CoA-independent Transacylase Activity Research Articles

Altered Arachidonate Distribution in Macrophages from Caveolin-1 Null Mice Leading to Reduced Eicosanoid Synthesis

In this work we have studied the effect of caveolin-1 deficiency on the mechanisms that regulate free arachidonic acid (AA) availability. The results presented here demonstrate that macrophages from caveolin-1-deficient mice exhibit elevated fatty acid incorporation and remodeling and a constitutively increased CoA-independent transacylase activity. Mass spectrometry-based lipidomic analyses reveal stable alterations in the profile of AA distribution among phospholipids, manifested by reduced levels of AA in choline glycerophospholipids but elevated levels in ethanolamine glycerophospholipids and phosphatidylinositol. Furthermore, macrophages from caveolin-1 null mice show decreased AA mobilization and prostaglandin E(2) and LTB(4) production upon cell stimulation. Collectively, these results provide insight into the role of caveolin-1 in AA homeostasis and suggest an important role for this protein in the eicosanoid biosynthetic response.

Influence of cellular arachidonic acid levels on phospholipid remodeling and CoA-independent transacylase activity in human monocytes and U937 cells

The availability of free arachidonic acid (AA) constitutes a limiting step in the synthesis of biologically active eicosanoids. Free AA levels in cells are regulated by a deacylation/reacylation cycle of membrane phospholipids, the so-called Lands cycle, as well as by further remodeling reactions catalyzed by CoA-independent transacylase. In this work, we have comparatively investigated the process of AA incorporation into and remodeling between the various phospholipid classes of human monocytes and monocyte-like U937 cells. AA incorporation into phospholipids was similar in both cell types, but a marked difference in the rate of remodeling was appreciated. U937 cells remodeled AA at a much faster rate than human monocytes. This difference was found not to be related to the differentiation state of the U937 cells, but rather to the low levels of esterified arachidonate found in U937 cells compared to human monocytes. Incubating the U937 cells in AA-rich media increased the cellular content of this fatty acid and led to a substantial decrease of the rate of phospholipid AA remodeling, which was due to reduced CoA-independent transacylase activity. Collectively, these findings provide the first evidence that cellular AA levels determine the amount of CoA-independent transacylase activity expressed by cells and provide support to the notion that CoA-IT is a major regulator of AA metabolism in human monocytes.

Regulation of Arachidonate Remodeling Enzymes Impacts Eosinophil Survival during Allergic Asthma

Although the role of arachidonic acid (AA) metabolism to eicosanoids has been well established in allergy and asthma, recent studies in neoplastic cells have revealed that AA remodeling through phospholipids impacts cell survival. This study tests the hypothesis that regulation of AA/phospholipid-remodeling enzymes, cytosolic phospholipase A(2) alpha(cPLA(2)-alpha, gIValphaPLA(2)) and CoA-independent transacylase (CoA-IT), provides a mechanism for altered eosinophil survival during allergic asthma. In vitro incubation of human eosinophils (from donors without asthma) with IL-5 markedly increased cell survival, induced gIValphaPLA(2) phosphorylation, and increased both gIValphaPLA(2) and CoA-IT activity. Furthermore, treatment of eosinophils with nonselective (ET18-O-CH(3)) and selective (SK&F 98625) inhibitors of CoA-IT triggered apoptosis, measured by changes in morphology, membrane phosphatidylserine exposure, and caspase activation, completely reversing IL-5-induced eosinophil survival. To determine if similar activation occurs in vivo, human blood eosinophils were isolated from either normal individuals at baseline or from subjects with mild asthma, at both baseline and 24 hours after inhaled allergen challenge. Allergen challenge of subjects with allergic asthma induced a marked increase in cPLA(2) phosphorylation, augmented gIValphaPLA(2) activity, and increased CoA-IT activity. These findings indicate that both in vitro and in vivo challenge of eosinophils activated gIValphaPLA(2) and CoA-IT, which may play a key role in enhanced eosinophil survival.

Differential effects of arachidonoyl trifluoromethyl ketone on arachidonic acid release and lipid mediator biosynthesis by human neutrophils. Evidence for different arachidonate pools.

The goal of this study was to determine the effects of a putative specific cytosolic phospholipase A2 inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3), on arachidonic acid (AA) release and lipid mediator biosynthesis by ionophore-stimulated human neutrophils. Initial studies indicated that AACOCF3 at concentrations 0-10 micro m did not affect AA release from neutrophils. In contrast, AACOCF3 potently inhibited leukotriene B4 formation by ionophore-stimulated neutrophils (IC50 approximately 2.5 micro m). Likewise, AACOCF3 significantly inhibited the biosynthesis of platelet activating factor. In cell-free assay systems, 10 micro m AACOCF3 inhibited 5-lipoxygenase and CoA-independent transacylase activities. [3H]AA labeling studies indicated that the specific activities of cell-associated AA mimicked that of leukotriene B4 and PtdCho/PtdIns, while the specific activities of AA released into the supernatant fluid closely mimicked that of PtdEtn. Taken together, these data argue for the existence of segregated pools of arachidonate in human neutrophils. One pool of AA is linked to lipid mediator biosynthesis while another pool provides free AA that is released from cells. Additionally, the data suggest that AACOCF3 is also an inhibitor of CoA-independent transacylase and 5-lipoxygenase. Thus, caution should be exercised in using AACOCF3 as an inhibitor of cytosolic phospholipase A2 in whole cell assays because of the complexity of AA metabolism.

A Decrease in Remodeling Accounts for the Accumulation of Arachidonic Acid in Murine Mast Cells Undergoing Apoptosis

The goal of this study was to examine arachidonic acid (AA) metabolism by murine bone marrow-derived mast cells (BMMC) during apoptosis induced by cytokine depletion. BMMC deprived of cytokines for 12-48 h displayed apoptotic characteristics. During apoptosis, levels of AA, but not other unsaturated fatty acids, correlated with the percentage of apoptotic cells. A decrease in both cytosolic phospholipase A(2) expression and activity indicated that cytosolic phospholipase A(2) did not account for AA mobilization during apoptosis. Free AA accumulation is also unlikely to be due to decreases in 5-lipoxygenase and/or cyclooxygenase activities, since BMMC undergoing apoptosis produced similar amounts of leukotriene B(4) and significantly greater amounts of PGD(2) than control cells. Arachidonoyl-CoA synthetase and CoA-dependent transferase activities responsible for incorporating AA into phospholipids were not altered during apoptosis. However, there was an increase in arachidonate in phosphatidylcholine (PC) and neutral lipids concomitant with a 40.7 +/- 8.1% decrease in arachidonate content in phosphatidylethanolamine (PE), suggesting a diminished capacity of mast cells to remodel arachidonate from PC to PE pools. Further evidence of a decrease in AA remodeling was shown by a significant decrease in microsomal CoA-independent transacylase activity. Levels of lyso-PC and lyso-PE were not altered in cells undergoing apoptosis, suggesting that the accumulation of lysophospholipids did not account for the decrease in CoA-independent transacylase activity or the induction of apoptosis. Together, these data suggest that the mole quantities of free AA closely correlated with apoptosis and that the accumulation of AA in BMMC during apoptosis was mediated by a decreased capacity of these cells to remodel AA from PC to PE.

The regulation of CoA-independent transacylation reactions in neuronal nuclei by lysophospholipid, free fatty acid, and lysophospholipase: the control of nuclear lyso platelet-activating factor metabolism.

CoA-independent transacylase activities generating alkylacylglycerophosphocholine (AAGPC) from alkylglycerophosphocholine (1-alkyl GPC) were considerably enriched in neuronal nuclei isolated from rabbit cerebral cortex. Specific nuclear transacylation activities were 13 times the corresponding microsomal values. Several lysophospholipids, notably 1-acyl glycerophosphocholine (1-acyl GPC), 1-alkenyl GPC and 1-alkenyl GPE (1-alkenyl glycerophosphoethanolamine) inhibited the transacylation of 1-alkyl GPC. The inhibitory effects of 1-acyl GPC were seen in the presence of MAFP (methyl arachidonoylfluorophosphonate) or free oleate, compounds that inhibit neuronal nuclear lysophospholipase. When neuronal nuclei were preincubated with 1-alkyl GPC, the radioactive AAGPC product served as donor in transacylation reactions, to generate 1-alkyl GPC. In these nuclear reactions, 1-palmitoyl GPE and 1-palmitoyl GPC appeared to be poor acceptor substrates, when compared with corresponding 1-alkyl and 1-alkenyl analogues. The presence of free oleate or MAFP in the reactions containing 1-acyl GPC boosted the release of 1-alkyl GPC from AAGPC. These observations are of particular relevance to brain ischemia in which lysophospholipid, free fatty acid, and platelet-activating factor (PAF) levels rise dramatically. PAF can be made by the nuclear acetylation of 1-alkyl GPC, which is formed by nuclear transacylation mechanisms. Yet transacylase also removes 1-alkyl GPC, and thus this enzyme activity can regulate 1-alkyl GPC availability. Our observations indicate that lysophospholipids promote the formation of 1-alkyl GPC from nuclear AAGPC via transacylation, while free fatty acid likely prolongs the lifetime of 1-acyl lysophospholipids substrates by lysophospholipase inhibition. Similarly, once 1-alkyl GPC is formed, other lysophospholipids effectively compete with this 1-alkyl analogue and reduce its conversion back to AAGPC by transacylation. Free oleate, in this case, sustains 1-acyl lysophospholipid inhibitors of 1-alkyl GPC transacylation. Thus the cycle of transacylation may favour 1-alkyl GPC formation during ischemia, increasing levels of 1-alkyl GPC for nuclear acetylation reactions and PAF formation. The nuclear generation of PAF is of considerable importance as PAF can play regulatory roles in transcription events associated with inflammation.

Regulation of leukotriene and platelet-activating factor synthesis in human alveolar macrophages

It has been suggested that phospholipase A 2 (PLA 2) contributes to the regulation of leukotriene (LT) and platelet-activating factor (PAF) synthesis by controlling the release of their precursors, arachidonic acid (AA) and lysophosphatidylcholine (IysoPC), from membrane phospholipids. In rat alveolar macrophages (AMs), PLA 2 appears to have a major role in LT synthesis but a more limited role in PAF synthesis. The present study was designed to define the role of PLA 2 in LT and PAF synthesis in human AMs and determine whether differences exist between AMs obtained from normal subjects and those from patients with asthma. In the normal subjects, the calcium ionophore A23187 (Cal) increased AM PAF synthesis (percent incorporation of tritiated acetate) by 135% ( p < 0.01) and LTB 4 synthesis 88-fold ( p < 0.001). Phorbol myristate acetate (PMA) had little effect alone, but it had a synergistic effect with Cal, increasing PAF synthesis by 466% and LTB 4 synthesis to 229-fold above the control values ( p < 0.001 for both). Ro 25-4331, a combined cytosolic (c) and secretary (s) PLA 2 inhibitor, had little effect on the Cal-stimulated PAF synthesis, but it completely blocked the effect of PMA. It also blocked the Cal− and Cal+PMAstimulated LTB 4 synthesis. AACOCF 3, a cPLA 2 inhibitor, had no effect on either Cal or Cal+PMA-stimulated PAF synthesis. It reduced LTB 4 synthesis, but it did so less effectively than Ro 25-4331. CoA-independent transacylase (CoAl-TA) activity in the AMs increased after stimulation and exposure to Ro 25-4331. SK&F 45905, a CoAl-TA inhibitor, reduced stimulated PAF synthesis by 30% to 40%. Patients with asthma had similar results except that cPLA 2 had a greater role in stimulated LTB 4 synthesis. These data indicate that PLA 2 plays a direct role in human AM LT synthesis; both the cytosolic and secretary forms contribute to LT synthesis; PLA 2 appears to have a more limited role in PAF synthesis, although it mediates the synergistic effect of PMA, probably via sPLA 2 and CoAl-TA contributes to PAF synthesis during PLA 2 inhibition. With the exception of the greater role for cPLA 2 in stimulated LTB 4 synthesis in the patients with asthma, the contributions of PLA 2 and CoAl-TA to AM LT and IPAF synthesis appear to be similar in normal subjects and patients with asthma.

Evidence that 85 kDa phospholipase A 2 is not linked to CoA-independent transacylase-mediated production of platelet-activating factor in human monocytes

Platelet-activating factor (PAF) production is carefully controlled in inflammatory cells. The specific removal of arachidonate (AA) from l-O-alkyl-2-arachidonoyl- sn-glycero-3-phosphocholine (GPC), thought to be mediated by CoA-independent transacylase (CoA-IT), is required to generate the PAF precursor 1-O-alkyl-2-lyso-GPC in human neutrophils. Exposure of A23187-stimulated human monocytes to the CoA-IT inhibitors SK&F 98625 and SK&F 45905 inhibited PAF formation (IC 50S of 10 and 12 µM, respectively), indicating that these cells also need CoA-IT activity for PAF production. Because CoA-IT activity transfers arachidonate to a 2-lyso phospholipid substrate, its activity is obligated to an sn-2 acyl hydrolase to form the 2-lyso phospholipid substrate. SB 203347, an inhibitor of 14 kDa phospholipase A 2 (PLA 2), and AACOCF 3, an inhibitor of 85 kDa PLA 2, both inhibited AA release from A23187-stimulated human monocytes. However, AACOCF 3 had no effect on A23187-induced PAF formation at concentrations as high as 3 µM. Further, depletion of 85 kDa PLA 2 using antisense (SB 7111, 1 µM) had no effect on PAF production, indicating a lack of a role of 85 kDa PLA 2 in PAF biosynthesis. Both SB 203347 and the 14 kDa PLA 2 inhibitor scalaradial blocked PAF synthesis in monocytes (IC 50s of 2 and 0.5 µM, respectively), suggesting a key role of 14 kDa PLA 2 in this process. Further, A23187-stimulated monocytes produced two forms of PAF: 80% 1-O-alkyl-2-acetyl-GPC and 20% 1-acyl-2-acetyl-GPC, which were both equally inhibited by SB 203347. In contrast, inhibition of CoA-IT using SK&F 45905 (20 µM) had a greater effect on the production of 1-O-alkyl (−80%) than of 1-acyl (−14%) acetylated material. Finally, treatment of U937 cell membranes with exogenous human recombinant (rh) type II 14 kDa PLA 2, but not rh 85 kDa PLA 2, induced PAF production. Elimination of membrane CoA-IT activity by heat treatment impaired the ability of 14 kDa PLA 2 to induce PAF formation. Taken together, these results suggest that a 14 kDa PLA 2-like activity, and not 85 kDa PLA 2, is coupled to monocyte CoA-IT-induced PAF production.

Chronic hyperinsulinernia inhibits platelet-activating factor (PAF) biosynthesis in the rat kidney

A number of risk factors for cardiovascular disease, including hypertension, are associated with the insulin resistance syndrome. The hallmark of this syndrome is an impairment in insulin action which provokes a compensatory increase in pancreatic β-cell insulin secretion leading to chronic hyperinsulinemia. Indirect studies show that platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine, PAF), a potent antihypertensive lipid produced by the kidney, may be decreased by hyperinsulinemia. The present study was designed to evaluate the effect of chronic hyperinsulinemia on renal PAF metabolism, arterial blood pressure and whole body insulin sensitivity. Chronic catheterized, unstressed rats were infused with saline or insulin plus glucose to create a chronic condition of sustained euglycemic (∼ 130 mg/dl) hyperinsulinemia (∼ 90 mU/1, or 3-fold over basal levels). PAF is a metabolically unstable compound being susceptible to rapid degradation to the biologically inactive lyso-PAF, a metabolite which also serves as a precursor for PAF synthesis. PAF synthesis and counter-regulatory prostaglandins may be derived from the same arachidonate precursor. The enzymes which catalyze these reactions were measured in plasma and in the subcellular fractions of the kidneys. Compared to saline-treated rats, sustained physiologic hyperinsulinemia for 7 days: (i) decreased insulin-mediated glucose disposal by 30%; (ii) caused an increased plasma PAF:acetylhydrolase, which degrades PAF to lyso-PAF, without any change in cytosolic PA F: acetyl hydrolase activity; and (iii) completely inhibited microsomal lyso-PAF:acetyl CoA acetyltransferase activity which catalyzes the conversion of lyso-PAF back to bioactive PAF. The increased catabolism of PAF in plasma, combined with decreased renal PAF biosynthesis, would be expected to decrease circulating PAF levels, leading to a rise in blood pressure. However, blood pressure remained unchanged. The sustained hyperinsulinemia stimulated plasma membrane CoA-independent transacylase activity, which is responsible for the mobilization of arachidonates into lyso-PAF, to form 1-alkylarachidonoyl-glycerophosphocholine. The latter is the stored precursor for the synthesis of PAF and vasodilatory prostaglandins, which may have offset the effect of decreased PAF. We hypothesize that hyperinsulinemia may alter the blood pressure only if the balance between the synthesis /catabolism of PAF and vasodilatory Prostaglandins is disrupted.

Irreversible inhibition of Ca 2+-independent phospholipase A 2 by methyl arachidonyl fluorophosphonate

Methyl arachidonyl fluorophosphonate (MAFP) has been recently reported to be a selective, active-site directed, irreversible inhibitor of the Group IV 85 kDa cytosolic phospholipase A 2 (cPLA 2). We have now shown that this compound also potently inhibits the Ca 2+-independent cytosolic phospholipase A 2 (iPLA 2). MAFP inhibited iPLA 2 in a concentration-dependent manner with half-maximal inhibition observed at 0.5 μM after a 5 min preincubation at 40°C. This inhibition was not reversed upon extensive dilution of the enzyme into the assay mixture. Preincubation of iPLA 2 with MAFP resulted in a linear, time-dependent inactivation of enzyme activity, and the enzyme was protected from inactivation by the reversible inhibitor PACOCF 3. The ability of MAFP to inhibit the iPLA 2 suggests that this enzyme proceeds through an acyl-enzyme intermediate as has been proposed for the cPLA 2. Further testing indicated that MAFP did not inhibit the arachidonyl-CoA synthetase, CoA-dependent acyltransferase, or CoA-independent transacylase activities from P388D 1 cells. Thus, MAFP is not a general inhibitor for enzymes which act on arachidonoyl substrates. Instead, the inhibitor appears to show some selectivity for PLA 2, although it does not discriminate between cPLA 2 and iPLA 2. Particular caution must be exercised to distinguish these activities if this inhibitor is used in intact cells.

Transacylase-mediated alkylacyl-GPC synthesis and its hydrolysis by phospholipase D occur in separate cell compartments in the human neutrophil.

Subcellular localizations of CoA-independent transacylase and phospholipase D enzymes have been investigated in human neutrophils performing a two-step gradient system to separate plasma membranes from internal membranes and from the bulk of granules. The internal membranes were constituted by endoplasmic reticulum and by a subpopulation of specific and tertiary granules. The enzymes activities were assayed in vitro on gradient fractions using exogenous substrates. Following cell prelabelling with [3H]alkyllyso-GPC, we also analyzed the in situ localization of labelled products involving the action of both enzymes. The CoA-independent transacylase activity, together with the CoA-dependent transacylase and acyltransferase activities were only located in the internal membranes. Following 15 min cell labelling, part of the [3H]alkylacyl-GPC was recovered in plasma membranes indicating a rapid redistribution of the acylated compound. Very high contents in arachidonate containing [3H]alkylacyl-GPC were recovered both in plasma membranes and internal membranes. Phospholipase D activity being assayed in the presence of cytosol, GTP gamma S and gradient fractions, only the plasma membrane fractions from resting or stimulated cells allowed the enzyme to be active. The [3H]alkylacyl-GP and [3H]alkylacyl-GPethanol, phospholipase D breakdown products from [3H]alkylacyl-GPC, obtained after neutrophil prelabelling and activation by phorbol myristate acetate, were exclusively present in the plasma membranes. In contrast, the secondary generated [3H]alkylacylglycerols were equally distributed between plasma and internal membranes. No labelled product was recovered on azurophil granules. These data demonstrate that internal membranes are the site of action of the CoA-independent transacylase and plasma membranes are the site of action of the phospholipase D. This topographical separation between CoA-independent transacylase which generated substrate and phospholipase D which degraded it, suggested that subcellular localisation and traffic of substrates within the cell can be important to regulate the enzymes.

Comparison of acceptor and donor substrates in the CoA-independent transacylate reaction in human neutrophils

In human neutrophils (PMN) the ethanolamine-containing phosphoglyceride fraction (PE), principally plasmalogen-linked PE (1- O-alk-1′-enyl-2-acyl- sn-glycero-3-phosphoethanolamine), is the major store of arachidonic acid (AA). Exogenous AA is initially incorporated into 1-acyl-linked phosphoglycerides and is believed to be transferred into the 1-ether-linked phosphoglycerides via the action of a CoA-independent transacylase (CoA-IT). We have investigated the selectivity for both the ‘acceptor’ lysophospholipids and ‘donor’ AA-containing phospholipid substrates in the CoA-IT reaction. Evidence suggests CoA-IT may also participate in the synthesis of platelet activating factor. The transfer of [ 3H]AA from endogenously labeled choline-containing phosphoglycerides (PC) to exogenously added alkenyl-lyso-PE (0–50 μM) was examined in saponin-permeabilized PMN. In these ‘donor’ studies, we observed that [ 3H]AA was transferred from both alkyl- and diacyl-linked PC in a proportional manner. More detailed molecular species analysis showed that [ 3H]AA was deacylated from all the major AA-containing molecular species in both the alkyl and diacyl subclasses with no selectivity for either subclass. To investigate the ‘acceptor’ selectivity, membrane fractions prelabeled with either [ 3H]alkyl-arachidonoyl-PE or -PC were utilized as donor substrates. Various unlabeled lysophospholipids (10 μM) were added and the generation of [ 3H]lyso-PE or -PC was monitored as a measure of CoA-IT activity. Significant subclass preference was observed upon addition of lyso-PE species (1-alkenyl > 1-alkyl > 1-acyl) however, little selectivity was seen with the corresponding lyso-PC species. On the other hand, lysophosphatidylserine, lysophosphatidylinositol, and lysophosphatidic acid all served as poor acceptor substrates in the reaction. These data from PMN are consistent with other evidence that the CoA-IT plays a pivotal role in the enrichment of AA into plasmalogen-linked PE.

Stimulation of platelet-activating factor synthesis in polymorphonuclear leukocytes from streptozotocin-induced diabetic rats.

The platelet-activating factor (PAF)-synthesizing capacity was investigated and compared in peritoneal polymorphonuclear leukocytes (PMN) from streptozotocin-induced diabetic and normal rats. PAF synthesis was significantly enhanced in the PMN from diabetic rats compared with that from normal rats stimulated with fMLP. This was manifested as the increased incorporation of [3H]acetate into PAF. Selected ion monitoring/GC/MS analysis revealed that the molecular species of PAF synthesized were mostly of the 1-hexadecyl type, and the amount synthesized in fMLP-stimulated diabetic rat PMN was 1.5 times higher than that in normal rat PMN. The fMLP-induced arachidonic acid liberation resulting from phospholipase A2 activation, was facilitated with a concomitant increase in the cytosolic Ca2+ concentration in diabetic rat PMN. The CoA-independent transacylase activity was similar in both PMN lysates, whereas acetyl-CoA:lyso-PAF acetyltransferase activity was accelerated in the diabetic rat PMN lysate. These results revealed that diabetic rat PMN has more ability to synthesize PAF, presumably due to the large increase in activated phospholipase A2 and acetyltransferase, as well as the increased cytosolic Ca2+ concentration.

The CoA-independent transacylase in PAF biosynthesis: tissue distribution and molecular species selectivity

Microsomal membranes from six different rat tissues (spleen, lung, kidney, brain, testis, and liver) were found to possess CoA-independent transacylase activity that could both acylate lyso-[ 3 H] PAF (1-[ 3 H] hexadecyl-2- lyso-sn- glycero-3- phosphocholine) and then deacylate the 1-[ 3 H] hexadecyl-2- acyl-sn- glycero-3- phosphocholine product via the transacylation of added exogenous 1-alk-1′-enyl-2-lyso- sn-glycero-3-phosphoethanolamine. Platelet-activating factor ( 1-[ 3 H] hexadecyl-2- acetyl-sn- glycero-3- phosphocholine) was produced when acetyl-CoA was added to the spleen microsomes during generation of lyso-[ 3H]PAF by the transacylases. More extensive studies with subcellular fractions from spleen revealed that, in addition to microsomes, the transacylase activities were also present in the 15 000 × g membrane fraction but not in the cytosol. Analysis of molecular species of 1-[ 3 H] hexadecyl-2- acyl-sn- glycero-3- phosphocholine before and after addition of 1-alk-1′-enyl-2-lyso- sn-glycero-3-phosphoethanolamine as the acyl acceptor demonstrated a high selectivity for polyunsaturated fatty acids (> 3 double bonds/acyl group) in both the acylation and deacylation processes that occurred in testicular microsomal membranes. The transfer of acyl groups by the transacylase appeared to be equally effective for either arachidonic or docosapentaenoic( n − 6) fatty acids, whereas linoleic and oleic fatty acids were not transferred from 1-[ 3 H] hexadecyl-2- acyl-sn- glycero-3- phosphocholine following the addition of 1-alk-1′-enyl-2-yso- sn-glycero-3-phosphoethanolamine. Similar experiments with the membrane fraction of undifferentiated HL-60 cells showed that arachidonic acid supplementation of intact cells enhanced both the CoA-independent transacylation of lyso-[ 3H]PAF and the subsequent deacylation of 1-[ 3 H] hexadecyl-2- acyl-sn- glycero-3- phosphocholine caused by addition of 1-alk-1′-enyl-2-acyl- sn-glycero-3-phosphoethanolamine. Differentiation of the HL-60 cells into a neutrophil-like form had no effect on the transacylase activity. Our results indicate the PAF-related transacylase is widely distributed among tissues and, although highly selective for polyunsaturated acyl groups, does not discriminate selectively among the polyunsaturates.

CoA-independent transacylase activity is increased in human neutrophils after treatment with tumor necrosis factor α

CoA-independent transacylase (CoA-IT) appears to play a critical role in lipid mediator generation by rapidly moving arachidonate (AA) between phospholipid pools during cell activation. Tumor necrosis factor-α (TNF) pretreatment of human neutrophils increases agonist-induced production of inflammatory mediators. The current study tested if the TNF-induced increase in lipid mediator production may be, in part, due to altered CoA-IT activity. Neutrophils were treated with TNF (250 U/ml, 30 min), homogenates prepared, and CoA-IT activity measured by the ability of these homogenates to acylate 1-[ 3H]alkyl-2-lyso- sn-glycero-3-phosphocholine (GPC). There was an increased CoA-IT activity, from 9.1 ± 1.1 to 13.7 ± 1.4 pmol/ mg per min in control vs. TNF-treated samples, respectively. Varying the concentration of 1-alkyl-2-lyso-GPC revealed an increased CoA-IT activity in microsomes that was due to an increased V max , from 26 to 54 pmol/mg per min. The ability of TNF to increase CoA-IT activity was concentration-dependent, with maximal response observed at 25 U/ml. This effect on CoA-IT appears to be specific, in that TNF treatment of neutrophils had no effect on CoA-dependent acylation of 1-acyl-2-lyso- sn-glycero-3-phosphocholine, using either AA-CoA or linolenoyl-CoA as substrates. In the intact cell, the movement of [ 3H]AA from other phospholipids into PE in fMLP-stimulated neutrophils was greatly enhanced after TNF treatment, demonstrating a functional consequence of increased CoA-IT activity. In addition, TNF treatment doubled platelet-activating factor production in response to the chemotactic peptide fMLP, as measured by [ 3H]acetate incorporation, while the response to A23187 remained unchanged. Taken together, these results provide the first evidence of modulation of CoA-IT activity by a proinflammatory cytokine and suggest that one mechanism for augmented lipid mediator formation is through increases in CoA-IT activity.

Molecular species of ethanolamine plasmalogens and transacylase activity in rat tissues are altered by fish oil diets

Effects of dietary fish oil ethyl esters and alkyldiacetylglycerols (an ether-linked lipid) on the distribution of subclasses of choline- and ethanolamine-glycerophospholipids as well as effects on highly unsaturated molecular species of ethanolamine plasmalogens from brain, spleen, kidney, lung, and testis of rats were examined. Supplementation of ethyl ester concentrates of n − 3 fatty acids had no effect on the distribution of subclasses in any of the tissues. However, the supplements of 1-0-octadec-9'-enyl-2,3-diacetyl-OT-glycerol (diacetates of selachyl alcohol) caused significant increases in the alkylacylglycerophosphocholine and alkylacylglycerophosphoethanolamine subclasses from spleen and lung and in the alkylacylglycerophosphoethanolamine subclass from kidney. Dietary supplements of fish oil ethyl esters reduced the arachidonate-containing species of ethanolamine plasmalogens whereas molecular species having 20:5( n − 3), 22:6( n − 3), and/or 22:5( n − 3) acyl groups were increased in the spleen, lung, and kidneys, but not brain. In testicular tissue from rats fed the fish oil diets, the molecular species of ethanolamine plasmalogens containing 22:5( n − 6) acyl groups were reduced. An increase of ethanolamine plasmalogens with 18:1 alk-1-enyl moieties paired with highly unsaturated sn-2 acyl groups were found in the tissues of rats fed the fish oil plus selachyl alcohol diacetate supplements. Rats on the diet containing fish oil ethyl esters had significantly lower [ 3H]alkyllysoglycerophosphocholine CoA-independent transacylase activity in spleen microsomes than controls. This suggests that supplements of n − 3 fatty acids interferes with the transacylation of arachidonate, an event that could seriously impair the release of arachidonate and lysophospholipids (e.g., lyso-PAF) that are precursors of potent bioactive lipid derivatives.

Fatty acid and phospholipid selectivity of different phospholipase A2 enzymes studied by using a mammalian membrane as substrate

Previous studies using phospholipid mixed vesicles have demonstrated that several types of phospholipase A2 (PLA2) enzymes exhibit different selectivity for fatty acids at the sn-2 position, for the type of chemical bond at the sn-1 position or for the phosphobase moiety at the sn-3 position of phospholipids. In the present study, we have utilized natural mammalian membranes from U937 monocytes to determine whether two purified 14 kDa PLA2 isoenzymes (Type I, Type II) and a partially purified 110 kDa PLA2 exhibit substrate selectivity for certain fatty acids or phospholipids. In these studies, arachidonic acid (AA) release from membranes was measured under conditions where the remodelling of AA mediated by CoA-independent transacylase (CoA-IT) activity has been eliminated. In agreement with the mixed-vesicle models, AA was the major unsaturated fatty acid hydrolysed from membranes by the 110 kDa PLA2, suggesting that this PLA2 is selective in releasing AA from natural membranes. By contrast, Type I and Type II PLA2s were less selective in releasing AA from phospholipids and released a variety of unsaturated fatty acids at molar ratios that were proportional to the ratios of these fatty acids in U937 microsomal membranes. Examination of AA release from phospholipid classes indicated that all three enzymes released AA from the major AA-containing phospholipid classes (phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol) of U937 membranes. The 110 kDa PLA2 released AA from phospholipid subclasses in ratios that were proportional to the AA content within phospholipid classes and subclasses of U937 membranes. These data suggested that the 110 kDa PLA2 shows no preference either for the sn-1 linkage or for the sn-3 phosphobase moiety of phospholipids. By contrast, Type I and Type II PLA2s preferentially released AA from ethanolamine-containing phospholipids and appeared to prefer the 1-acyl-linked subclass. Taken together, these data indicate that the 110 kDa PLA2 selectively releases AA from U937 membranes, whereas Type I and Type II PLA2 release a variety of unsaturated fatty acids. Furthermore, the 110 kDa PLA2 releases the same molar ratios of AA from all major phospholipid subclasses, whereas Type I and Type II PLA2s show some specificity for phosphatidylethanolamine when these enzymes are incubated with a complex mammalian membrane substrate.

Vitamin E enhances the acylation of 1-O-alkyl-sn-glycero-3-phosphocholine in human endothelial cells.

1-O-Alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC) is the precursor of platelet-activating factor. It is formed via the CoA-independent transacylase reaction, which transfers the polyenoyl acyl group from the sn-2 position of a diacyl phospholipid to the sn-2 position of 1-O-alkyl-sn-glycero-3-phosphocholine (alkyl-GPC). We have reported previously that vitamin E alters phospholipid turnover in the endothelial cells by increasing arachidonic acid release and prostacyclin synthesis. In the present study, the role of vitamin E in the formation of alkylacyl-GPC was investigated. Incubation of endothelial cells with vitamin E resulted in an increase in the formation of [3H]alkylacyl-GPC from [3H]alkyl-GPC. The effect of vitamin E was dose-dependent at concentrations below 23 microM. However, vitamin E did not have a direct effect on the transacylase activity. When endothelial cells were incubated with vitamin E, the CoA-independent transacylase activity in the cell homogenate was found to be enhanced. Kinetic analysis of the transacylase activity in the pre-incubated cells showed that the enhancement of enzyme activity was at the enzyme-substrate level. When endothelial cells were incubated with vitamin E analogues (Trolox, tocol and tocopherol acetate), only limited enhancement of the transacylation process was detected. It is clear that vitamin E enhanced the synthesis of alkylacyl-GPC from alkyl-GPC in a very specific manner by an indirect stimulation of the CoA-independent transacylase activity. The regulation by vitamin E of the formation of alkylacyl-GPC may mediate the transfer of arachidonate from the diacyl phospholipid pool into the ether-linked phospholipid pool.

Metal ion and salt effects on the phospholipase A 2, lysophospholipase, and transacylase activities of human cytosolic phospholipase A 2

Human cytosolic phospholipase A 2 (cPLA 2) is an arachidonic acid specific enzyme which may play a role in arachidonic acid release, eicosanoid production, and signal transduction. The PLA 2 activity of this enzyme is stimulated by μM levels of Ca 2+. Using a pure recombinant enzyme, we have confirmed that cPLA 2 is not absolutely dependent on Ca 2+, since Sr 2+, Ba 2+ and Mn 2+ also gave full enzyme activity. Heavy metals, in contrast, inhibited enzyme catalysis suggesting the involvement of an essential cysteine residue. In the absence of Ca 2+, high salt concentrations overcame the requirement for divalent metals, indicating that Ca 2+ is not required for PLA 2 catalytic activity. cPLA 2 also displays a lysophospholipase (lyso PLA) activity with lysophosphatidylcholine micelles as a substrate. Unlike the PLA 2 activity, the lyso PLA activity toward these micelles is not stimulated by Ca 2+. However, upon the addition of glycerol or Triton X-100 to the assay, Ca 2+ activation is observed, indicating that substrate presentation can affect the apparent Ca 2+ dependence. Glycerol was found to be a potent stimulator of lyso PLA activity and specific activities up to 50 μmol min −1 mg −1 were observed. In addition to the PLA 2, and lyso PLA activities, we report that cPLA 2 displays a relatively low, CoA-independent transacylase activity which produces phosphatidylcholine from lysophosphatidylcholine substrate. The observation of this novel transacylase activity is consistent with the formation of an acyl-enzyme intermediate.

Evidence for different mechanisms involved in the formation of lyso platelet-activating factor and the calcium-dependent release of arachidonic acid from human neutrophils

Recent studies suggest that the first step in platelet-activating factor (PAF) biosynthesis, 1-alkyl-2-lyso-GPC (lyso PAF) formation, may be initiated by the selective transfer of arachidonate from 1-alkyl-2-arachidonoyl-GPC to an acceptor lyso phospholipid by a CoA-independent transacylase activity (CoA-IT). The present study was designed to determine whether the formation of 1-alkyl-2-lyso-GPC and the release of arachidonic acid can occur by different mechanisms. These experiments examined both the formation of 1-[ 3H]alkyl-2-lyso-GPC from 1-[ 3H]alkyl-2-acyI-GPC and the release of arachidonic acid from membrane phospholipids as determined by GC/MS in neutrophil homogenates under various conditions. The addition of unlabelled lyso phospholipids to neutrophil homogenates stimulated the time-dependent formation of 1-[ 3H]alkyl-2-lyso-GPC from 1-[ 3H]alkyl-2-acyl-GPC. Without exogenous lyso phospholipids, little 1-[ 3H]alkyl-2-lyso-GPC was formed in this reaction. The activity which catalyzed the formation of 1-[ 3H]alkyl-2-lyso-GPC had characteristics identical to CoA-IT as indicated by the fact that both reactions were: independent of Ca 2+, Mg 2+, CoA and CoA fatty acids, located in microsomal fractions, and stable in 10 mM dithiothreitol. In sharp contrast to the aforementioned reaction, addition of lyso phospholipids did not affect the quantity of arachidonic acid released from membrane phospholipids. Furthermore, there was a Ca 2+-independent release of arachidonic acid from membrane phospholipid that was increased 4 to 5-fold after the addition of 5 mM Ca 2+. Finally, Ca 2+-dependent arachidonic acid release was inhibited by putative phospholipase A 2 inhibitors, aristolochic acid and scalaradial, at concentrations where neither the production of 1-[ 3H]alkyl-2-lyso-GPC nor Ca 2+-independent arachidonic acid release was altered. Together these data imply that there may be different mechanisms involved in the formation of 1-alkyl-2-lyso-GPC and arachidonic acid from membrane phospholipids.