)-butoxy]-2-desoxy-2-amino-carbamoyl-me thyl-rac(-glycero-3-phosphocholine 13 could be used for enzyme labeling and to demonstrate an inhibitor-enzyme stoichiometry of 0.7:1. At 8 degrees C, the compound accumulated in the membranes of U937 cells, at 37 degrees C it was internalized into intracellular compartments. Structure activity studies in a mixed micelle assay indicated that the inhibition power of reversible and irreversible inhibitors increases along with the (sn)-1-chain length similar to the structure-dependent binding of ether phospholipids to the PAF-receptor. Unlike the situation at the (sn)-1-position, increasing chain length at the sn-2-position, or an alkyl branching of the glycerol backbone significantly reduced the inhibitory potency.

Single and multiple desipramine exposures of cultured cells. Changes in cellular anisotropy and in lipid composition of whole cells and of plasma membranes.

Effects of the antidepressant drug desipramine (DMI) on fluorescence anisotropy were studied in living cultured human fibroblasts, rat brain astrocytes and rat ROC-1 hybridoma cells (oligodendrocytes x C6). Fluorescence anisotropy, a measure for fluidity, was measured by means of a fluorescence polarization technique using a set of n-(9-anthroyloxy) fatty acids as markers. Apparent fluorescence anisotropies were determined in cells following single or multiple dose exposures to 5 microM DMI at 37 degrees and compared to control cells. In all three cell types single doses of DMI led to significant decreases in anisotropies of the deeper layers (12-AS) of the membranes only, suggesting increases in fluidity. Repeated exposures to 5 microM DMI led to cell specific, significant changes in anisotropies of the superficial membrane layers, as determined by 2-AP, 6-, 7- and 9-AS. The resulting anisotropy values of the three different cell types became more alike than prior to DMI exposure. Alterations in anisotropies were accompanied with changes in the phospholipid patterns of whole cells and isolated plasma membrane vesicles. The changes of PC/PE ratios were consistent with changes observed in fluorescence anisotropies. Such alterations may be individual regulatory responses of the cells to the chronic presence of the drug within the membranes.

Oxidation of fluorescent glycero- and sphingophospholipids in human plasma lipoproteins: alkenylacyl subclasses are preferred targets.

OBJECTIVES: Oxidation of polyunsaturated fatty acids in lipoproteins is supposed to play a crucial role at the early stages of atherogenesis. The polyunsaturated lipids (PUFAs) become oxidized and, thus, the degree and rate of lipid oxidation depend on their concentration and, probably, on the lipid moiety to which they are attached. DESIGN AND METHODS: To determine the relative oxidation susceptibilities of sphingo- and glycerolipid-bound fatty acyl chains, we used fluorescent analogs of the respective compounds, in which one natural fatty acyl chain was replaced by fluorescent diphenylhexatriene propionic acid. RESULTS: Oxidation susceptibilities of the fluorescent acyl chains in the presence of Cu2+ or AAPH depended, in general, on the phospholipid to which they were bound and the lipoprotein. Phospholipids were oxidized faster in HDL than in LDL or Lp(a). Plasmalogens were more susceptible to oxidation than phosphatidylcholine and sphingomyelin. CONCLUSION: Thus, HDL and plasmalogens may be considered as preferred targets of lipid oxidation before the bulk of polyunsaturated phospholipids (mainly phosphatidylcholine) in LDL is subject to free radical attack.