Rapid remodeling of arachidonate from phosphatidylcholine to phosphatidylethanolamine pools during mast cell activation.

The objective of the present study was to better understand the remodeling of arachidonic acid (AA) in phospholipids of the mouse bone marrow-derived mast cell (BMMC) during Ag and ionophore A23187 activation. Initial studies were designed to understand the movement of AA in phospholipid classes under resting conditions. BMMC pulse labeled with AA incorporated greater than 95% of the label into the major phospholipid classes. Phosphatidylcholine (PC) subclasses, 1-acyl-2-arachidonoyl-(sn-glycero-3-phosphocholine (GPC)) in particular, initially accounted for most of the label incorporated into the cells with phosphatidylinositol/phosphatidylserine (PI/PS) and phosphatidylethanolamine (PE) subclasses containing much smaller quantities. Prolonged incubation of labeled BMMC resulted in a decrease in the radioactivity in PC with a concomitant increase in PE such that 1-alk-1-enyl-2-arachidonoyl-(sn-glycero-3-phosphoethanolamine (GPE)) became the single largest labeled AA pool by 24 h. Further experiments indicated that 24 h was the time required to reach isotopic equilibrium among AA-containing phospholipids of the BMMC. In the next series of experiments, BMMC phospholipids were labeled to different specific activities by either labeling the cells for 0.5 h or for 24 h followed by stimulation. Under isotopic equilibrium conditions (24 h), stimulation resulted in AA release from PE greater than PC much greater than PI/PS with 1-alk-1-enyl-2-arachidonoyl-GPE providing the bulk of AA released from the BMMC. By contrast, cells labeled for 0.5 h released AA from PC much greater than PI/PS, with 1-acyl-2-arachidonoyl-GPC accounting for most of the AA released from BMMC phospholipids. Label associated with PE subclasses under nonequilibrium conditions remained unchanged or slightly increased throughout a 10-min stimulation period. Finally, BMMC were double labeled with [14C]-AA for 24 h and then with [3H]-AA for 0.5 h. Cell stimulation resulted in a decrease in the [3H]/[14C] ratio in PC and PI and an increase in the ratio in PE. The decrease in [3H]/[14C] ratio in PC was mainly in 1-acyl-2-arachidonoyl-GPC, whereas the increase in PE subclasses was primarily in 1-alk-1-enyl-2-arachidonoyl-GPE. The [3H]/[14C] ratio in cellular neutral lipids and in supernatant fluid products were at values between PC and PE subclasses. Taken together, these data suggest that during Ag activation, the release of free arachidonic acid is from predominantly PE subclasses. Concomitant with the release of AA, there is a rapid remodeling of AA from PC subclasses into PE subclasses (1-alk-1-enyl-2-acyl-GPE).     

Evidence that increasing the cellular content of eicosapentaenoic acid does not reduce the biosynthesis of platelet-activating factor

Our study has examined platelet-activating factor (PAF) biosynthesis in neutrophils from individuals on a fish oil-enriched diet and in mast cells enriched with eicosapentaenoic acid (EPA) in vitro. Neutrophils isolated from males who were fed fish oil supplement (EPA; 2.8 g/day) for 5 wk contained large quantities of eicosapentaenoate in phosphatidylcholine (PC) and phosphatidylethanolamine and less in phosphatidylinositol. The ratio arachidonate/eicosapentaenoate in PC and phosphatidylethanolamine decreased from greater than 10 before the enriched diet to approximately 3 after the diet. The putative precursor of PAF, 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acyl-GPC) contained the bulk of eicosapentaenoate in PC subclasses with smaller quantities found in 1-acyl and 1-alk-1'-enyl linked species. Ionophore A23187-stimulated neutrophils produced similar quantities of PAF before and after enriched diet. Neutrophils during normal diet acylated 1-O-alkyl-2-lyso-GPC only with arachidonate whereas neutrophils from individuals on enriched diet transferred both arachidonate and eicosapentaenoate into exogenously-provided 1-O-alkyl-2-lyso-GPC. This allowed for the labeling of neutrophils with 1-O-[3H]-alkyl-2-arachidonoyl-GPC (before diet) as well as neutrophils with 1-O-[3H]-alkyl-2-eicosapentaenoyl-GPC and 1-O-[3H]-alkyl-2-arachidonoyl-GPC (after diet). Neutrophils after diet converted similar quantities of these labeled precursors to labeled PAF upon stimulation as those before the diet. Analysis of the nature of the long chain acyl residue remaining in the sn-2 position of 1-alkyl-2-acyl-GPC after cell stimulation indicated that arachidonate and eicosapentaenoate were both released from 1-O-alkyl-2-acyl-GPC at comparable rates. Finally, in vitro supplementation of murine mast cells (PT-18) with arachidonic acid or EPA caused a marked increase in the amount of PAF produced by the cell without having any effect on histamine release. Data from these experiments suggest that EPA is incorporated into a PAF precursor pool. However, this appears not to inhibit PAF production because phospholipase A2 can use eicosapentaenoate- as well as arachidonate-containing phospholipids in the initial step of PAF biosynthesis.     

1-ether-linked phosphoglycerides. Major endogenous sources of arachidonate in the human neutrophil

This study has quantitated changes in the content of labeled and unlabeled arachidonate of neutrophil phosphoglyceride classes and subclasses during cell activation with ionophore A23187. The predominant pools of endogenous arachidonate in the resting neutrophil were found in ethanolamine (68%)-, choline (19%)-, and inositol (12.0%)-containing glycerolipids. Upon stimulation, endogenous arachidonate was lost from primarily ethanolamine (PE) greater than choline (PC) greater than inositol (PI)-linked phosphoglycerides. Released leukotriene B4 and 20-hydroxyleukotriene B4 accounted for 10-35% of the total arachidonate lost from all phosphoglyceride classes. In contrast to the mass loss, ionophore induced a decrease of labeled arachidonate from primarily PC and PI. In the resting neutrophil, 66% of the total arachidonate in PC was found in the 1-alkyl-linked fraction. Furthermore, loss of endogenous arachidonate from 1-alkyl-2-arachidonoyl sn-glycero-3-phosphocholine accounted for 62% of the decrease of arachidonate from choline-linked phosphoglycerides. In contrast, 60% of the release of labeled arachidonate from PC subclasses originated from 1-acyl molecular species. 1-Alk-1'-enyl-2-acyl-sn-glycero-3-PE contained 71% of the arachidonate in ethanolamine-linked phosphoglycerides and was the major PE subclass which was degraded during neutrophil activation with ionophore A23187. These findings demonstrate that human neutrophils contain large ether-linked stores of arachidonate and the capacity to mobilize these stores. In addition, this study points out major discrepancies between using mass or label to determine sources of arachidonate for eicosanoids.     

Cytosolic phospholipase A2 shows burst kinetics consistent with the slow, reversible formation of a dead-end complex.

Cytosolic phospholipase A2 catalyzes the hydrolysis of the sn-2 ester of arachidonate-containing phospholipids. In the present research, a "burst" of arachidonate which precedes a somewhat slower, linear rate (upsilon) of product formation was observed and characterized using covesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DMPM) containing <10 mol% 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine as substrate. The magnitude of the burst (pi) was enzyme dependent, in both the presence and absence of glycerol. Upon subsequent addition of enzyme after the primary burst was complete, a second burst of arachidonate production was observed. This is consistent with the effect resulting from an enzyme effect and not from changes in the substrate. The use of 1,2-dioleoyl-sn-glycero-3-phosphomethanol as the carrier phospholipid instead of DMPM greatly reduced the rate of hydrolysis without a large effect on the pi/upsilon ratio, consistent with the burst not being the result of limitations in the lateral diffusion rate of phospholipids within the covesicles. When the assay is performed in the presence of glycerol, the burst phenomenon was also observed with the monoarachidonoyl glycerol transacylase product which shows that the effect occurs through a common mechanism. The burst and subsequent linear rate of hydrolysis are highly temperature dependent, with a pronounced increase in the pi/upsilon ratio as the temperature is increased from 35 to 45 degrees C. A mechanism in which a slow equilibrium between an active and less active (inactive) state of substrate-bound enzyme is proposed. This may provide a means by which the enzyme is switched off after a few hundred turnovers in order to prevent unabated phospholipid hydrolysis in cells which may be deleterious to membrane integrity.     

Evidence for a highly asymmetric arrangement of ether- and diacyl-phospholipid subclasses in the plasma membrane of Krebs II ascites cells

(1) Krebs II ascites cells were taken as a model of the neoplastic cells to investigate the transverse distribution of phospholipids in the plasma membrane. The experimental procedure was based on non-lytic degradation of phospholipids in the intact cell by Naja naja phospholipase A2 and Staphylococcus aureus sphingomyelinase C and on phospholipid analysis of purified plasma membranes. It was shown that the three major phospholipids, i.e., phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, are randomly distributed between the two halves of the membranes, whereas phosphatidylserine remains located in the inner leaflet. (2) The membrane localization of phosphatidylcholine and phosphatidylethanolamine subclasses (diacyl, alkylacyl and alkenylacyl) was also examined, using a new procedure of ether-phospholipid determination. The method involves a selective removal of diacyl species by guinea pig pancreas phospholipase A1 and of alkenylacyl species by acidolysis. This analysis revealed a 50% increase of ether phospholipids in the plasma membrane as compared to the whole cell (36.5 and 23.1% of total phospholipid, respectively). Furthermore, a strong membrane asymmetry was demonstrated for the three phosphatidylcholine subclasses, since 1-alkyl-2-acyl-sn-glycerol-3-phosphocholine (alkylacyl-GPC) was entirely found in the inner leaflet, whereas both diacyl- and alkenylacyl-GPC displayed an external localization. The same pattern was observed for phosphatidylethanolamine subclasses, except for 1-alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine, which was found randomly distributed. These results are discussed in relation to the process of cell malignant transformation and to the biosynthesis of platelet-activating factor (PAF-acether or 1-alkyl-2-acetyl-GPC).     

Synthesis of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine by an enriched preparation of the human lung mast cell

Our study has examined the synthesis of platelet activating factor (PAF; 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and of structurally related molecules by an enriched preparation (greater than 70%) of the human lung mast cell (HLMC) in response to immunologic stimulation. Upon activation with anti-IgE, HLMC incorporated exogenously provided acetate into a phospholipid that migrated with authentic PAF on TLC. The formation of this product in HLMC occurred concomitantly with histamine and leukotriene C4 release. Further analysis of this phospholipid revealed that 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (GPC) and not 1-alkyl-2-acetyl-GPC was the major 1-radyl-2-acetyl-GPC subclass formed during cell activation. The presence of 1-alkyl-2-acetyl-GPC was confirmed by negative ion chemical ionization mass spectrometry. In addition to this product, anti-IgE-stimulated HLMC synthesized relatively small quantities of another 2-acetylated phospholipid migrating on TLC between phosphatidylcholine and phosphatidylinositol. The chromatographic characteristics of this product suggested that it is a subclass of 1-radyl-2-acetyl-sn-glycero-3-phosphoethanolamine. The catabolism of both 1-acyl-2-acetyl-GPC and 1-alkyl-2-acetyl-GPC was next examined to determine if the predominant formation of 1-acyl-2-acetyl-GPC over 1-alkyl-2-acetyl-GPC were metabolized by the HLMC at similar rates. There was, however, a qualitative difference in the metabolic products derived from the two phospholipids. 1-Alkyl-2-acetyl-GPC was rapidly inactivated by removal of the acetate moiety at the sn-2 position followed by rapid reacylation with arachidonate. By contrast, 1-acyl-2-acetyl-GPC was catabolized mainly by removal of the fatty acyl moiety at the sn-1 position. These data demonstrate the natural occurrence of PAF and at least two structurally similar molecules in anti-IgE stimulated HLMC. Furthermore, an analog containing an ester linkage at the sn-1 position, 1-acyl-2-acetyl-GPC, appears to be the major acetylated product synthesized under these conditions.     

Oxidatively fragmented phosphatidylcholines activate human neutrophils through the receptor for platelet-activating factor.

Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) activates neutrophils (polymorphonuclear leukocytes, PMN) through a receptor that specifically recognizes short sn-2 residues. We oxidized synthetic [2-arachidonoyl]phosphatidylcholine to fragment and shorten the sn-2 residue, and then examined the phospholipid products for the ability to stimulate PMN. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine was fragmented by ozonolysis to 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine. This phospholipid activated human neutrophils at submicromolar concentrations, and is effects were inhibited by specific PAF receptor antagonists WEB2086, L659,989, and CV3988. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine next was fragmented by an uncontrolled free radical-catalyzed reaction: it was treated with soybean lipoxygenase to form its sn-2 15-hydroperoxy derivative (which did not activate neutrophils) and then allowed to oxidize under air. The secondary oxidation resulted in the formation of numerous fragmented phospholipids (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103), some of which activated PMN. Hydrolysis of sn-2 residues with phospholipase A2 destroyed biologic activity, as did hydrolysis with PAF acetylhydrolase. PAF acetylhydrolase is specific for short or intermediate length sn-2 residues and does not hydrolyze the starting material (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103). Neutrophil activation was completely blocked by L659,989, a specific PAF receptor antagonist. We conclude that diacylphosphatidylcholines containing an sn-2 polyunsaturated fatty acyl residue can be oxidatively fragmented to species with sn-2 residues short enough to activate the PAF receptor of neutrophils. This suggests a new mechanism for the appearance of biologically active phospholipids, and shows that PAF receptor antagonists block the action of both PAF and these PAF-like lipids.     

Evidence that activation of a common G-protein by receptors for leukotriene B4 and N-formylmethionyl-leucyl-phenylalanine in HL-60 cells occurs by different mechanisms.

Using cultured human umbilical vein endothelial cells, in which phosphatidylcholine (PC) is equally pulse-labelled by various eicosanoid precursor fatty acids (EPFAs), we have studied the remodelling of EPFAs among the phospholipid classes and subclasses with and without activation, and the relationship of this remodelling process to the selective release of arachidonic acid (AA) by phospholipase A2-mediated cell stimulation. When endothelial cells are pulse-incubated with radiolabelled EPFA for 15 min, greater than 80% of cell-associated radioactivity is present in phospholipids, among which greater than 60% is found in 1,2-diacyl-sn-glycero-3-phosphocholine (diacyl PC). After removing unincorporated radioactivity, reincubation of the pulse-labelled cells for up to 6 h results in progressive decrease in EPFA-labelled diacyl PC, increase in AA- or eicosapentaenoic acid (EPA)-labelled 1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (plasmalogen PE) and increase only in AA-labelled 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl PC). This redistribution of radiolabelled phospholipids is not altered by the presence of excess non-radiolabelled EPFAs. When aspirin-treated EPFA-labelled endothelial cells are stimulated with ionophore A23187, a very selective release of AA is noted in comparison with eicosatrienoate (ETA) or EPA, accompanied by an equivalent decrease in AA-labelled diacyl PC and specific increase in AA-labelled plasmalogen PE and alkyl PC. These selective changes in AA radioactivity induced by A23187 are enhanced 2-fold by pretreating the AA-labelled cells with phorbol 12-myristate 13-acetate, which by itself induces no changes. The changes in radioactivity induced by A23187 without and with phorbol ester among the released AA, the diacyl PC and the plasmalogen PE are significantly correlated with each other. These results indicate that human endothelial cells incorporate EPFAs (AA, ETA, EPA) equally into diacyl PC but selectively release AA esterified into diacyl PC with specific remodelling into plasmalogen PE and alkyl PC.     

Time-dependent utilization of platelet arachidonic acid by the neutrophil in formation of 5-lipoxygenase products in platelet-neutrophil co-incubations.

The biosynthesis of leukotrienes is known to occur through a series of complex processes which, in part, can be influenced by cell-cell interactions. Several studies have suggested that arachidonic acid availability is a major limiting step for leukotriene biosynthesis and that its transfer between cells can represent a significant source of this precursor. Accordingly, effect of time and source of arachidonic acid on transcellular leukotriene synthesis was studied in mixed platelet/neutrophil populations challenged with the calcium ionophore A23187. A time-dependent contribution of platelet-derived as well as neutrophil-derived arachidonate was found in the selective formation of neutrophil 5-lipoxygenase metabolites. Utilization of platelet or neutrophil arachidonate was followed by incorporation of radiolabeled arachidonic acid into platelet or neutrophil phospholipids prior to stimulation. Specific activity of liberated arachidonic acid along with numerous 5-lipoxygenase products (including LTB4, 20-hydroxy-LTB4, 5-HETE and LTC4) was determined in order to follow mass and radiolabel. A large amount of platelet-derived arachidonic acid was released in the first 1.5 min, whereas 10 min platelet-derived arachidonate was much lower in amount but significantly higher in specific activity, suggesting different precursor pools. The platelet-derived arachidonate was heavily utilized by the neutrophils at the early time points for formation of 5-HETE and delta 6-trans-LTB4 isomers, but appeared to contribute only marginally to the constitutive metabolism of neutrophil arachidonate into LTB4. Results from these experiments suggest different pools of 5-lipoxygenase in the neutrophil and indicate a time and source dependent modulation of arachidonate metabolism in mixed cell interactions.     

Evidence for a highly asymmetric arrangement of ether- and diacyl-phospholipid subclasses in the plasma membrane of Krebs II ascites cells

(1) Krebs II ascites cells were taken as a model of the neoplastic cells to investigate the transverse distribution of phospholipids in the plasma membrane. The experimental procedure was based on non-lytic degradation of phospholipids in the intact cell by Naja naja phospholipase A₂ and Staphylococcus aureus sphingomyelinase C and on phopholipid analysis of purified plasma membranes. It was shown that the three major phospholipids, i.e., phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, are randomly distributed between the two halves of the membranes, whereas phosphatidylserine remains located in the inner leaflet. (2) The membrane localization of phosphatidylcholine and phosphatidylethanolamine subclasses (diacyl, alkylacyl and alkenylacyl) was also examined, using a new procedure of ether-phospholipid determination. The method involves a selective removal of diacyl species by guinea pig pancreas phospholipase A₁ and of alkenylacyl species by acidolysis. This analysis revealed a 50% increase of ether phospholipids in the plasma membrane as compared to the whole cell (36.5 and 23.1% of total phospholipid, respectively). Furthermore, a strong membrane asymmetry was demonstrated for the three phosphatidylcholine subclasses, since 1-alkyl-2-acyl-sn-glycerol-3-phosphocholine (alkylacyl-GPC) was entirely found in the inner leaflet, whereas both diacyl- and alkenylacyl-GPC displayed an external localization. The same pattern was observed for phosphatidylethanolamine subclasses, except for 1-alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine, which was found randomly distributed. These results are discussed in relation to the process of cell malignant transformation and to the biosynthesis of platelet-activating factor (PAF-acether or 1-alkyl-2-acetyl-GPC).     

Metabolism of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by human promyelocytic leukemic HL60 cells. Stimulated expression of phospholipase A2 and acetyltransferase requires differentiation

Human promyelocytic leukemia (HL60) cells can be induced to differentiate into mature granulocytes by exposure to dimethyl sulfoxide. The addition of N-formylMet-Leu-Phe or the Ca2+ ionophore A23187 to these differentiated cells generated 15-30 pmol of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC)/10(6) cells as quantified by platelet aggregation assays. Under identical conditions, uninduced cells produced little alkylacetyl-GPC. Upon the addition of ionophore A23187, differentiated cells, and not uninduced ones, released [14C]arachidonate from prelabeled phospholipids including ether-linked phosphatidylcholines, formed both 3H-labeled 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (alkyllyso-GPC) and [3H]alkylacetyl-GPC from endogenous 3H-labeled 1-O-alkyl-2-(long chain) acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC), and incorporated exogenously added [3H]acetate or [3H]alkyllyso-GPC into alkylacetyl-GPC. These results are suggestive that both phospholipase A2 and acetyltransferase activities are involved in alkylacetyl-GPC biosynthesis by HL60 cells and that these activities appear during differentiation. However, when measured in cell extracts, the activities of phospholipase A2 and acetyltransferase of uninduced cells were virtually indistinguishable from those of differentiated cells. Uninduced cells exhibited enhanced incorporation of [3H]alkyllyso-GPC or [3H]alkylacetyl-GPC into alkylacyl-GPC and of [14C]arachidonate and [14C]oleate into various phospholipids including phosphatidylcholine. However, such enhanced expression of acylation reactions could not account for the lack of accumulation of arachidonate or of alkylacetyl-GPC by uninduced cells. Furthermore, analyses of phospholipid classes by phosphorus determination showed no significant alterations in phospholipid composition of HL60 cells during differentiation. Together these data are suggestive that mechanisms regulating the activation of phospholipase A2 and acetyltransferase activities are defective in uninduced cells and that an increased concentration of cytosolic free Ca2+ alone is not a sufficient requirement for these mechanisms.     

Heterogeneity of arachidonate and paf-acether precursor pools in mast cells.

In mammalian cells, arachidonate release and paf-acether formation are frequently associated. The alkyl-acyl-GPC has been proposed as an important source for released arachidonic acid and arachidonate-containing alkylacyl-GPC species as unique precursor for paf-acether. However, the specificity of precursor pools either concerning arachidonic acid or paf-acether is still a matter of controversy. We studied the relationship between the precursor pools for both autacoids in antigenically-stimulated cultured mast cells. We took advantage of the particular arachidonate turnover rate in each phospholipid to investigate the role of alkyl-arachidonyl-GPC in the supply of arachidonic acid by using newly and previously [14C]arachidonate-labeled cells. The specific activity of the released arachidonate was reduced 2-fold following overnight cell incubation, whereas labeling in alkyl-arachidonoyl-GPC was only slightly modified and never corresponded to that of released arachidonate when newly or previously labeled cells were triggered with the antigen. These results are not in favor of a major role for alkyl-arachidonoyl-GPC in supplying arachidonate. In contrast, by using previously labeled cells, we demonstrated that all arachidonate-containing phospholipids were involved in the release of arachidonic acid. The pattern of alkyl chains in alkyl-arachidonoyl-GPC, as well as in total alkylacyl-GPC, is unique since it consists mainly of 18:1 (more than 55%), whereas the 16:0 represents only about 30% of total alkyl chains. Therefore, we analyzed paf-acether molecular composition in order to compare it to the alkyl composition of the precursor pools. The content in 18:1 species of paf-acether, as measured by bioassay (aggregation of rabbit platelets), was always lower than that of 16:0 species and then did not correspond to the alkyl composition of the precursor. These data suggest that the enzymes involved in paf synthesis might be specific for 16:0 alkyl chains of precursor pool.     

Structural identification of a novel pro-inflammatory epoxyisoprostane phospholipid in mildly oxidized low density lipoprotein.

One of the earliest steps in the development of the atherosclerotic lesion is the accumulation of monocyte/macrophages within the vessel wall. Oxidized lipids present in minimally modified-low density lipoproteins (MM-LDL) contribute to this process by activating endothelial cells to express monocyte-specific adhesion molecules and chemoattractant factors. A major focus of our group has been the isolation and characterization of the biologically active oxidized lipids in MM-LDL. We have previously characterized three oxidized phospholipids present in MM-LDL, atherosclerotic lesions of fat fed rabbits, and autoxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) that induced human aortic endothelial cells to adhere human monocytes in vitro. We have used sequential normal and reverse phase-high performance liquid chromatography to isolate various isomers of an oxidized phospholipid from autoxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine. The fatty acid in the sn-2 position of this biologically active isomer and its dehydration product was released by phospholipase A(2) and characterized. Hydrogenation with platinum(IV) oxide/hydrogen suggested a cyclic moiety, and reduction with sodium borohydride suggested two reducible oxygen-containing groups in the molecule. The fragmentation pattern produced by electrospray ionization-collision induced dissociation-tandem mass spectrometry was consistent with a molecule resembling an E-ring prostaglandin with an epoxide at the 5,6 position. The structure of this lipid was confirmed by proton nuclear magnetic resonance spectroscopy analysis of the free fatty acid isolated from the dehydration product of m/z 828.5. Based on these studies, we arrived at the structure of the biologically active oxidized phospholipids as 1-palmitoyl-2-(5, 6-epoxyisoprostane E(2))-sn-glycero-3-phosphocholine. The identification of this molecule adds epoxyisoprostanes to the growing list of biologically active isoprostanes.     

Inhibition of arachidonic acid incorporation into erythrocyte phospholipids by peracetic acid and other peroxides. Role of arachidonoyl-CoA: 1-palmitoyl-sn-glycero-3-phosphocholine acyl transferase

To explore possible mechanisms of the arachidonic acid deficiency of the red blood cell membrane in alcoholics, we compared the effect of ethanol and its oxidized products, acetaldehyde and peracetic acid, with other peroxides on the accumulation of [14C]arachidonate into RBC membrane lipids in vitro. Incubation of erythrocytes with 50 mM ethanol or 3 mM acetaldehyde had no effect on arachidonate incorporation. Pretreatment of erythrocytes with 10 mM hydrogen peroxide, 0.1 mM cumene hydroperoxide or 0.1 mM t-butyl hydroperoxide had little effect on [14C]arachidonate incorporation in the absence of azide. However, pretreatment of cells with N-ethylmaleimide, 0.1 mM peracetic acid or performic acid, with or without azide, inhibited arachidonate incorporation into phospholipids but not neutral lipids. In chase experiments, peracetate also inhibited transfer of arachidonate from neutral lipids to phospholipids. To investigate a possible site of this inhibition of arachidonate transfer into phospholipids by percarboxylic acids, we assayed a repair enzyme, arachidonoyl CoA: 1-palmitoyl-sn-glycero-3-phosphocholine acyl transferase (EC 2.3.1.23). As in intact cells, phospholipid biosynthesis was inhibited more by N-ethylmalemide and peracetic acid than by hydrogen peroxide, cumene hydroperoxide, and t-butyl hydroperoxide. Peracetic acid was the only active inhibitor among ethanol and its oxidized products studied and may deserve further examination in ethanol toxicity.     

Remodeling of arachidonate-containing phosphoglycerides within the human neutrophil

Initial incorporation and subsequent remodeling of 16 phosphoglyceride molecular species containing arachidonate in the human neutrophil have been studied. Neutrophils were pulse-labeled with [3H]arachidonic acid (AA) for 5 min, then phospholipids were analyzed either at this time point or after a subsequent 120-min incubation. [3H]AA was found to be incorporated into phosphoglycerides phosphatidylinositol (PI) greater than phosphatidylcholine (PC) greater than phosphatidylethanolamine (PE) by 5 min. Incorporation of [3H]AA was not related to pool size, but reflected an increase in phosphoglyceride turnover. Following the 120-min incubation, only PE gained a significant amount of labeled arachidonate. Specific activity analysis revealed that PI contained the highest labeled/unlabeled ratio at both 5 min and 120 min. After the initial 5-min pulse, the majority of [3H]arachidonate was incorporated into 1-acyl-2-[3H]arachidonoyl-sn-glycero-3-PC, -PE, and -PI showing no preference for fatty acyl chains at the sn-1 position. However, [3H]AA was remodeled into 1-alkyl-acyl-and 1-alk-1-enyl-acyl-sn-glycero-3-PC and -PE molecular species in those neutrophils incubated for the additional 120 min. Specific activities of [3H]AA within all diacyl molecular species were initially higher relative to those alkyl-acyl and alk-1-enyl-acyl molecular species, but for PC and PE became more uniform as label shifted into ether and plasmalogen pools during the additional 120-min incubation. In contrast, the specific activity of 1-stearoyl-2-arachidonoyl-sn-glycero-3-PI remained constant throughout the 120-min incubation.     

Multiple substrates for paraoxonase-1 during oxidation of phosphatidylcholine by peroxynitrite.

Paraoxonase (PON-1) is a high-density lipoprotein (HDL)-bound enzyme with activity toward multiple substrates. It hydrolyzes organic phosphate and aromatic carboxylic acid esters. It also inhibits accumulation of oxidized phospholipids in plasma lipoproteins by a mechanism yet to be determined. Therefore, we subjected apolipoprotein A-I proteoliposomes containing either 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine or 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine to oxidation by a peroxynitrite generator, SIN-1, in the presence and absence of purified PON-1. PON-1 modified the proportion of oxidation products without affecting the overall extent of PC oxidation. However, in the presence of PON-1, phosphatidylcholine isoprostanes were hydrolyzed to lysophosphatidylcholine. In addition, PON-1 hydrolyzed the phosphatidylcholine core aldehydes 1-palmitoyl-2-(9-oxo)nonanoyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-(5-oxo)valeroyl-sn-glycero-3-phosphocholine to lysophosphatidylcholine. This hydrolysis was not affected by pefabloc, a serine esterase inhibitor. There was no detectable release of linoleate, arachidonate, or their hydroperoxy or hydroxy derivatives in the presence of PON-1. We conclude that PON-1 minimizes the accumulation of phosphatidylcholine oxidation products by the hydrolysis of phosphatidylcholine isoprostanes and core aldehydes to lysophosphatidylcholine with a serine esterase-independent mechanism.     

Identification of a membrane-associated 1-0-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine hydrolyzing phospholipase A2 in guinea pig 1 epidermis. Implications in the cutaneous biosynthesis of platelet-activating factor.

A membrane-associated 1-0-alkyl-2-arachidonoyl-GPC hydrolyzing phospholipase A2 was identified in guinea pig epidermis. It is regio-specific (associated with the particulate microsomal fraction) and specific for the hydrolysis of 1-0-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine. It is sensitive to low calcium concentrations suggesting that it may be activated by increasing intracellular calcium. Since ether-linked phospholipids are known to exist in the epidermis, further understanding of the properties of this 1-0-alkyl-arachidonoyl-hydrolyzing PLA2 may allow us to control the generation of 1-0-alkyl-2-lyso-sn-glycero-3-phosphocholine, a key substrate for the generation of the platelet-activating factor in the tissue.     

Lipid metabolism and signal transduction in endothelial cells

Endothelial cells have the capacity to metabolize several important lipids; this includes the ability to store and then metabolize arachidonate, as well as the capacity to synthesize platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). Arachidonate is predominantly metabolized via cyclooxygenase to PGI2 although the spectrum of prostaglandins may vary depending upon the source of the endothelial cell. Biosynthesis of eicosanoids and PAF are likely to be an important physiologic function of the endothelial cell as these potent lipids appear to have a role in maintaining vascular tone and mediating interactions of the endothelium with circulating inflammatory cells. In addition to production of eicosanoids and PAF, endothelial cells metabolize exogenous arachidonate and arachidonate metabolites and other fatty acids such as linoleate to bioactive compounds (HODEs). There is also evidence that small amounts of arachidonate are metabolized via a lipoxygenase. The physiologic significance of these minor lipid pathways is not known at this time. Production of eicosanoids and PAF is not a constitutive function of the endothelial cell. Lipid biosynthesis by endothelial cells is one component of the early activation response that occurs in response to stimulation with pro-inflammatory and vasoactive hormones or to pathologic agents such as oxidants and bacterial toxins. A central mechanism for activation of the relevant pathways is a rise in cellular calcium concentrations that can be mediated by hormone-receptor-binding or by direct permeabilization of the cell membrane to calcium (Fig. 3). Regulatory mechanisms distal to the calcium signal are unknown, but current evidence suggests that calcium directly or indirectly activates phospholipases that release arachidonate from phospholipids and hydrolyze a specific phospholipid to the immediate precursor of PAF. There is evidence that protein kinase C may, in part, regulate this process, but the role of other potential regulatory components, such as other protein kinases or G-proteins is not known. As noted above, the most direct mechanism for initiation of PAF biosynthesis and arachidonate release would be activation of a phospholipase A2 as shown in Fig. 3. Activation of other phospholipases (e.g. phospholipase C) may contribute to the total amount of arachidonate released, although the magnitude of that contribution is not yet known. In addition to generation of PAF and eicosanoids, activation of endothelial cell phospholipases generates second messengers that are important in intracellular signaling (Fig. 4). Activation of phospholipase C, in response to hormonal stimulation, generates diacylglycerol and inositol phosphates from phosphatidylinositol. Each of these is a potent intracellular second messenger.(ABSTRACT TRUNCATED AT 400 WORDS)     

Leukotriene B formation by neutrophils from essential fatty acid-deficient rats

Analysis of neutrophil phospholipids from rats fed an essential fatty acid-deficient diet revealed a 33% reduction in arachidonate and a 90% reduction in linoleate compared to neutrophil phospholipids of rats fed a normal diet. The neutrophil phospholipids from rats fed the essential fatty acid-deficient diet also contained significant amounts of 5,8,11-eicosatrienoate, a fatty acid not found in the neutrophils of rats fed a normal diet. Analysis of the production of leukotrienes of the B series by ionophore-stimulated neutrophils from rats fed an essential fatty acid-deficient diet revealed a 87% reduction in leukotriene B4 compared to neutrophils from rats fed a normal diet even though the arachidonate content was reduced by only 34%. Essential fatty acid-deficient neutrophils converted endogenous 5,8,11-eicosatrienoic acid to leukotriene A3 and its nonenzymatic degradation products, but little or no leukotriene B3 was formed. Neutrophils from rats fed a normal diet incubated with ionophore and exogenous 5,8,11-eicosatrienoate also produced leukotriene A3 and its nonenzymatic degradation products but little or no leukotriene B3. Exogenous 5,8,11-eicosatrienoate incubated with ionophore-stimulated normal neutrophils caused a dose-dependent inhibition of leukotriene A hydrolase resulting in diminished production of leukotriene B4 from endogenous arachidonate. Assays of leukotriene A hydrolase in the 10,000 X g supernatant fraction of a homogenate of RBL-1 cells revealed that a lipoxygenase metabolite of 5,8,11-eicosatrienoate rather than 5,8,11-eicosatrienoate itself is the inhibitor of leukotriene A hydrolase. Thus the finding that leukotriene B4 production by neutrophils from essential fatty acid-deficient rats is diminished out of proportion to the decrease in arachidonate content appears to be due to inhibition of leukotriene A hydrolase by a lipoxygenase metabolite.     

Brain Arachidonic Acid Incorporation and Precursor Pool Specific Activity During Intravenous Infusion of Unesterified [3H]Arachidonate in the Anesthetized Rat

Abstract: Brain fatty acid incorporation into phospholipids can be measured in vivo following intravenous injection of fatty acid tracer. However, to calculate a cerebral incorporation rate, knowledge is required of tracer specific activity in the final brain precursor pool. To determine this for one tracer, unesterified [³H]arachidonate was infused intravenously in pentobarbital-anesthetized rats to maintain constant plasma specific activity for 1–10 min. At the end of infusion, animals were killed by microwave irradiation and analyzed for tracer specific activity and concentration in brain phospholipid, neutral lipid, and lipid precursor, i.e., unesterified arachidonate and arachidonoyl-CoA, pools. Tracer specific activity in brain unesterified arachidonate and arachidonoyl-CoA rose quickly ( t _1/2 < 1 min) to steady-state values that averaged <5% of plasma specific activity. Incorporation was rapid, as >85% of brain tracer was present in phospholipids at 1 min of infusion. The results demonstrate that unesterified arachidonate is rapidly taken up and incorporated in brain but that brain phospholipid precursor pools fail to equilibrate with plasma in short experiments. Low brain precursor specific activity may result from (a) dilution of label with unlabeled arachidonate from alternate sources or (b) precursor pool compartmentalization. The results suggest that arachidonate turnover in brain phospholipids is more rapid than previously assumed.