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.     

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.     

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.     

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.     

Biosynthesis and metabolism of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine in rat glomerular mesangial cells

The ability of rat mesangial cells to synthesize 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acetyl-GPC), also known as platelet activating factor (PAF), was studied in mesangial cell cultures originating from isolated rat glomeruli. In response to the phospholipase A2 agonist A23187 mesangial cells synthesized PAF primarily via an acetyltransferase utilizing either [3H]lyso-PAF or [3H]acetate/[3H]acetyl-CoA substrates. The major PAF species synthesized was 1-O-hexadecyl-2-acetyl-GPC. PAF was also synthesized from 1-O-[3H]alkyl-2-acetyl-sn-3-glycerol, indicating the presence of a CDP-cholinephosphotransferase. Mesangial cells incorporated [3H]lyso-PAF to 1-O-[3H]alkyl-2-acyl-GPC. Subsequent stimulation with A23187 (2 microM) resulted in formation and release of [3H]PAF following 3 h, and this was associated with concomitant decrements in intracellular 1-O-[3H]alkyl-2-acyl-GPC and [3H]lyso-PAF levels, indicating a precursor-product relationship among these alkyl ether lipids. Mesangial cells rapidly converted exogenous [3H]PAF to [3H]lyso-PAF and 1-O-[3H]alkyl-2-acyl-GPC, and this process was inhibited by diisopropyl fluorophosphate (10 microM). The demonstration of PAF activation-inactivation pathways in mesangial cells may be of importance in regulating their function and in glomerular injury.     

Heterogeneity in the sn-1 carbon chain of platelet-activating factor glycerophospholipids determines pro- or anti-apoptotic signaling in primary neurons

The platelet-activating factor (PAF) family of glycerophospholipids accumulates in damaged brain tissue following injury. Little is known about the role of individual isoforms in regulating neuronal survival. Here, we compared the neurotoxic and neuroprotective activities of 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C(16)-PAF) and 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphocholine (C(18)-PAF) in cerebellar granule neurons. We find that both C(16)-PAF and C(18)-PAF cause PAF receptor-independent death but signal through different pathways. C(16)-PAF activates caspase-7, whereas C(18)-PAF triggers caspase-independent death in PAF receptor-deficient neurons. We further show that PAF receptor signaling is either pro- or anti-apoptotic, depending upon the identity of the sn-1 fatty acid of the PAF ligand. Activation of the PAF G-protein-coupled receptor (PAFR) by C(16)-PAF stimulation is anti-apoptotic and inhibits caspase-dependent death. Activation of PAFR by C(18)-PAF is pro-apoptotic. These results demonstrate the importance of the long-chain sn-1 fatty acid in regulating PAF-induced caspase-dependent apoptosis, caspase-independent neurodegeneration, and neuroprotection in the presence or absence of the PAF receptor.     

Inactivation of platelet activating factor by rabbit platelets. Lyso-platelet activating factor as a key intermediate with phosphatidylcholine as the source of arachidonic acid in its conversion to a tetraenoic acylated product

[3H]PAF (platelet activating factor or 1-alkyl-2-acetyl-GPC) is converted to 1-alkyl-2-lyso-GPC and 1-alkyl-2-acyl-GPC by rabbit platelets (GPC is sn-glycero-3-phosphocholine). The deacetylation reaction does not involve the transfer of the acetate of PAF to any other lipid class and added exogenous lyso-PAF readily mixes with the cellular pool of the [3H]lyso-PAF intermediate formed from [3H]PAF. [3H]1-Alkyl-2-acyl-GPC produced during the inactivation of [3H]PAF contained primarily the tetraenoic acyl species (approximately 80% of the 3H in this fraction). The source of the arachidonic acid used for the reacylation of the lyso-PAF intermediate is the diacyl species, phosphatidylcholine.     

C20 polyunsaturated fatty acids and phorbol myristate acetate enhance agonist-stimulated synthesis of 1-radyl-2-acetyl-sn-glycero-3-phosphocholine in vascular endothelial cells

This study has investigated the effect of supplementation of vascular endothelial cells with arachidonate and other polyunsaturated fatty acids on the agonist-stimulated synthesis of platelet activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; 1-alkyl-2-acetyl-GPC). Incubation of calf pulmonary artery endothelial cells for 48 h in medium containing 40 microM arachidonate resulted in a 2-3-fold enhancement of [3H]acetate incorporation into 1-radyl-2[3H]acetyl-GPC in response to either bradykinin or calcium ionophore A23187. The effects of arachidonate supplementation were both dose- and time-dependent, requiring a minimum exogenous arachidonate concentration of 2.5 microM and an incubation time of 4-6 h. Eicosapentaenoate and docosahexaenoate also enhanced the synthesis of 1-radyl-2-[3H]acetyl-GPC, but were less potent than arachidonate; alpha-linolenate, linoleate and oleate were without effect. Although not effective as an agonist, phorbol myristate acetate potentiated A23187- and bradykinin-stimulated synthesis of 1-radyl-2-[3H]acetyl-GPC. The effects of arachidonate supplementation were synergistic with potentiation by phorbol myristate acetate. Sphingosine inhibited agonist-stimulated incorporation of [3H]acetate into 1-radyl-2-[3H]acetyl-GPC both in the presence and absence of PMA. Characterization of the radiolabeled material indicated that the primary product was the acyl analogue of PAF (1-acyl-2-acetyl-GPC) rather than PAF. The results from this study suggest that agonist-stimulated synthesis of 1-radyl-2-acetyl-GPC in vascular endothelial cells is modulated both by cellular fatty acyl composition and activation of protein kinase C. Enrichment of vascular endothelial cells with fatty acids, which are mobilized by agonist-stimulated phospholipase A2, may enhance subsequent deacylation of choline phospholipids and, thus, increase synthesis of both 1-acyl-2-acetyl-GPC and PAF.     

Metabolism of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine in the human neutrophil

The biosynthesis of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-2-acetyl-GPC) together with that of 1-alkyl-2-acetyl-GPC (platelet-activating factor) has been demonstrated in a variety of inflammatory cells and tissues. It has been hypothesized that the relative proportion of these phospholipids produced upon cell activation may be influenced by their rates of catabolism. We studied the catabolism of 1-acyl-2-acetyl-GPC in resting and activated human neutrophils and compared it to that of 1-alkyl-2-acetyl-GPC. Neutrophils rapidly catabolize both 1-alkyl-2-acetyl-GPC and 1-acyl-2-acetyl-GPC; however, the rate of catabolism of 1-acyl-2-acetyl-GPC is approximately 2-fold higher than that of 1-alkyl-2-acetyl-GPC. In addition, most of 1-acyl-2-acetyl-GPC is catabolized through a pathway different from that of 1-alkyl-2-acetyl-GPC. The main step in the catabolism of 1-acyl-2-acetyl-GPC is the removal of the long chain at the sn-1 position; the long chain residue is subsequently incorporated either into triglycerides or into phosphatidylcholine. The 1-lyso-2-acetyl-GPC formed in this reaction is then further degraded to glycerophosphocholine, choline, or phosphocholine. 1-Acyl-2-acetyl-GPC is also catabolized, to a lesser extent, through deacetylation at the sn-2 position and reacylation with a long chain fatty acid. Stimulation of neutrophils by A23187 results in a higher rate of catabolism of 1-acyl-2-acetyl-GPC by increasing both the removal of the long chain at the sn-1 position and the deacetylation-reacylation at the sn-2 position. In a broken cell preparation, the cytosolic fraction of the neutrophil was shown to contain an enzyme activity which cleaved the sn-1 position of 1-acyl-2-acetyl-GPC and 1-acyl-2-lyso-GPC but not of 1,2-diacyl-GPC. Taken together, these data demonstrate that the human neutrophil is able to catabolize 1-acyl-2-acetyl-GPC in a manner both quantitatively and qualitatively different from that of platelet-activating factor. The differential catabolism may regulate the relative proportion of these two bioactive phospholipids in the neutrophil.     

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.     

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.     

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).     

Binding of platelet activating factor by isolated membranes from U937 cells

Platelet activating factor (PAF), 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acetyl-GPC) has been known to have biological effect on cells. The mechanisms of the effect of the potent phospholipid on cells has not been established. We have used 1-O-[3H]alkyl-2-acetyl-GPC [( 3H]PAF) to study the interaction on the isolated membranes of U937 cells. The binding process was time, protein concentration, temperature dependent and reversible. The binding of [3H]PAF to the U937 cell membranes was slightly inhibited by the addition of PAF analogue, 3-O-Hexadecyl-2-acetyl-sn-glycerol-1-phosphorylcholine. U937 cell membranes showed high affinity binding sites for PAF with equilibrium dissociation constant (Kd) of 5 x 10(-9) M. The displacement of bound [3H]PAF with 500-fold excess of nonlabeled PAF was not altered suggesting that the bound [3H]PAF was not degraded during the binding. Binding of [3H]PAF on U937 cell membranes was inhibited by PAF antagonist, 59227RP. The kinetic of the inhibition by PAF antagonist is competitive suggesting that PAF and PAF antagonist bind at the same site.     

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.     

Species-specific variations in the molecular heterogeneity of the platelet-activating factor

The molecular heterogeneity of platelet-activating factor (PAF) synthesized by unstimulated and Ca2+ ionophore (A23187)-stimulated PMN from rat, mouse, and guinea pig and by rat basophilic leukemia (RBL) cells was investigated by gas chromatography-negative ion chemical ionization mass spectrometry. Several molecular species of PAF ranging from C14:0 to C19:0 were detected in all of the cells studied. PAF produced by each cell type exhibited a unique pattern of molecular species distribution. Although C16:0 was the major PAF molecular species of rat PMN and RBL cells representing 96% and 85% of the total PAF, respectively, PAF from mice PMN contained 81% of C16:0, 10% of C18:1, and 6% of C18:0. Alternatively, A23187-stimulated guinea pig PMN yielded PAF molecular species 35% in C16:0, 35% in C17:0, 8% in C18:1, and 3% in C18:0. Small but significant differences in the PAF molecular species distribution of resting and ionophore stimulated cells were also observed. In contrast to the PAF molecular species composition, the precursor 1-O-alkyl-2-acyl-glycero-3-phosphocholine of all the cell types was predominantly hexadecyl (C16:0) alkyl chain in the sn-1 position, representing 60 to 80% of the total 1-O-alkyl-2-acyl-glycero-3-phosphocholine. Thus, these results not only indicate a high degree of selectivity for utilization of precursor substrates for PAF biosynthesis, but also demonstrate that the selectivity is species specific.     

Synthesis of 1-acyl-2-[3H]acetyl-SN-glycero-3-phosphocholine, a structural analog of platelet activating factor, by vascular endothelial cells

Human umbilical vein endothelial cells (HUVECS) were challenged with thrombin in the presence of [3H]acetate to stimulate the production of radiolabeled platelet activating factor (PAF, 1-O-alkyl-2-[3H]acetyl-sn-glycero-3-phosphocholine, 1-O-alkyl-2-[3H]acetyl-GPC). The 3H-product was isolated by thin-layer chromatography, and 1-radyl-2[3H],3- diacetylglycerols were prepared by phospholipase C digestion and subsequent acetylation at the sn-3 position. When the 1-radyl-2[3H],3-diacetylglycerols were analyzed by zonal thin-layer chromatography, 96-97% of the radiolabeled derivative migrated with 1-acyl-2,3-diacetylglycerol standard. Only minor amounts (3-4%) of 1-alkyl-2[3H],3-diacetylglycerol were observed, demonstrating that the predominant acetylated product synthesized by thrombin-stimulated HUVECS was 1-acyl-2-[3H]acetyl-GPC. This relative abundance of 1-acyl-2-[3H]-acetyl-GPC was not significantly affected by thrombin dose, incubation time, or cell passage, and was also observed in HUVECS challenged with ionophore A23187. In addition, the acetylated product from ionophore A23187- or bradykinin-stimulated bovine aortic endothelial cells contained 90% 1-acyl-2-[3H]acetyl-GPC, suggesting that the synthesis of the 1-acyl PAF analog is not unique to HUVECS. These findings demonstrate that PAF is a minor synthetic component of HUVECS and bovine aortic endothelial cells. In light of the integral role which the vascular endothelial cell plays in the regulation of thrombosis, these findings also suggest that the production of 1-acyl-2-acetyl-GPC may be biologically important.     

The metabolism of platelet-activating factor in human T-lymphocytes

The metabolism of 1-[3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine (1-[3H]alkyl-2-acetyl-GPC; platelet-activating factor; PAF) was investigated in purified human peripheral blood T-lymphocytes and a human leukemia cell line of T-cell origin (MOLT-4). The major metabolic products of T-lymphocyte PAF metabolism are 1-alkyl-2-acyl-GPC, 1-alkyl-2-lyso-GPC and neutral lipid. The pattern of PAF metabolism in peripheral blood T-lymphocytes and MOLT-4 lymphoblasts was similar, although MOLT-4 lymphoblasts transformed PAF to 1-alkyl-2-acyl-GPC faster than peripheral blood T-lymphocytes (67% vs. 21% of added label after 64 min at 37 degrees C, respectively). Pre-exposure of MOLT-4 lymphoblasts to 1 mM of the serine hydrolase inhibitor phenylmethylsulfonyl fluoride resulted in an inhibition of PAF metabolism. Our results indicate that intact T-lymphocytes actively metabolize this biologically active phospholipid by the deacetylation-transacylation pathway.     

Metabolic conversion of platelet-activating factor into ethanolamine plasmalogen in an amnion-derived cell line.

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) labeled with 3H in the alkyl side chain was taken up rapidly by amnion-derived WISH cells in culture. The radioactivity was found in a number of cellular metabolites, principally 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-acyl-GPC) which was labeled at a rapid rate. No intracellular accumulation of lyso-PAF was detected. At longer time periods, a substantial proportion of the radioactivity was found in association with the phosphatidylethanolamine fraction extracted from the cells. This fraction contained a high proportion of the corresponding 1',2'-alkenyl derivative (plasmalogen), as judged by the formation of long-chain fatty aldehyde after exposure to acid. The magnitude of the conversion of PAF into ethanolamine plasmalogen is suggestive of a correlation between plasmalogen content and exposure to PAF in some tissues. The exact sequence of reactions leading from alkyl-acyl-GPC to the ethanolamine derivatives is yet to be established.     

Selective acylation of lyso platelet activating factor by arachidonate in human neutrophils

Human polymorphonuclear leukocytes (PMN) incubated with 1-O-[3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine (1-[3H]alkyl-2-acetyl-GPC; platelet activating factor) inactivated the compound by removing the acetyl group and replacing it with a long chain acyl residue. The nature of the acyl group added at the 2-position of the 1-O-[3H]alkyl-2-acyl-GPC formed was examined by argentation chromatography and by reverse phase high performance liquid chromatography. A striking selectivity for arachidonate was observed in the acylation reaction. The major labeled component of the starting material was the 1-O-hexadecyl-linked species; high performance liquid chromatography analysis revealed that 75 to 80% of this component was acylated by arachidonate. Similarly, based on argentation thin layer chromatography, approximately 80% of the total starting material was acylated by tetraenoic acyl residues. The incorporation of 1-O-[3H]alkyl-2-lyso-GPC into 1-O-alkyl-2-acyl-GPC by the PMN was compared; no difference in the acylation pattern was observed with the 2-acetyl and 2-lyso precursors. Thus, activation of the PMN does not appear to be required to elicit the selectivity for arachidonate. When labeled 1-palmitoyl-2-lyso-GPC was compared in the system under the same conditions, it was also preferentially acylated by arachidonate; thus, it is not clear at this time whether or not the selectivity for arachidonate is physiologically limited to platelet activating factor. Our findings suggest a close relationship exists between the metabolism of platelet activating factor and arachidonate in human PMN.     

Substrate specificity and partial characterization of the PAF-acylhydrolase in human serum that rapidly inactivates platelet-activating factor

The structure of the potent inflammatory mediator, platelet-activating factor, is 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC, PAF-acether). Human sera contain an acid labile factor (ALF) that is a Ca+2-independent 2-acylhydrolase-specific for AGEPC and AGEPC-like molecules. The enzyme functions by catalytically removing the sn-2 acetyl moiety from AGEPC, producing the biologically inactive sn-2 hydroxy form or 2-lyso-GEPC. Incubation of ALF with sn-2 acyl PAF analogs indicated that the enzyme hydrolyzes the sn-2 fatty acid only if the chain length is five carbons or less, the sn-1 position fatty acid length is greater than 10 carbon units, and at least one methyl group is present on the terminal amine of the choline group. The enzyme was active with either an ether or ester linkage at the sn-1 position. ALF is inactivated by heating to 65 degrees C for 30 min. It is pronase and trypsin sensitive but resistant to papain and papain with dithiothreitol. Further characteristics of human ALF indicated a broad pH range of activity with an optimum of pH 6.2 and an isoelectric point of 6.2 to 6.7. The specificity and Ca+2 independence of human ALF sets it apart from phospholipase A2. It is proposed that human ALF be called human serum PAF-acylhydrolase to distinguish it from other hydrolases currently known to exist.