Inflammatory platelet-activating factor-like phospholipids in oxidized low density lipoproteins are fragmented alkyl phosphatidylcholines.

Oxidation of human low density lipoprotein (LDL) generates proinflammatory mediators and underlies early events in atherogenesis. We identified mediators in oxidized LDL that induced an inflammatory reaction in vivo, and activated polymorphonuclear leukocytes and cells ectopically expressing human platelet-activating factor (PAF) receptors. Oxidation of a synthetic phosphatidylcholine showed that an sn-1 ether bond confers an 800-fold increase in potency. This suggests that rare ether-linked phospholipids in LDL are the likely source of PAF-like activity in oxidized LDL. Accordingly, treatment of oxidized LDL with phospholipase A(1) greatly reduced phospholipid mass, but did not decrease its PAF-like activity. Tandem mass spectrometry identified traces of PAF, and more abundant levels of 1-O-hexadecyl-2-(butanoyl or butenoyl)-sn-glycero-3-phosphocholines (C(4)-PAF analogs) in oxidized LDL that comigrated with PAF-like activity. Synthesis showed that either C(4)-PAF was just 10-fold less potent than PAF as a PAF receptor ligand and agonist. Quantitation by gas chromatography-mass spectrometry of pentafluorobenzoyl derivatives shows the C(4)-PAF analogs were 100-fold more abundant in oxidized LDL than PAF. Oxidation of synthetic alkyl arachidonoyl phosphatidylcholine generated these C(4)-PAFs in abundance. These results show that quite minor constituents of the LDL phosphatidylcholine pool are the exclusive precursors for PAF-like bioactivity in oxidized LDL.     

Thermotropic and barotropic phase transitions in bilayer membranes of ether-linked phospholipids with varying alkyl chain lengths

The bilayer phase transitions of a series of ether-linked phospholipids, 1,2-dialkylphosphatidylcholines containing linear saturated alkyl chain (C_n = 12, 14, 16 and 18), were observed by differential scanning calorimetry (DSC) under ambient pressure and light-transmittance measurements under high pressure. The thermodynamic quantities of the pre- and main-transitions for the ether-linked PC bilayer membranes were calculated and compared with those of a series of ester-linked PCs, 1,2-diacylphosphatidylcholines. The thermodynamic quantities of the main transition for the ether-linked PC bilayers showed distinct dependence on alkyl-chain length and were slightly different from those of the ester-linked PC bilayers. From the comparison of thermodynamic quantities for the main transition between both PC bilayers, we revealed that the attractive interaction in the gel phase for the ether-linked PC bilayers is weaker than that for the ester-linked PC bilayers. Regarding the pretransition, although changes in enthalpy and entropy for both PC bilayers were comparable to each other, the volume changes of the ether-linked PC bilayers roughly doubled those of the ester-linked PC bilayers. The larger volume change results from the smallest partial molar volume of the ether-linked PC molecule in the interdigitated gel phase. Further, we constructed the temperature-pressure phase diagrams for the ether-linked PC bilayers by using the phase-transition data. The region of the interdigitated gel phase in the phase diagrams was extended by applying pressure and by increasing the alkyl-chain length of the molecule. Comparing the phase diagrams with those for the ester-linked PC bilayers, it was proved that the phase behavior of the ester-linked PC bilayers under high temperature and pressure is almost equivalent to that of the ether-linked PC bilayers in the vicinity of ambient pressure.     

Synthesis of platelet-activating factor by human blood platelets and leucocytes. Evidence against selective utilization of cellular ether-linked phospholipids

Synthesis of platelet activating factor (PAF) in blood platelet suspensions may be due to leucocyte contamination. We therefore investigated PAF synthesis in human blood platelet suspensions and granulocyte- (PMN)-enriched leucocyte suspensions upon stimulation by thrombin and Ca2+-ionophore A23187, both in the presence and absence of the presumed PAF catabolism inhibitor phenylmethylsulfonyl fluoride (PMSF). PAF synthesis was measured by aggregation of washed rabbit platelets and by [3H]acetate incorporation. In contrast to A23187, thrombin was unable to stimulate PAF synthesis by leucocytes. As thrombin did induce PAF synthesis by platelet suspensions, this was evidently not due to leucocyte contamination. A23187 also induced PAF synthesis by platelets, but this was dependent upon the platelet isolation method and possibly associated activation. The ratio of [3H]acetate incorporation into 1-alkyl- versus 1-acyl-2-acetylglycerophosphocholine upon stimulation of non-PMSF-treated leucocytes and platelets amounted to 12.8 and 1.2, respectively. These values are at least 10-fold higher than the ratio of 1-alkyl versus 1-acyl species in the cellular phosphatidylcholine precursor for PAF. By PMSF pretreatment, the distribution of incorporated [3H]acetate between 1-ether- and 1-ester-linked species became similar to that in the precursor phosphatidylcholines of the respective cell type, due to increased recovery of [3H]acetate in the acyl compounds. Both leucocyte and platelet homogenates rapidly degraded acylacetylglycerophosphocholine to (acetyl)glycerophosphocholine, and this deacylation was inhibited by PMSF pretreatment of the cells. We conclude that upon cell stimulation a phospholipase A2 converts both alkylacylglycerophosphocholine and diacylglycerophosphocholine to the 2-lysoanalogs in a ratio similar to the occurrence of the parent compounds. The acetyltransferase subsequently acetylates both compounds to acylacetylglycerophosphocholine and alkylacetylglycerophosphocholine (PAF), respectively. Deacylation of the 1-ester-linked species, either before or after acetylation, gives the impression of selective utilization of 1-ether-linked species for PAF production. It is only after inhibition of the deacylation by pretreatment of the cells with PMSF that a mainly nondiscriminative use of 1-ether- and 1-ester-linked species by both phospholipase A2 and acetyltransferase becomes evident.     

Thermotropic and barotropic phase transitions in bilayer membranes of ether-linked phospholipids with varying alkyl chain lengths

The bilayer phase transitions of a series of ether-linked phospholipids, 1,2-dialkylphosphatidylcholines containing linear saturated alkyl chain (C(n)=12, 14, 16 and 18), were observed by differential scanning calorimetry (DSC) under ambient pressure and light-transmittance measurements under high pressure. The thermodynamic quantities of the pre- and main-transitions for the ether-linked PC bilayer membranes were calculated and compared with those of a series of ester-linked PCs, 1,2-diacylphosphatidylcholines. The thermodynamic quantities of the main transition for the ether-linked PC bilayers showed distinct dependence on alkyl-chain length and were slightly different from those of the ester-linked PC bilayers. From the comparison of thermodynamic quantities for the main transition between both PC bilayers, we revealed that the attractive interaction in the gel phase for the ether-linked PC bilayers is weaker than that for the ester-linked PC bilayers. Regarding the pretransition, although changes in enthalpy and entropy for both PC bilayers were comparable to each other, the volume changes of the ether-linked PC bilayers roughly doubled those of the ester-linked PC bilayers. The larger volume change results from the smallest partial molar volume of the ether-linked PC molecule in the interdigitated gel phase. Further, we constructed the temperature-pressure phase diagrams for the ether-linked PC bilayers by using the phase-transition data. The region of the interdigitated gel phase in the phase diagrams was extended by applying pressure and by increasing the alkyl-chain length of the molecule. Comparing the phase diagrams with those for the ester-linked PC bilayers, it was proved that the phase behavior of the ester-linked PC bilayers under high temperature and pressure is almost equivalent to that of the ether-linked PC bilayers in the vicinity of ambient pressure.     

Synergism between platelet-activating factor-like phospholipids and peroxisome proliferator-activated receptor gamma agonists generated during low density lipoprotein oxidation that induces lipid body formation in leukocytes

Oxidized low density lipoprotein (LDL) has an important proinflammatory role in atherogenesis. In this study, we investigated the ability of oxidized LDL (oxLDL) and its phospholipid components to induce lipid body formation in leukocytes. Incubation of mouse peritoneal macrophages with oxidized, but not with native LDL led to lipid body formation within 1 h. This was blocked by platelet-activating factor (PAF) receptor antagonists or by preincubation of oxLDL with rPAF acetylhydrolase. HPLC fractions of phospholipids purified from oxLDL induced calcium flux in neutrophils as well as lipid body formation in macrophages. Injection of the bioactive phospholipid fractions or butanoyl and butenoyl PAF, a phospholipid previously shown to be present in oxLDL, into the pleural cavity of mice induced lipid body formation in leukocytes recovered after 3 h. The 5-lipoxygenase and cyclooxygenase-2 colocalized within lipid bodies formed after stimulation with oxLDL, bioactive phospholipid fractions, or butanoyl and butenoyl PAF. Lipid body formation was inhibited by 5-lipoxygenase antagonists, but not by cyclooxygenase-2 inhibitors. Azelaoyl-phosphatidylcholine, a peroxisome proliferator-activated receptor-gamma agonist in oxLDL phospholipid fractions, induced formation of lipid bodies at late time points (6 h) and synergized with suboptimal concentrations of oxLDL. We conclude that lipid body formation is an important proinflammatory effect of oxLDL and that PAF-like phospholipids and peroxisome proliferator-activated receptor-gamma agonists generated during LDL oxidation are important mediators in this phenomenon.     

Parallel synthesis and antileishmanial activity of ether-linked phospholipids

The synthesis and antileishmanial activity of 18 edelfosine analogues are described. Compounds were obtained in parallel combining solid phase and solution phase synthesis. The most active analogue is characterized by the octadecyl group in position 2 of the glycerol chain. Considering that this substitution determines the loss of antitumor activity, a different mechanism of antileishmanial action can be hypothesized.     

Turnover rates of the molecular species of alkenyl ether phospholipids of Ehrlich ascites tumor cells.

[1-14C]Glycerol was injected into the peritoneal cavity of mice bearing Ehrlich ascites tumor cells and the lipids were extracted from the cells after selected periods. 1-O-Alkenyl-2-acyl-3-acetylglycerols were prepared from the ethanolamine phosphoglycerides and fractionated by AgNO3-impregnated thin-layer chromatography into five molecular species. Bands 1--5 obtained in this way can be designated as saturated, monoene, diene, tetraene and hexaene species, respectively. Band 5, composed mainly of hexaene species was predominant among those derived from alkenyl ether phospholipids. The specific radioactivities of the fractionated species were determined. Hexaene molecular species turned over most rapidly and disaturated and tetraene species turned over at a high rate, while monoene and diene molecular species turned over very slowly.     

Synthesis of long-chain alkyl and alkenyl bromides

Long-chain alkyl and alkenyl bromides are obtained in quantitative yields by the reaction of methane-sulfonates with anhydrous magnesium bromide in absolute ether; cis-trans isomerization of double bonds does not occur.     

Packing characteristics of two-component bilayers composed of ester- and ether-linked phospholipids.

The miscibility properties of ether- and ester-linked phospholipids in two-component, fully hydrated bilayers have been studied by differential scanning calorimetry (DSC) and Raman spectroscopy. Mixtures of 1,2-di-O-hexadecyl-rac-glycero-3-phosphocholine (DHPC) with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DHPE) and of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) with 1,2-di-O-hexadecyl-sn-glycero-3-phosphoethanolamine (DHPE) have been investigated. The phase diagram for the DPPC/DHPE mixtures indicates that these two phospholipids are miscible in all proportions in the nonrippled bilayer gel phase. In contrast, the DHPC/DPPE mixtures display two regions of gel phase immiscibility between 10 and 30 mol% DPPE. Raman spectroscopic measurements of DHPC/DPPE mixtures in the C-H stretching mode region suggest that this immiscibility arises from the formation of DHPC-rich interdigitated gel phase domains with strong lateral chain packing interactions at temperatures below 27 degrees C. However, in the absence of interdigitation, our findings, and those of others, lead to the conclusion that the miscibility properties of mixtures of ether- and ester-linked phospholipids are determined by the nature of the phospholipid headgroups and are independent of the character of the hydrocarbon chain linkages. Thus it seems unlikely that the ether linkage has any significant effect on the miscibility properties of phospholipids in biological membranes.     

Arachidonic acid mobilization among phospholipids in murine mastocytoma P-815 cells: role of ether-linked phospholipids

The ethanolamine-containing glycerophospholipids, choline-containing glycerophospholipids, and phosphatidylinositol fractions are major sources of arachidonic acid in murine mastocytoma P-815 cloned cells. The choline-linked fraction contained high arachidonic acid contents in 1-O-alkyl-2-acyl- (18%) and 1,2-diacyl-sn-glycero-3-phosphocholine (11%), with smaller amounts in 1-O-alk-1'-enyl-2-acyl species, whereas the arachidonic acid content of the ethanolamine-linked fraction was high in 1-O-alk-1'-enyl-2-acyl (26%) and 1,2-diacyl species (15%) and low in 1-O-alkyl-2-acyl species. The uptake and transfer of [3H]arachidonic acid into the 1,2-diacyl and ether classes of choline-containing glycerophospholipids and ethanolamine-containing glycerophospholipids in mastocytoma cells were examined. There was very rapid incorporation of radioactive arachidonic acid into mastocytoma cells that leveled off after 30 min. By labeling cells with [3H]arachidonic acid for 7.5 min, the radioactivity was recovered in the choline-containing glycerophospholipids (43%), phosphatidylinositol (32%), and ethanolamine-containing glycerophospholipids (20%) with little in other phospholipids, neutral lipid, or free fatty acid fractions. Upon reincubation of the mastocytoma cells in the radiolabel-free medium, the [3H]arachidonate radioactivity was gradually lost from the choline-containing glycerophospholipids fraction and, concomitantly, increased in ethanolamine-containing glycerophospholipids. At the zero time of reincubation, most of the radioactivity was recovered in the 1,2-diacyl species of both choline-containing glycerophospholipids and ethanolamine-containing glycerophospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of sulfated mucopolysaccharides in invertebrates.

The sulfated mucopolysaccharide composition of 22 species of invertebrates belonging to the phyla Arthropoda, Mollusca, Annelida, Tunicata, Echinodermata, Coelenterata, and Porifera was analyzed. It is shown that all the species contain variable amounts of one or more types of sulfated mocopolysaccharides, most of which similar to the ones found in vertebrates. It is shown also that each species has a characteristic composition, differing from each other regarding the relative amount and type of chondroitin sulfates A, B, and C, heparitin sulfate, and heparin. The possible biological role of the sulfated mucopolysaccharides in cell recognition or aggregation or both is discussed in view of the present findings.     

Molecular dynamics of antitumor ether-linked phospholipids in model membranes: a spin-label study

We have synthesized a spin-labeled derivative of ET-18-OCH3, a known antitumor ether-linked phospholipid. The spin-labeled analog was shown to be as potent as ET-18-OCH3 in inhibiting 3H-thymidine uptake of HL60 leukemic cells. Electron spin resonance (ESR) studies showed that the mobility of this ether-linked phospholipid in the membrane is more restricted when compared to its ester-linked counterparts. It is probable that the absence of the bulky carbonyl oxygens allows closer packing of the two alkyl chains in the ether-linked phospholipid, thereby reducing the angular amplitude of the motion of the alkyl chains. These findings may be of importance in elucidating mechanisms by which the antitumor ether-linked phospholipids perturb the structure of cellular membranes.     

Molecular and mechanistic characterization of platelet-activating factor-like bioactivity produced upon LDL oxidation

Oxidation of LDL is thought to be involved in both initiating and sustaining atherogenesis through the formation of proinflammatory lipids and the covalent modification of LDL particles. Platelet-activating factor (PAF; 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent phospholipid mediator involved in inflammation. Upon oxidation of LDL, oxidized phospholipids with PAF-like structure are generated, and some of them may act via the PAF receptor. We evaluated the contribution of 1-0-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16:0 PAF) and of other PAF analogs on the PAF-like bioactivity formed upon Cu2+-initiated oxidation of LDL. Reverse-phase HPLC purification and electrospray ionization-MS analyses showed that upon oxidation of LDL with inactivated PAF-acetylhydrolase (PAF-AH), C16:0 PAF accounted for >30% of PAF-like biological activity and its sn-2 butenoyl analog accounted for >50%. However, upon LDL oxidation in the presence of exogenous 1-0-alkyl-sn-glycero-3-phosphocholine (lyso-PAF) without PAF-AH inactivation, C16:0 PAF formation accounted for >90% of the biological activity recovered. We suggest that the C16:0 PAF, despite being a minor constituent of the LDL peroxidation products, may contribute substantially to the bioactivity formed in oxidized LDL. The higher bioactivity of C16:0 PAF, and the higher selectivity of the LDL-attached lyso-PAF transacetylase toward very short acyl chains [acetate (C2) vs. butanate (C4)], may explain the contribution described above.     

Influence of cholesterol on bilayers of ester- and ether-linked phospholipids. Permeability and 13C-nuclear magnetic resonance measurements

13C-NMR and permeability studies are described for sonicated vesicles of phosphatidylcholines bearing two 16-carbon saturated hydrocarbon chains with (a) one ether linkage at carbon 1 (3) or 2 of glycerol and one ester linkage at carbon 2 or 1 (3) of glycerol; (b) two ether linkages and (c) two ester linkages at carbons 1 (3) and 2 of glycerol. The results of 13C-NMR relaxation enhancement measurements using cholesterol enriched with 13C at the 4 position indicate that no significant relocation of the cholesterol molecules takes place in the bilayer when a methylene group is substituted for a carbonyl group in phosphatidylcholine. The 4-13C atom of cholesterol undergoes similar fast anisotropic motions in diester- and diether -phosphatidylcholine bilayers, as judged by spin-lattice relaxation time measurements in the liquid-crystalline phase; although the fast motions are unaltered, linewidth and spin-spin relaxation time measurements suggested some restriction of the slow motions of cholesterol molecules in bilayers from phosphatidylcholines containing an O-alkyl linkage at the sn-2 position instead of an acyl linkage. At temperatures above the gel to liquid-crystal phase transition, the kinetics of ionophore A23187-mediated 45Ca2+ efflux from vesicles prepared from each type of phosphatidylcholine molecule were the same; the kinetics of spontaneous carboxyfluorescein diffusion from diester- and diether -phosphatidylcholine vesicles were the same, whereas mixed ether/ester phosphatidylcholine molecules gave bilayers which are less permeable. The rate constants were reduced on cholesterol incorporation into the bilayers of each type of phosphatidylcholine molecule. The reductions were not statistically significant for 45Ca2+ release. The rate constants for carboxyfluorescein release were also reduced by cholesterol to the same extent in vesicles from diester-, diether -, and 1-ether, and 1-ether-2-ester-phosphatidylcholines; however, a smaller reduction was noted in bilayers from the 1-ester-2-ether analog. The results provide further evidence that there are no highly specific requirements for ester or ether linkages in phosphatidylcholine for cholesterol to reduce bilayer permeability. This is a reflection of the fact that in both diester- and diether -phosphatidylcholine bilayers, the 4-13C atom of cholesterol is located in the region of the acyl carboxyl group or the glyceryl ether oxygen atom.     

Comparable interaction of doxorubicin with various acidic phospholipids results in changes of lipid order and dynamics

We have characterized the interaction of the antitumor drug doxorubicin with model membranes of the anionic phospholipids dioleoylphosphatidic acid (DOPA), dioleoylphosphatidylserine (DOPS), cardiolipin and dioleoylphosphatidylglycerol (DOPG) as compared to the zwitterionic dioleoylphosphatidylcholine (DOPC) or dioleoylphosphatidylethanolamine (DOPE). The saturating binding levels were: 2.4 (DOPA), 1.3 (cardiolipin), 1.5 (DOPS, DOPG) and 0.02 (DOPC) doxorubicin per lipid phosphorus (mol/mol). The half-saturating free drug concentrations were comparable for DOPA, cardiolipin, DOPS and DOPG: 20, 16, 35 and 18 microM, respectively. Doxorubicin fluorescence revealed the simultaneous existence of at least two populations of bound drug in the various anionic phospholipids: (1) fluorescent molecules with chromophores that reside between the lipid molecules and (2) above 0.01-0.02 doxorubicin bound per lipid phosphorus: non-fluorescent drug-stacks that are closer to the aqueous phase than the fluorescent molecules. Small-angle X-ray scattering indicated that doxorubicin can reorganize anionic phospholipid dispersions into closely-packed multilamellar structures. Addition of the drug caused leakage of entrapped 6-carboxyfluorescein. Neither 2H-NMR on [2-2H]serine-labelled DOPS nor 31P-NMR revealed any significant effect of doxorubicin on headgroup conformation, but 2H-NMR on di[11,11-2H2]oleoyl-labelled phospholipids showed that the drug had a strong acyl chain-disordering effect on anionic phospholipids. 2H-NMR relaxation measurements indicated that the drug immobilized the headgroups and acyl chains of anionic phospholipids. The implications of these observations for the cellular activity of the drug are indicated.     

Temporary membrane distortion of vascular smooth muscle cells is responsible for their apoptosis induced by platelet-activating factor-like oxidized phospholipids and their degradation product, lysophosphatidylcholine

To obtain information about the mechanism of apoptosis induced by oxidized low density lipoproteins (oxLDL) in atherosclerotic plaques, we examined the effects of lysophosphatidylcholine (LPC) and platelet-activating factor (PAF)-like lipids (PAF-LL), which can be derived from oxLDL, on rat vascular smooth muscle cells (VSMC). All the lipids with different structures examined induced apoptosis of VSMC, so we studied the mechanism of induction of apoptosis by LPC. LPC-induced apoptosis was inhibited by alpha-tocopherol (alpha-T) and cholesterol (Chol), but not by other antioxidants such as palmitoyl ascorbic acid and PAF receptor antagonist. The cells temporarily became spherical and highly permeable before induction of apoptosis, and their change in shape was prevented by alpha-T and Chol. From these results, we suggest that the apoptosis induced by oxLDL-derived phospholipids in VSMC is caused by temporary membrane distortion, not through specific receptors.