Human plasma platelet-activating factor acetylhydrolase. Oxidatively fragmented phospholipids as substrates

Human plasma platelet-activating factor (PAF) acetylhydrolase hydrolyzes the sn-2 acetyl residue of PAF, but not phospholipids with long chain sn-2 residues. It is associated with low density lipoprotein (LDL) particles, and is the LDL-associated phospholipase A2 activity that specifically degrades oxidatively damaged phospholipids (Stremler, K. E., Stafforini, D. M., Prescott, S. M., Zimmerman, G. A., and McIntyre, T. M. (1989) J. Biol. Chem. 264, 5331-5334). To identify potential substrates, we synthesized phosphatidylcholines with sn-2 residues from two to nine carbon atoms long, and found the V/k ratio decreased as the sn-2 residue was lengthened: the C5 homolog was 50%, the C6 20%, while the C9 homolog was only 2% as efficient as PAF. However, the presence of an omega-oxo function radically affected hydrolysis: the half-life of the sn-2 9-aldehydic homolog was identical to that of PAF. We oxidized [2-arachidonoyl]phosphatidylcholine and isolated a number of more polar phosphatidylcholines. We treated these with phospholipase C, derivatized the resulting diglycerides for gas chromatographic/mass spectroscopic analysis, and found a number of diglycerides where the m/z ratio was consistent with a series of short to medium length sn-2 residues. We treated the polar phosphatidylcholines with acetylhydrolase and derivatized the products for analysis by gas chromatography/mass spectroscopy. The liberated residues were more polar than straight chain standards and had m/z ratios from 129 to 296, consistent with short to medium chain residues. Therefore, oxidation fragments the sn-2 residue of phospholipids, and the acetylhydrolase specifically degrades such oxidatively fragmented phospholipids.     

Novel leukocyte agonists are released by endothelial cells exposed to peroxide.

Reactive oxygen species do not activate isolated neutrophils, yet in vivo, such oxidants promote their adhesion to, and subsequent migration through, the vascular wall. We show human endothelial cells exposed to t-butylhydroperoxide shed large, sealed membrane vesicles that contained potent neutrophil agonists. This activity migrated on TLC like platelet-activating factor (PAF). Since neutrophils have a receptor for this phospholipid, which recognizes its unique characteristics including the short sn-2 acetyl residue, we examined the effect of PAF receptor antagonists and PAF acetylhydrolase on this activity. Structurally unrelated PAF receptor antagonists blocked neutrophil stimulation by vesicular phospholipids, and digestion with PAF acetylhydrolase, which is specific for short sn-2 residues, destroyed this activity. However, metabolic labeling, inhibition of synthesis, phospholipase A1 digestion, and high performance liquid chromatographic studies demonstrated that the vesicles did not contain PAF. Instead, the bioactivity migrated on high performance liquid chromatography like the phospholipids generated by oxidative fragmentation of synthetic arachidonoyl phosphatidylcholine that we have shown previously (Smiley, P. L., Stremler, K. E., Prescott, S. M., Zimmerman, G. A., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11104-11110) to stimulate neutrophils through their receptor for PAF. Thus, peroxide treatment of endothelial cells fragments cellular phosphatidylcholines, forming novel PAF-like phospholipids, and induces the shedding of membrane vesicles that contain these bioactive phospholipids.     

Human macrophages secret platelet-activating factor acetylhydrolase

When monocytes mature to macrophages, their ability to accumulate the pro-inflammatory lipid autacoid, platelet-activating factor (PAF), is markedly decreased (Elstad, M. R. Stafforini, D. M., McIntyre, T. M., Prescott, S. M., and Zimmerman, G. A. (1989) J. Biol. Chem. 264, 8467-8470) in conjunction with a 260-fold increase in the activity of intracellular PAF acetylhydrolase (PAF-AH). We now demonstrate that macrophages also secrete PAF-AH and that the secreted enzyme is biochemically and immunologically identical to the human plasma PAF-AH. It is sensitive to the same active-site-directed inhibitors, has the same electrophoretic mobility, is associated with lipoprotein particles, and transfers between low density lipoprotein and high density lipoprotein in a pH-dependent manner like the plasma PAF-AH. In addition, both activities hydrolyze oxidatively fragmented phospholipids and PAF. These data indicate that macrophages are a cellular source of the plasma PAF-AH. Thus, macrophages secrete an enzyme that inactivates lipid mediators at sites of inflammation and in plasma. These changes during the maturation of monocytes to macrophages may serve to limit the acute inflammatory response.     

An oxidized derivative of phosphatidylcholine is a substrate for the platelet-activating factor acetylhydrolase from human plasma

Platelet-activating factor (PAF) is a glycerophospholipid that has diverse potent biological actions. A plasma enzyme catalyzes the hydrolysis of the sn-2 acetoyl group of PAF and thereby abolishes its bioactivity. This PAF acetylhydrolase is specific for phospholipids, such as PAF, with a short acyl group at the sn-2 position. The majority of it (60-70%) is associated with low density lipoprotein (LDL), and the remainder is with high density lipoprotein (HDL). LDL also has a phospholipase A2 activity that is specific for oxidized polyunsaturated fatty acids, which may be important in determining how LDL is recognized by cellular receptors. We previously have purified and characterized the PAF acetylhydrolase from human plasma. We now have found that the purified PAF acetylhydrolase catalyzes the hydrolysis of the oxidized fragments of arachidonic acid from the sn-2 position of phosphatidylcholine. One of the preferred substrates appeared by mass spectrometry to have 5-oxovalerate at the sn-2 position. We synthesized 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine and found that the PAF acetylhydrolase had the same apparent Km for it (11.3 microM) as for PAF (12.5 microM), with Vmax values of 100 and 167 mumol/h/mg of protein, respectively. We also conclude that the PAF acetylhydrolase is the sole activity in LDL that degrades oxidized phospholipids since we found co-localization of the activity against both substrates to LDL and HDL, and precipitation of enzyme activity with an antibody to the PAF acetylhydrolase. Thus, the PAF acetylhydrolase in human plasma degrades oxidized phospholipids, which may be involved in the modification of apolipoprotein B100 and other pathological processes.     

Ether lysophospholipid-induced production of platelet-activating factor in human polymorphonuclear leukocytes

Human polymorphonuclear leukocytes (PMN) produced considerable amounts of platelet-activating factor (PAF) when exposed to various concentrations of lyso-PAF, especially in the absence of albumin. The amount of produced PAF in the presence of 5 microM lyso-PAF (without albumin) was 1.1 pmol/10 min per 2.5 X 10(6) cells, which was close to the level in the case of opsonized zymosan stimulation. We found that the activity of neither acetyltransferase nor acetylhydrolase was affected markedly by the treatment of cells with lyso-PAF, suggesting that the increased availability of lyso-PAF could be responsible for the induction of PAF synthesis. We also found that PAF synthesis was induced not only by lyso-PAF but also by ether-containing ethanolamine lysophospholipids, 1-alkenyl(alkyl)-sn-glycero-3-phosphoethanolamine (GPE). The addition of 1-alkenyl(alkyl)-GPE caused the degradation of pre-existing 1-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine (GPC) and an increased level of lyso-PAF, followed by the formation of PAF. By contrast, 1-acyl-GPC and 1-acyl-GPE failed to induce PAF production. These results suggest a possible key role of the availability of lyso-PAF in triggering the biosynthesis of PAF in human PMN.     

Lysophosphatidylcholine and lyso-PAF display PAF-like activity derived from contaminating phospholipids

Lysophosphatidylcholine is an abundant component of plasma and oxidized LDL that displays several biological activities, some of which may occur through the platelet-activating factor (PAF) receptor. We find that commercial lysophosphatidylcholine, its alkyl homolog (lyso-PAF), and PAF all induce inflammation in a murine model of pleurisy. Hydrolysis of PAF to lyso-PAF by recombinant PAF acetylhydrolase abolished this eosinophilic infiltration, implying that lyso-PAF should not have displayed inflammatory activity. Saponification of lyso-PAF or PAF acetylhydrolase treatment of lyso-PAF or lysophosphatidylcholine abolished activity; neither lysolipid should contain susceptible sn-2 residues, suggesting contaminants account for the bioactivity. Lyso-PAF and to a lesser extent lysophosphatidylcholine stimulated Ca(2+) accumulation in 293 cells stably transfected with the human PAF receptor, and this was inhibited by specific PAF receptor antagonists. Again, treatment of lyso-PAF or lysophosphatidylcholine with recombinant PAF acetylhydrolase, a nonselective phospholipase A(2), or saponification of lyso-PAF destroyed the PAF-like activity, a result incompatible with lyso-PAF or lysophosphatidylcholine being the actual agonist.We conclude that neither lyso-PAF nor lysophosphatidylcholine is a PAF receptor agonist, nor are they inflammatory by themselves. We suggest that PAF or a PAF-like mimetic accounts for inflammatory effects of lysophosphatidylcholine and lyso-PAF.     

Effect of dietary alpha-linolenate on platelet-activating factor production in rat peritoneal polymorphonuclear leukocytes.

The effects of dietary alpha-linolenate (18:3, n-3) and linoleate (18:2, n-6) on platelet-activating factor (PAF) production were examined. Rats were fed an alpha-linolenic acid-rich (perilla oil) diet or a linoleic acid-rich (safflower oil) diet for 6 wk, and polymorphonuclear leukocytes (PMN) were elicited by peritoneal injection of casein. The overall phospholipid content and composition as well as the subclass distribution of choline and ethanolamine glycerophospholipids in PMN were not altered by these diets. However, with the perilla oil diet their content of a putative precursor of PAF, 1-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine was approximately 50% of that with safflower oil diet. On exposure to various concentrations of FMLP, PAF formation by PMN in the perilla oil group was less than 50% of that by PMN in the safflower oil group. A larger difference in PAF productions by PMN in the two dietary groups was observed on their stimulation with calcium ionophore A23187. These results demonstrate that PAF production is modulated in some as yet unknown way by changing the alpha-linolenate/linoleate balance of the diet.     

Human plasma platelet-activating factor acetylhydrolase. Purification and properties

Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a biologically active phospholipid synthesized by a variety of cell types upon appropriate stimulation. PAF is a potent hypotensive factor and it activates platelets and inflammatory cells at concentrations as low as 10(-10) M. Removal of the acetyl moiety at the sn-2 position abolishes the biological activity and this reaction is catalyzed by a specific acetylhydrolase present in plasma and animal tissues. Ultracentrifugation in density gradients showed that 30% of the activity is associated with high density lipoproteins and 70% with low density lipoproteins. We have purified the plasma low density lipoprotein-associated activity to near homogeneity using a rapid assay based on the separation of [3H]acetate from 1-O-alkyl-2-[3H]acetyl-sn-glycerol-3-phosphocholine on disposable reversed-phase columns. The enzyme was purified by 25,000-fold and approximately 10% of the starting activity was recovered. Plasma PAF-acetylhydrolase has an apparent molecular weight of 43,000, does not require calcium, has preference for micellar versus monomeric substrate, and exhibits surface dilution kinetics. The purified protein has an apparent Km of 13.7 microM and a Vmax of 568 mumol/h/mg with micellar PAF. It can act both on 1-O-alkyl and 1-acyl substrates and on ethanolamine analogs of PAF. However, the enzyme has a marked preference for the sn-2 acetyl residue and therefore can be considered as a specific PAF-acetylhydrolase.     

Comparison of 1-O-alkyl-, 1-O-alk-1'-enyl-, and 1-O-acyl-2-acetyl-sn-glycero-3-phosphoethanolamines and -3-phosphocholines as agonists of the platelet-activating factor family

Four naturally occurring platelet-activating factor (PAF) analogs, 1-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphocholine, 1-hexade-canoyl-2-acetyl-sn-glycero-3-phosphocholine, 1-octadecanoyl-2-acetyl-sn-glycero-3-phosphocholine, and 1-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamine, stimulated human neutrophils (PMN) to mobilize Ca²⁺, degranulate, and produce Superoxide anion. They were, respectively, 5-, 300-, 500-, and 4000-fold weaker than PAF in each assay; inhibited PMN-binding of [³H]PAF at concentrations paralleling their biological potencies; and showed sensitivity to the inhibitory effects of PAF antagonists. PAF and the analogs, moreover, desensitized PMN responses to each other but not to leukotriene B₄ and actually increased (or primed) PMN responses to N-formyl-MET-LEU-PHE. Finally, 5-hydroxyicosatetraenoate-enhanced PMN responses to PAF and the analogs without enhancing the actions of other stimuli. It stereospecifically raised each analog's potency by as much as 100-fold and converted a fifth natural analog, 1-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine from inactive to a weak stimulator of PMN. PAF and its analogs thus represent a structurally diverse family of cell-derived phospholipids which can activate, prime, and desensitize neutrophils by using a common, apparently PAF receptor-dependent mechanism.     

Activation of guinea pig peritoneal macrophages by platelet activating factor (PAF) and its agonists

The platelet activating factor (PAF: 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine) and its analogs were examined to determine their effects on guinea pig peritoneal macrophages. PAF activated macrophages, but its effect on macrophages was much weaker than that observed on platelets: the concentration required for 50% maximum activation was 8.5 X 10(-6) M for macrophages and 2.9 X 10(-10) M for platelets. Three PAF agonists, 1-O-octadecyl-2-O-(N,N-dimethylcarbamoyl)-glycero-3-phosphocholine (Compound I), 1-O-octadecyl-2-acetamido-2-deoxy-glycero-3-phosphocholine (Compound II), and 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine (Compound III), showed higher activity in stimulating macrophage function than PAF. The abilities of these non-metabolizable PAF agonists to activate macrophage paralleled their relative potency to induce platelet activation. The sn-3 enantiomers of PAF and Compound III exhibited activity, while the sn-1 did not. By comparing the activities of derivatives of Compound III, it was shown that the long-chain alkyl-ether group in the glycerol-1 position, a relatively small size of the substituent on the hydroxy group at the sn-2 position, and the choline moiety in the glycerol-3 position must play critical roles in the process of macrophage activation. A specific PAF antagonist, CV3988, which inhibits PAF-induced platelet activation and hypotension, inhibited the activation of macrophages caused by PAF and its agonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of platelet-activating factor by arachidonic acid-depleted rat polymorphonuclear leukocytes

1-O-[3H]Alkyl-2-acetyl-sn-glycero-3-phosphocholine ([3H]PAF) and 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphocholine ([3H]lyso-PAF) when incubated with rat polymorphonuclear leukocytes (PMN) were rapidly metabolized to 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine ([3H]alkyl-acyl-GPC) containing long chain acyl groups in the sn-2 position. The specificity and the absolute requirements of arachidonate (20:4) for acylation into PAF and lyso-PAF were investigated by comparing the rate of [3H]PAF and [3H]lyso-PAF metabolism by control rat PMN with that by rat PMN depleted of 20:4. Comparable rates of metabolism of [3H]PAF and [3H]lyso-PAF by both control and 20:4-depleted PMN were observed at all the concentrations of PAF and lyso-PAF studied. The nature of the fatty acyl group incorporated into the sn-2 position of the [3H]alkyl-acyl-GPC formed was analyzed by argentation chromatography. Dienoic fatty acids were the major fatty acid incorporated into the alkyl-acyl-GPC by both control and 20:4-depleted PMN at all the incubation times studied. At 3 min of incubation with [3H]PAF and [3H]lyso-PAF, control PMN had small but significant amounts of [3H]alkyl-acyl-GPC containing tetraenoic fatty acids, the concentration of which gradually increased as the incubation time progressed. On the other hand, under similar conditions, 20:4-depleted PMN had only trace amounts of the [3H]alkyl-acyl-GPC with tetraenoic fatty acid and the concentration of which remained at the low level throughout the incubation time. At 3 min of incubation, the 20:4-depleted PMN had small but significant amounts of [3H]alkyl-acyl-GPC with saturated fatty acids, the amount of which declined by 10 min and remained at that level as the incubation time progressed. While the concentration of [3H]alkyl-acyl-GPC with dienoic fatty acids in the 20:4-depleted cells gradually increased with the progress of incubation time, these molecular species of GPC in the control PMN remained more or less constant. In spite of a very high concentration (equivalent to that of 20:4 in control PMN) of eicosatrienoic acid (20:3 delta 5,8,11) in the 20:4-depleted PMN, no significant amounts of [3H]alkyl-acyl-GPC with trienoic fatty acid were formed by these cells. The rate of metabolism of [3H]PAF and [3H]lyso-PAF by the resident macrophages isolated from control and 20:4-depleted rats was similar.(ABSTRACT TRUNCATED AT 400 WORDS)     

Crystal structure of human plasma platelet-activating factor acetylhydrolase: structural implication to lipoprotein binding and catalysis

Human plasma platelet-activating factor (PAF) acetylhydrolase functions by reducing PAF levels as a general anti-inflammatory scavenger and is linked to anaphylactic shock, asthma, and allergic reactions. The enzyme has also been implicated in hydrolytic activities of other pro-inflammatory agents, such as sn-2 oxidatively fragmented phospholipids. This plasma enzyme is tightly bound to low and high density lipoprotein particles and is also referred to as lipoprotein-associated phospholipase A2. The crystal structure of this enzyme has been solved from x-ray diffraction data collected to a resolution of 1.5 angstroms. It has a classic lipase alpha/beta-hydrolase fold, and it contains a catalytic triad of Ser273, His351, and Asp296. Two clusters of hydrophobic residues define the probable interface-binding region, and a prediction is given of how the enzyme is bound to lipoproteins. Additionally, an acidic patch of 10 carboxylate residues and a neighboring basic patch of three residues are suggested to play a role in high density lipoprotein/low density lipoprotein partitioning. A crystal structure is also presented of PAF acetylhydrolase reacted with the organophosphate compound paraoxon via its active site Ser273. The resulting diethyl phosphoryl complex was used to model the tetrahedral intermediate of the substrate PAF to the active site. The model of interface binding begins to explain the known specificity of lipoprotein-bound substrates and how the active site can be both close to the hydrophobic-hydrophilic interface and at the same time be accessible to the aqueous phase.     

Endothelial cell-associated platelet-activating factor: a novel mechanism for signaling intercellular adhesion

The binding of neutrophils (polymorphonuclear leukocytes [PMNs]) to endothelial cells (ECs) presents special requirements in the regulation of intercellular adhesion. ECs that are stimulated by certain agonists, including thrombin and cytokines (tumor necrosis factor alpha, interleukin-1), generate molecular signals that induce the adhesion of PMNs (endothelial cell-dependent neutrophil adhesion). Our experiments demonstrate that the mechanism of binding induced by thrombin is distinct from that induced by the cytokines based on the time courses, the requirement for protein synthesis, and differential binding of HL60 promyelocytic leukemia cells to ECs activated by the two classes of agonists. The rapid EC-dependent PMN adhesion (initiated in minutes) that occurs when the ECs are stimulated by thrombin is temporally coupled with the accumulation of platelet-activating factor, a biologically active phosphoglyceride that remains associated with ECs and that activates PMNs by binding to a cell surface receptor. A portion of the newly synthesized platelet-activating factor (PAF) is on the EC surface, as demonstrated by experiments in which the rate of hydrolysis of PAF synthesized by activated ECs was accelerated by extracellular PAF acetylhydrolase. When ECs were treated with exogenous PAF they became adhesive for PMNs; the PMN binding was prevented by incubating the ECs with PAF acetylhydrolase or by treating the PMNs with competitive PAF receptor antagonists. Thus PAF associated with the EC plasma membrane induces PMN binding, an observation supported by experiments in which PAF in model membranes (liposomes) stimulated rapid PMN adhesion to ECs and to cell-free surfaces. In addition, competitive antagonists of the PAF receptor inhibited the binding of PMNs to ECs activated by thrombin and other rapidly acting agonists, but not to ECs activated by tumor necrosis factor alpha, indicating that PAF that is endogenously synthesized by ECs can mediate neutrophil adhesion. These experiments demonstrate a novel mechanism by which a cell-associated phospholipid, PAF, can serve as a signal for an intercellular adhesive event.     

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.     

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.     

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.     

Ultraviolet B radiation generates platelet-activating factor-like phospholipids underlying cutaneous damage

Ultraviolet B light (UVB) causes cutaneous inflammation and cell death, but the agents responsible are not defined. These studies examined the role of the platelet-activating factor (PAF) signaling system in UVB-mediated effects. Expression of the PAF receptor in the PAF receptor-negative epidermoid cell line KB augmented apoptosis in response to UVB irradiation. Overexpression of the PAF receptor in primary human keratinocytes also enhanced UVB-mediated apoptosis in vitro, and it enhanced apoptosis in an in vivo model of human keratinocytes grafted onto severe combined immune-deficient (SCID) mice. To define the mechanism by which UVB activates the PAF receptor, we used mass spectrometry to demonstrate significant amounts of the C4 PAF analogs 1-alkyl-2-(butanoyl and butenoyl)-sn-glycero-3-phosphocholine, as well as native PAF in an epidermal cell line after UVB irradiation. Supplementing the cells with the precursor phospholipid 1-hexadecyl-2-arachidonoyl-sn-glycero-3-phosphocholine (HAPC) increased the amount of C4 PAF analogs recovered after UVB exposure. We irradiated HAPC directly and found, even in the absence of a photosensitizer, fragmentation to C4-PAF receptor ligands. We conclude UVB photo-oxidizes cellular phospholipids, creating PAF analogs that stimulate the PAF receptor to induce further PAF synthesis and apoptosis. PAF signaling may participate in the cutaneous inflammation that occurs during photo-aggravated dermatoses.     

Characterization of the transacylase activity of rat liver 60-kDa lysophospholipase-transacylase. Acyl transfer from the sn-2 to the sn-1 position.

Rat liver 60-kDa lysophospholipase-transacylase catalyzes not only the hydrolysis of 1-acyl-sn-glycero-3-phosphocholine, but also the transfer of its acyl chain to a second molecule of 1-acyl-sn-glycero-3-phosphocholine to form phosphatidylcholine (H. Sugimoto, S. Yamashita, J. Biol. Chem. 269 (1994) 6252-6258). Here we report the detailed characterization of the transacylase activity of the enzyme. The enzyme mediated three types of acyl transfer between donor and acceptor lipids, transferring acyl residues from: (1) the sn-1 to -1(3); (2) sn-1 to -2; and (3) sn-2 to -1 positions. In the sn-1 to -1(3) transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1(3) positions of glycerol and 2-acyl-sn-glycerol, producing 1(3)-acyl-sn-glycerol and 1,2-diacyl-sn-glycerol, respectively. In the sn-1 to -2 transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to not only the sn-2 positions of 1-acyl-sn-glycero-3-phosphocholine, but also 1-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. 1-Acyl-sn-glycero-3-phospho-myo-inositol and 1-acyl-sn-glycero-3-phosphoserine were much less effectively transacylated by the enzyme. In the sn-2 to -1 transfer, the sn-2 acyl residue of 2-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1 position of 2-acyl-sn-glycero-3-phosphocholine and 2-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. Consistently, the enzyme hydrolyzed the sn-2 acyl residue from 2-acyl-sn-glycero-3-phosphocholine. By the sn-2 to -1 transfer activity, arachidonic acid was transferred from the sn-2 position of donor lipids to the sn-1 position of acceptor lipids, thus producing 1-arachidonoyl phosphatidylcholine. When 2-arachidonoyl-sn-glycero-3-phosphocholine was used as the sole substrate, diarachidonoyl phosphatidylcholine was synthesized at a rate of 0.23 micromol/min/mg protein. Thus, 60-kDa lysophospholipase-transacylase may play a role in the synthesis of 1-arachidonoyl phosphatidylcholine needed for important cell functions, such as anandamide synthesis.     

Intracellular PAF catabolism by PAF acetylhydrolase counteracts continual PAF synthesis

Stimulated inflammatory cells synthesize platelet-activating factor (PAF), but lysates of these cells show little enhancement in PAF synthase activity. We show that human neutrophils contain intracellular plasma PAF acetylhydrolase (PLA2G7), an enzyme normally secreted by monocytes. The esterase inhibitors methyl arachidonoylfluorophosphonate (MAFP), its linoleoyl homolog, and Pefabloc inhibit plasma PAF acetylhydrolase. All of these inhibitors induced PAF accumulation by quiescent neutrophils and monocytes that was equivalent to agonist stimulation. Agonist stimulation after esterase inhibition did not further increase PAF accumulation. PAF acetylhydrolase activity in intact neutrophils was reduced, but not abolished, by agonist stimulation. Erythrocytes, which do not participate in the acute inflammatory response, inexplicably express the type I PAF acetylhydrolase, whose only known substrate is PAF. Inhibition of this enzyme by MAFP caused PAF accumulation by erythrocytes, which was hemolytic in the absence of PAF acetylhydrolase activity. We propose that PAF is continuously synthesized by a nonselective acyltransferase activity(ies) found even in noninflammatory cells as a component of membrane remodeling, which is then selectively and continually degraded by intracellular PAF acetylhydrolase activity to modulate PAF production.     

A yeast PAF acetylhydrolase ortholog suppresses oxidative death

Phospholipids containing sn-2 polyunsaturated fatty acyl residues are primary targets of oxidizing radicals, producing proapoptotic and membrane perturbing fragmented phospholipids. The only known phospholipases that specifically select these oxidized and/or short-chained phospholipids as substrates are mammalian group VII phospholipases A2s that were purified and cloned as PAF acetylhydrolases. Platelet-activating factor (PAF) is a short-chained phospholipid, and whether these enzymes actually are PAF hydrolases or evolved as oxidized phospholipid phospholipases is unknown. The fission yeast Schizosaccharomyces pombe, which does not form or use PAF as a signaling molecule, contains an open-reading frame potentially homologous to mammalian group VII phospholipase A2s. We cloned this SPBC106.11c locus and expressed it in distantly related Saccharomyces cerevisiae that lack homologous sequences. The S. pombe locus encoded a functional phospholipase A2, now renamed plg7+, that hydrolyzed PAF and a synthetic oxidized phospholipid. Expression of human type II PAF acetylhydrolase or S. pombe Plg7p enhanced the viability of S. cerevisiae subjected to oxidative stress. We conclude that a single-celled organism with an exceedingly spare genome still expresses an unusually discriminating phospholipase A2, and that selective hydrolysis of phospholipid oxidation products is an early, and critical, way to overcome oxidative membrane damage and oxidant-induced cell death.