Conversion of 1-O-[3H]alkyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine to lyso platelet-activating factor by the CoA-independent transacylase in membrane fractions of human neutrophils

The first step in the synthesis of platelet-activating factor (PAF) in stimulated neutrophils is generally accepted to be hydrolysis of 1-O-alkyl-2-acyl-sn-glycero-3-phosphorylcholine (1-O-alkyl-2-acyl-GPC), with 1-O-alkyl-2-arachidonoyl-GPC being the preferred precursor. Characterization of the enzymatic activity responsible for the hydrolysis of 1-O-alkyl-2-arachidonoyl-GPC has been hampered by lack of an active and reliable cell-free system for study. In the present studies, membrane preparations containing 1-O-[3H]alkyl-2-arachidonoyl-GPC were prepared from intact human neutrophils that had been labeled using 1-O-[3H]hexadecyl-2-lyso-GPC. When the labeled membrane preparations were incubated in the presence of unlabeled 1-O-alkyl-2-lyso-GPC (5 microM), rapid deacylation (up to 25% of the label in 10 min) of the 1-O-[3H]alkyl-2-arachidonoyl-GPC to 1-O-[3H]alkyl-2-lyso-GPC (lyso-PAF) was observed. The deacylation activity appeared to be the same in preparations from resting or stimulated cells. No requirement for Ca2+, various nucleotides, or protein kinase activation could be demonstrated. A number of observations indicated that [3H]lyso-PAF is formed in the system by the action of the CoA-independent transacylase present in the cells rather than by phospholipase A2. Both 1-O-alkyl-2-lyso-GPC and 1-acyl-2-lyso-GPC elicited deacylation of 1-O-[3H]alkyl-2-arachidonoyl-GPC, whereas neither 3-O-alkyl-2-lyso-GPC nor 1-O-alkyl-2-O-methyl-rac-glycero-3-phosphorylcholine, which should act as detergents but are not transacylase substrates, effected deacylation. The deacylation activity and CoA-independent transacylase activities were blocked in parallel by a number of inhibitors and by heat inactivation. In preparations containing 1-O-alkyl-2-[3H]arachidonoyl-GPC, no release of free [3H]arachidonic acid was observed. However, a shift of the [3H]arachidonate into exogenous 1-O-tetradecyl-2-lyso-GPC was observed in the system. These findings are consistent with the generation of [3H]lyso-PAF by the CoA-independent transacylase activity.     

Reacylation of platelet activating factor with eicosapentaenoic acid in fish-oil-enriched monkey neutrophils

Platelet activating factor (PAF) is rapidly metabolized via a deacetylation: reacylation pathway which shows striking specificity for arachidonate at the sn-2 position of the 1-O-alkyl-2-acyl-GPC thus formed. We have now examined the effects of a diet enriched in fish oils on the metabolism of PAF and specificity for arachidonate in the reacylation reaction. [3H]PAF was incubated for various lengths of time with neutrophils from monkeys fed a control diet or one enriched in fish oils. The [3H]PAF added to the cell suspension was rapidly converted to 1-O-alkyl-2-acyl-GPC. Reverse-phase HPLC analysis of the acyl chains added at the sn-2 position revealed that arachidonate was the major fatty acid incorporated into the 1-O-alkyl-2-acyl-GPC formed by neutrophils from monkeys on the control diet. In contrast, both 1-O-alkyl-2-arachidonoyl-GPC and 1-O-alkyl-2-eicosapentaenoyl-GPC were formed by the fish-oil-enriched neutrophils. We also report on the fatty acid composition of neutrophil phospholipids during such a diet.     

Biologically active lipids. Semi-synthesis of 3H-labeled ether glycerophospholipids and ether glyceroglycolipids from ratfish liver oil

1-O-Alkyl-2,3-diacyl-sn-glycerols, the major constituents of ratfish (Chimaera monstrosa) liver oil, serve as starting material for the preparation of 1-O-alkyl-sn-glycero-3-phosphocholines, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholines, 1-O-alkyl-2-O-methyl-sn-glycero-3-phosphocholines, and 1-O-alkyl-2-O-methyl-sn-glycero-3-beta-D-glucopyranosides. Catalytic tritiation of the unsaturated alkyl moieties in these biologically active ether lipids affords the corresponding 3H-labeled substances.     

A facile synthesis of 1-O-alkyl-2-(R)-hydroxypropane-3-phosphonocholine (lyso-phosphono-platelet activating factor).

The synthesis of 1-O-alkyl-2-(R)-hydroxypropane-3-phosphonocholine is described. An efficient alkylation procedure using (NaH/DMSO) catalysis is also described and applied to the synthetic scheme. The key intermediate 1-O-alkyl-2-(R)-O-benzyl-3-bromopropane was phosphonylated using tris(methylsilyl)phosphite; the resulting phosphonic acid was coupled to choline using trichloroacetonitrile/pyridine or triisopropylbenzenesulfonyl chloride/pyridine followed by catalytic hydrogenation to yield 1-O-alkyl-2(R)-hydroxypropane-3-phosphonocholine.     

Interleukin-2 causes an increase in saturated/monounsaturated phosphatidic acid derived from 1,2-diacylglycerol and 1-O-alkyl-2-acylglycerol.

Phosphatidic acid generation through activation of diacylglycerol kinase alpha has been implicated in interleukin-2-dependent T-lymphocyte proliferation. To investigate this lipid signaling in more detail, we characterized the molecular structures of the diradylglycerols and phosphatidic acids in the murine CTLL-2 T-cell line under both basal and stimulated conditions. In resting cells, 1,2-diacylglycerol and 1-O-alkyl-2-acylglycerol subtypes represented 44 and 55% of total diradylglycerol, respectively, and both showed a highly saturated profile containing primarily 16:0 and 18:1 fatty acids. 1-O-Alk-1'-enyl-2-acylglycerol represented 1-2% of total diradylglycerol. Interleukin-2 stimulation did not alter the molecular species profiles, however, it did selectively reduce total 1-O-alkyl-2-acylglycerol by over 50% at 15 min while only causing a 10% drop in 1,2-diacylglycerol. When radiolabeled CTLL-2 cells were challenged with interleukin-2, no change in the cellular content of phosphatidylcholine nor phosphatidylethanolamine was observed thereby ruling out phospholipase C activity as the source of diradylglycerol. In addition, interleukin-2 failed to stimulate de novo synthesis of diradylglycerol. Structural analysis revealed approximately equal amounts of 1,2-diacyl phosphatidic acid and 1-O-alkyl-2-acyl phosphatidic acid under resting conditions, both containing only saturated and monounsaturated fatty acids. After acute (2 and 15 min) interleukin-2 stimulation the total phosphatidic acid mass increased, almost entirely through the formation of 1-O-alkyl-2-acyl species. In vitro assays revealed that both 1,2-diacylglycerol and 1-O-alkyl-2-acylglycerol were substrates for 1,2-diacylglycerol kinase alpha, the major isoform in CTLL-2 cells, and that the lipid kinase activity was almost totally inhibited by R59949. In conclusion, this investigation shows that, in CTLL-2 cells, 1,2-diacylglycerol kinase alpha specifically phosphorylates a pre-existing pool of 1-O-alkyl-2-acylglycerol to form the intracellular messenger 1-O-alkyl-2-acyl phosphatidic acid.     

Choline-linked phosphoglycerides. A source of phosphatidic acid and diglycerides in stimulated neutrophils

Stimulation of human polymorphonuclear leukocytes (PMN) may result in the metabolism of phospholipids other than phosphoinositides to generate second-messenger intermediary metabolites. We investigated agonist-induced breakdown of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acyl-GPC), which constitutes almost half the diradyl-GPC fraction in human PMN (Mueller, H. W., O'Flaherty, J. T., Green, D. G., Samuel, M. P., and Wykle, R. L. (1984) J. Lipid Res. 25: 383-388), in cells prelabeled with 1-O-[3H] alkyl-2-acyl-GPC. We also utilized normal-phase high pressure liquid chromatography to quantitate the accumulation of diradylglycerols (1-O-alkyl-2-acylglycerols and diacylglycerols) in stimulated PMN. Phorbol-12-myristate-13-acetate (PMA), 1-oleoyl-2-acetyl-sn-glycerol-, calcium ionophore A23187-, and f-methionyl-leucyl-phenylalanine (fMLP) stimulation of PMN resulted in a time- and concentration-dependent hydrolysis of 1-O-[3H]alkyl-2-acyl-GPC and the formation of 1-O-[3H]alkyl-2-acyl-phosphatidic acid (PA) and 1-O-[3H]alkyl-2-acylglycerol. In all cases formation of 1-O-[3H]alkyl-2-acyl-PA preceded that of 1-O-[3H]alkyl-2-acylglycerol. The times between addition of stimulus and appearance of 1-O-[3H] alkyl-2-acylglycerol varied for PMA (40 s at 1.6 microM), A23187 (5 min at 5 microM), and fMLP (30 sec at 1 microM). Preincubation of cells with 1 microgram/ml pertussis toxin (PT) inhibited the breakdown of 1-O-[3H]alkyl-2-acyl-GPC in cells stimulated with 1 microM fMLP, indicating a role for a PT-sensitive G protein with this stimulus. Quantitation of diglycerides as diradylglycerobenzoates in PMN stimulated with PMA (10 min), A23187 (10 min), or fMLP demonstrated marked accumulation of both 1-O-alkyl-2-acylglycerols and diacylglycerols. The highest increases over controls were observed for fMLP (33-fold for 1-O-alkyl-2-acylglycerols and 17-fold for diacylglycerols). In stimulated PMN prelabeled with 1-O-[3H]hexadecyl-2-acyl-GPC and 1-O-alkyl-2-acyl-sn-glycero-3-[32P]phosphocholine, the ratio of 3H to 32P in 1-O-alkyl-2-acyl-PA compared to 1-O-alkyl-2-acyl-GPC suggested the involvement of a phospholipase D in the hydrolysis of 1-O-[3H]-alkyl-2-acyl-GPC. Thus, stimulation of human PMN results in the hydrolysis of 1-O-[3H]alkyl-2-acyl-GPC to yield 1-O-[3H] alkyl-2-acyl-PA and 1-O-[3H]alkyl-2-acylglycerol possibly initiated by activation of a phospholipase D.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of immunologic activation and cellular fatty acid levels on the catabolism of platelet-activating factor within the murine mast cell (PT-18)

The present study has examined the catabolism of 1-O-[3H]hexadecyl-2-acetyl-GPC (C16-PAF) and of 1-O-octadecyl-2-acetyl-GPC (C18-PAF) in spleen-derived PT-18 murine mast cells (mast cells). Mast cells catabolized exogenous PAF into two inactive metabolites, 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (lysoPAF) and 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acyl-GPC). The rate of conversion of C16-PAF to metabolites was more rapid than that of C18-PAF. Analysis of the acyl composition of 1-O-alkyl-2-acyl-GPC formed during the metabolism of PAF revealed that arachidonic acid (20:4) was the major fatty acyl chain incorporated at the sn-2 position. However, 25% of newly formed 1-O-alkyl-2-acyl-GPC was reacylated with docosahexaenoic acid (22:6). The influence of cellular fatty acid content on PAF catabolism was further explored in mast cells in which the ratio of fatty acids within cellular phosphoglycerides had been altered by supplementing the cells with various fatty acids in culture. Mast cells supplemented with 20:4 or 22:6 converted PAF to 1-O-alkyl-2-acyl-GPC at a significantly higher rate than non-supplemented cells. In contrast, cells supplemented with linoleic acid (18:2) metabolized PAF at rates similar to non-supplemented cells. Analysis of the acyl composition of 1-O-alkyl-2-acyl-GPC derived from the metabolism of PAF in 20:4-supplemented cells indicated that 20:4 was incorporated exclusively into the sn-2 position. Conversely, 22:6-supplemented cells incorporated predominantly 22:6 at the sn-2 position of 1-alkyl-2-lyso-GPC. Supplementation with 18:2 had no effect on the acylation pattern seen in newly formed 1-O-alkyl-2-acyl-GPC. Activation of passively sensitized mast cells with antigen or with ionophore A23187 significantly enhanced the rate of catabolism of exogenously-provided PAF but had no effect on the acylation pattern of 1-O-alkyl-2-acyl-GPC. Experiments performed with the soluble fraction of the cells showed that acetyl hydrolase activity was increased in mast cells stimulated with antigen. In addition, supernatant fluids from antigen or ionophore-treated mast cells converted PAF to lysoPAF, suggesting that acetyl hydrolase activity was released during cell activation. These data indicate that the ability of mast cells to catabolize PAF to inactive metabolites is influenced by cell activation and by the cellular levels of certain fatty acids.     

Studies on the mechanism of interleukin 1 stimulation of platelet activating factor synthesis in human endothelial cells in culture

Interleukin 1 promotes the conversion of the biologically inactive lyso-platelet activating factor (lyso-PAF) to the bioactive platelet activating factor (PAF) by an acetylation reaction in cultured human endothelial cells. After 2 h stimulation with interleukin 1, 1-O-alkyl-2-lysoglycero-3-phosphocholine (GPC): acetyl CoA acetyltransferase is activated, reaching a plateau after 6 h and then declining to the basal value within 24 h. This time course is comparable to that of PAF production. These cells are able to incorporate [3H]acetate and [3H]lyso-PAF into PAF. Synthetized [3H]PAF is then catabolized in [3H]alkylacyl phosphoglycerides. 1-O-alkyl-2-acetylglycerol: CDP-choline cholinephosphotransferase and 1-O-alkyl-2-acetyl-GPC: acetylhydrolase activities are both present in endothelial cells, but are not activated under our conditions of stimuli. These findings indicate that interleukin 1 induces the PAF synthesis by a deacylation/reacetylation mechanism into human endothelial cells.     

Relative amounts of 1-O-alkyl- and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine in stimulated endothelial cells.

The specific precursor for platelet-activating factor, 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine, constitutes 10 per cent of the 1-radyl-2-acyl-sn-glycero-3-phosphocholines in endothelial cells. Stimulation of endothelial cells results in accumulation of PAF and its sn-1-acyl- analog (acylPAF), with acylPAF the predominant product. Mass spectrometry confirmed these relative amounts and confirmed that stimulated endothelial cells accumulate 1-3 ng PAF per million cells. These data suggest that stimulated endothelial cells accumulate both PAF and acylPAF and that the PAF synthetic pathway in endothelial cells is not highly selective for the specific PAF precursor (1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine).     

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-O-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine. A common source of platelet-activating factor and arachidonate in human polymorphonuclear leukocytes

1-O-[3H]Alkyl-2-lyso-sn-glycero-3-phosphocholine (1-O-[3H]alkyl-2-lyso-GPC) incubated with human polymorphonuclear leukocytes (PMN) for 30 min is metabolized to 1-O-alkyl-2-acyl-GPC containing greater than 80% arachidonate at the 2 position (Chilton, F. H., O'Flaherty, J. T., Ellis, J. M., Swendsen, C. L., and Wykle, R. L. (1983) J. Biol. Chem. 258, 7268-7271). PMN containing 1-O-[3H]alkyl-2-arachidonoyl-GPC incorporated into their cellular phospholipids in this manner were stimulated with Ca2+ ionophore (A23187). Within 5 min after stimulation, 14%, 7%, and 7% of the total 1-O-[3H]alkyl-2-arachidonoyl-GPC in the cells had been converted to 1-O-[3H]alkyl-2-acetyl-GPC (platelet-activating factor), 1-O-[3H]alkyl-2-lyso-GPC, and 3H-labeled neutral lipid, respectively. Stimulation by opsonized zymosan yielded similar results. In related studies, cells were labeled with 1-O-hexadecyl-2-arachidonoyl-GPC containing a [methyl-14C] choline moiety. The nature of the long-chain acyl residues in the sn-2 position of the labeled 1-O-hexadecyl-2-acyl-GPC remaining after stimulation with A23187 was examined. Analysis by high-performance liquid chromatography using synthetic 1-O-hexadecyl-2-acyl-GPC standards indicated there is a time-dependent loss of arachidonate from the 2 position of the labeled 1-O-hexadecyl-2-arachidonoyl-GPC followed by reacylation by other fatty acids (primarily linoleic and oleic). This shift in the acylation pattern exhibited after Ca2+ ionophore stimulation was further examined in PMN preincubated with A23187 and subsequently incubated with labeled 1-O-alkyl-2-lyso-GPC; the stimulated cells produced 1-O-[3H]alkyl-2-acetyl-GPC (greater than 15% of total label) and 1-O-[3H]alkyl-2-acyl-GPC containing linoleic acid and oleic acid, rather than arachidonic acid in the sn-2 position. The findings demonstrate that upon stimulation of PMN, 1-O-alkyl-2-arachidonoyl-GPC can yield arachidonate and 1-O-alkyl-2-lyso-GPC; the 1-O-alkyl-2-lyso-GPC formed may be acetylated producing platelet-activating factor or reacylated with fatty acyl residues other than arachidonate.     

Subcellular localization of enzyme activities involved in the metabolism of platelet-activating factor in rainbow trout leukocytes

The subcellular distribution of an alkyllyso-GPC: acetyl-CoA acetyltransferase (EC 2.3.1.67) and transacylase, two important enzyme activities involved in the remodeling pathway for the biosynthesis of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) have been examined in leukocytes isolated from the pronephros of the rainbow trout, Oncorhynchus mykiss. Contrary to mammalian systems, in which the acetyltransferase is localized to intracellular membranes, the subcellular distribution of an acetyltransferase activity in rainbow trout leukocytes was localized to the plasma membrane. Analysis of the acetyltransferase products by thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) confirmed synthesis of two subclasses of PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine. The transacylase activity in this study was detected in membrane fractions in two domains of the intermediate density region which also contained the NADH dehydrogenase activity, a marker enzyme for the endoplasmic reticulum. Acylation of lysoPAF (1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine) exhibited approximately 95% specificity for omega-3 fatty acids. Acylation patterns were not significantly different in either domain of the endoplasmic reticulum. A model is proposed herein for the metabolism of PAF in rainbow trout leukocytes.     

The enantiomer and the positional isomer of platelet-activating factor

We have compared for rabbit platelet aggregating and desensitizing activity two different preparations of platelet-activating factor (PAF-acether) (1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine) and of its enantiomer (3-O-alkyl-2-O-acetyl-sn-glycero-1-phosphocholine). After phospholipase A2 treatment, the unnatural enantiomers appeared about 3000-fold less active than PAF-acether, a result which definitively establishes the stereospecificity of the mode of action of this mediator. A new structural analog of PAF-acether, 1-O-hexadecyl-3-O-acetyl-sn-glycero-2-phosphocholine, was isolated and characterized. It was a weak platelet agonist, stressing further the importance for PAF-acether activity of the acetyl group at position 2 of the glycerol.     

The neutral glyceroglucolipids of alveolar lavage from rabbit.

Three individual glycolipids have been isolated from the neutral lipid fraction of rabbit alveolar lavage. All three glycolipids contained glucose, glyceryl monoethers and fatty acids, and differed from each other primarily with respect to the number of glucose residues. The structures of these glycolipids were identified by mild alkaline methanolysis, oxidation with periodate and CrO3, and methylation studies, as: Glc(alpha 1 leads to 3)-1,(3)-O-alkyl-2-O-acylglycerol, Glc(alpha 1 leads to 6)Glc(alpha 1 leads to 6)Glc(alpha 1 leads to 6)Glc(alpha 1 lead to 6)Glc(alpha 1 leads to 3)-1,(3)-O-alkyl-2-O-acyglycerol, and Glc(alpha 1 leads to 6)Glc(alpha 1 leads to 6)Glc(alpha 1 leads to 6)Glc(alpha 1 leads to 6)Glc(alpha 1 leads to 6)Glc(alpha 1 leads to 3)-l,(3)-O-alkyl-2-O-acylglycerol.     

The regulation of platelet-activating factor production in endothelial cells. The role of calcium and protein kinase C

Endothelial cells (EC) synthesize platelet-activating factor (PAF) when stimulated with agonists that bind to cell-surface receptors. We examined events that link receptor binding to synthesis of PAF by EC. Bovine EC stimulated with agonists that interact with specific cell-surface receptors accumulated PAF only in the presence of extracellular calcium. Hormonal stimulation of EC resulted in Ca2+ entry characteristic of that seen with receptor-operated calcium channels; Indo-1 measurements demonstrated that this inward flux of Ca2+ caused prolonged elevated levels of intracellular Ca2+. EC were exposed to melittin or theta toxin from Clostridium perfringens (pore-forming peptides that increase the permeability of the plasma membrane for small molecules) resulting in an inward flux of Ca2+ and accumulation of PAF. Ca2+ appears to be regulatory for PAF production at the level of phospholipase A2-mediated production of the PAF precursor 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine, as Ca2+ was required for the stimulated hydrolysis of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine. PAF accumulation in EC is also regulated by protein kinase C. Pretreatment of EC with phorbol esters that activate protein kinase C or with dioctanoylglycerol, followed by stimulation, resulted in a 2-fold increase in stimulated PAF production. The regulatory effect of protein kinase C also appears to be at a phospholipase A2-mediated hydrolysis of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine.     

Dietary lipid modification of renal disorders and ether phospholipid metabolism.

The formation of arachidonic acid derived eicosanoids, including thromboxane A2 and leukotriene B4, as well as platelet-activating factor (1-O-alkyl-2-acetyl-glycerophosphocholine), has been implicated in various renal pathophysiologies. Alteration of the fatty acid composition of membrane phospholipids in platelets, the glomerulus, and inflammatory cells, and of 1-O-alkyl-2-acyl-glycerophosphocholine (platelet-activating factor precursor) can be attained by dietary lipid modifications (e.g., consumption of fish oil containing n - 3 polyunsaturated fatty acids). These changes have been associated with an attenuation in renal disease progression and modifications in the synthesis and actions-interactions of eicosanoids, cytokines, and platelet-activating factors.     

An anti-inflammatory protein secreted from the rat seminal vesicle epithelium inhibits the synthesis of platelet-activating factor and the release of arachidonic acid and prostacyclin

Platelet-activating factor (PAF), a 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, is a mediator of inflammation and endotoxic shock produced by a variety of stimulated cells. Since the main biosynthetic pathway of PAF involves acetylation of 1-O-alkyl-sn-glycero-3-phosphocholine (lyso-PAF) generated from 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine by phospholipase A2, we suggest a general physiological role played by steroid-induced anti-(phospholipase A2) proteins in the modulation of PAF synthesis. The results of the present study support this hypothesis since an androgen-induced anti-inflammatory protein, SV-IV, secreted from rat seminal vesicles, inhibits PAF synthesis in stimulated polymorphonuclear neutrophils, macrophages and endothelial cells. SV-IV impairs PAF synthesis by inhibiting the activation of phospholipase A2, that also results in the inhibition of arachidonic acid and prostacyclin release, and of acetyl-CoA:lyso-PAF acetyltransferase.     

Turnover of arachidonic acid in the major diacyl and ether phospholipids of human platelets

In this work, the uptake and release of [3H]arachidonic acid by the diacyl and ether species of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in human platelets were studied. Uptake of [3H]arachidonic acid into 1,2-diacyl-PC and 1,2-diacyl-PE was much greater than into the ether phospholipids of the same class. In [3H]arachidonoyl-labeled platelets stimulated by thrombin, there was a decrease in total [3H] arachidonoyl-PC. This was accounted for mostly by a decrease in 1-acyl-2-[3H]arachidonoyl-PC while the level of 1-O-alkyl-2-[3H]arachidonoyl-PC (a precursor for platelet-activating factor) increased slightly. However, in ionophore A23187-stimulated platelets, the reduction of total [3H]arachidonoyl-PC was due to a decrease in both 1-acyl-2-[3H]arachidonoyl-PC and 1-O-alkyl-2-[3H] arachidonoyl-PC, suggesting that ionophore should yield more platelet-activating factor than thrombin. In both thrombin- and ionophore-stimulated platelets, there was a net increase in total [3H]arachidonoyl-PE. This consisted of a decrease in 1,2-diacyl-PE, which was essentially complete by 1 min, followed by an increase in 1-O-alk-1'-enyl-2-[3H]arachidonoyl-PE, which was slower and not apparent until 3-5 min after thrombin. During reincubation of labeled platelets with saline, the 1-O-alkyl-2-[3H]arachidonoyl-PC increased by a factor of 2, between 0 and 4 h, with no significant change in the radioactivity of any other phospholipid. Thus, upon stimulation of human platelets, arachidonic is released from both 1,2-diacyl-PC and 1,2-diacyl-PE for metabolism by platelet cyclooxygenase and lipoxygenase, while certain ether pools of PC and PE also collect arachidonic acid.     

Partial purification and characterization of 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase from rat spleen.

The enzyme 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine: acetyl-CoA acetyltransferase (EC 2.3.1.67) was purified from rat spleen approx. 1500-fold in 1.6% yield. The specific activity of the purified enzyme was 0.317 +/- 0.089 mumol/min per mg of protein (mean +/- S.D., n = 6). The Km for the substrate acetyl-CoA was 137 +/- 13 microM and the pH optimum was about 8. Incubation of the purified enzyme was 1-O-[3H]octadecyl-2-lyso-sn-glycero-3-phosphocholine followed by electrophoresis resulted in the incorporation of radioactivity into a protein of Mr 29,000. The enzyme was most active towards 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine as substrate, 1-palmitoyl-2-lyso-glycero-3-phosphocholine being a poor substrate. In addition, the enzyme preferred acetyl-CoA to palmitoyl-CoA or oleoyl-CoA as substrate.     

Metabolism of 1-O-alkyl-2-acetyl-sn-glycerol by washed rabbit platelets: formation of platelet activating factor

A new type of neutral lipid, 1-O-alkyl-2-acetyl-sn-glycerol (AAG), induced a delayed aggregation pattern on interaction with washed rabbit platelets. Although far less potent on a molar basis than platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, AGEPC, nevertheless this compound caused an aggregation, albeit delayed in time, remarkably similar to that exhibited by AGEPC. In view of the possible formation of AGEPC in this reaction, AAG was incubated with washed rabbit platelets, and a lipid corresponding in chromatographic behavior to AGEPC was isolated and identified as such by a combined gas-liquid chromatography/mass spectrometry technique coupled with selected ion monitoring.