Human group V phospholipase A2 induces group IVA phospholipase A2-independent cysteinyl leukotriene synthesis in human eosinophils

We previously reported that exogenously added human group V phospholipase A2 (hVPLA2) could elicit leukotriene B4 biosynthesis in human neutrophils through the activation of group IVA phospholipase A2 (cPLA2) (Kim, Y. J., Kim, K. P., Han, S. K., Munoz, N. M., Zhu, X., Sano, H., Leff, A. R., and Cho, W. (2002) J. Biol. Chem. 277, 36479-36488). In this study, we determined the functional significance and mechanism of the exogenous hVPLA2-induced arachidonic acid (AA) release and leukotriene C4 (LTC4) synthesis in isolated human peripheral blood eosinophils. As low a concentration as 10 nm exogenous hVPLA2 was able to elicit the significant release of AA and LTC4 from unstimulated eosinophils, which depended on its ability to act on phosphatidylcholine membranes. hVPLA2 also augmented the release of AA and LTC4 from eosinophils activated with formyl-Met-Leu-Phe + cytochalasin B. A cellular fluorescent PLA2 assay showed that hVPLA2 had a lipolytic action first on the outer plasma membrane and then on the perinuclear region. hVPLA2 also caused the translocation of 5-lipoxygenase from the cytosol to the nuclear membrane and a 2-fold increase in 5-lipoxygenase activity. However, hVPLA2 induced neither the increase in intracellular calcium concentration nor cPLA2 phosphorylation; consequently, cPLA2 activity was not affected by hVPLA2. Pharmacological inhibition of cPLA2 and the hVPLA2-induced activation of eosinophils derived from the cPLA2-deficient mouse corroborated that hVPLA2 mediates the release of AA and leukotriene in a cPLA2-independent manner. As such, this study represents a unique example in which a secretory phospholipase induces the eicosanoid formation in inflammatory cells, completely independent of cPLA2 activation.     

Mechanism of human group V phospholipase A2 (PLA2)-induced leukotriene biosynthesis in human neutrophils. A potential role of heparan sulfate binding in PLA2 internalization and degradation

Human group V phospholipase A(2) (hVPLA(2)) has been shown to have high activity to elicit leukotriene production in human neutrophils (Han, S. K., Kim, K. P., Koduri, R., Bittova, L., Munoz, N. M., Leff, A. R., Wilton, D. C., Gelb, M. H., and Cho, W. (1999) J. Biol. Chem. 274, 11881-11888). To determine the mechanism by which hVPLA(2) interacts with cell membranes to induce leukotriene formation, we mutated surface cationic residues and a catalytic residue of hVPLA(2) and measured the interactions of mutants with model membranes, immobilized heparin, and human neutrophils. These studies showed that cationic residues, Lys(7), Lys(11), and Arg(34), constitute a part of the interfacial binding surface of hVPLA(2), which accounts for its moderate preference for anionic membranes. Additionally, hVPLA(2) binds heparin with high affinity and has a well defined heparin-binding site. The site is composed of Arg(100), Lys(101), Lys(107), Arg(108), and Arg(111), and is spatially distinct from its interfacial binding surface. Importantly, the activities of the mutants to hydrolyze cell membrane phospholipids and induce leukotriene biosynthesis, when enzymes were added exogenously to neutrophils, correlated with their activities on phosphatidylcholine membranes but not with their affinities for anionic membranes and heparin. These results indicate that hVPLA(2) acts directly on the outer plasma membranes of neutrophils to release fatty acids and lysophospholipids. Further studies suggest that products of hVPLA(2) hydrolysis trigger the cellular leukotriene production by activating cellular enzymes involved in leukotriene formation. Finally, the temporal and spatial resolution of exogenously added hVPLA(2) and mutants suggests that binding to cell surface heparan sulfate proteoglycans is important for the internalization and clearance of cell surface-bound hVPLA(2).     

Mechanism of group IVA cytosolic phospholipase A(2) activation by phosphorylation

Group IVA cytosolic phospholipase A2 (cPLA2) has been shown to play a critical role in the agonist-induced release of arachidonic acid. To understand the mechanism by which phosphorylation of Ser505 and Ser727 activates cPLA2, we systematically analyzed the effects of S505A, S505E, S727A, S727E, S505A/S727A, S505A/S727E, and S505E/S727E mutations on its enzyme activity and membrane affinity. In vitro membrane binding measurements showed that S505A has lower affinity than the wild type or S505E for phosphatidylcholine membranes, which is exclusively due to faster desorption of the membrane-bound S505A. In contrast, neither S727A nor S727E mutation had a significant effect on the phosphatidylcholine vesicle binding affinity of cPLA2. The difference in in vitro membrane affinity between wild type (or S505E) and S505A increased with the decrease in Ca2+ concentration, reaching >60-fold at 2.5 microm Ca2+. When HEK293 cells transfected with cPLA2 and mutants were stimulated with ionomycin, the wild type and S505E translocated to the perinuclear region and caused the arachidonic acid release at 0.4 microm Ca2+, whereas S505A showed no membrane translocation and little activity to release arachidonic acid. Further mutational analysis of hydrophobic residues in the active site rim (Ile399, Leu400, and Leu552) indicate that a main role of the Ser505 phosphorylation is to promote membrane penetration of these residues, presumably by inducing a conformational change of the protein. These enhanced hydrophobic interactions allow the sustained membrane interaction of cPLA2 in response to transient calcium increases. On the basis of these results, we propose a mechanism for cPLA2 activation by calcium and phosphorylation.     

C2 domain membrane penetration by group IVA cytosolic phospholipase A2 induces membrane curvature changes

Group IVA cytosolic phospholipase A₂ (cPLA₂α) is an 85 kDa enzyme that regulates the release of arachidonic acid (AA) from the sn-2 position of membrane phospholipids. It is well established that cPLA₂α binds zwitterionic lipids such as phosphatidylcholine in a Ca²⁺-dependent manner through its N-terminal C2 domain, which regulates its translocation to cellular membranes. In addition to its role in AA synthesis, it has been shown that cPLA₂α promotes tubulation and vesiculation of the Golgi and regulates trafficking of endosomes. Additionally, the isolated C2 domain of cPLA₂α is able to reconstitute Fc receptor-mediated phagocytosis, suggesting that C2 domain membrane binding is sufficient for phagosome formation. These reported activities of cPLA₂α and its C2 domain require changes in membrane structure, but the ability of the C2 domain to promote changes in membrane shape has not been reported. Here we demonstrate that the C2 domain of cPLA₂α is able to induce membrane curvature changes to lipid vesicles, giant unilamellar vesicles, and membrane sheets. Biophysical assays combined with mutagenesis of C2 domain residues involved in membrane penetration demonstrate that membrane insertion by the C2 domain is required for membrane deformation, suggesting that C2 domain-induced membrane structural changes may be an important step in signaling pathways mediated by cPLA₂α.     

Transcellular secretion of group V phospholipase A2 from epithelium induces beta 2-integrin-mediated adhesion and synthesis of leukotriene C4 in eosinophils

We examined the mechanism by which secretory group V phospholipase A(2) (gVPLA(2)) secreted from stimulated epithelial cells activates eosinophil adhesion to ICAM-1 surrogate protein and secretion of leukotriene (LT)C(4). Exogenous human group V PLA(2) (hVPLA(2)) caused an increase in surface CD11b expression and focal clustering of this integrin, which corresponded to increased beta(2) integrin-mediated adhesion. Human IIaPLA(2), a close homolog of hVPLA(2), or W31A, an inactive mutant of hVPLA(2), did not affect these responses. Exogenous lysophosphatidylcholine but not arachidonic acid mimicked the beta(2) integrin-mediated adhesion caused by hVPLA(2) activation. Inhibition of hVPLA(2) with MCL-3G1, a mAb against gVPLA(2), or with LY311727, a global secretory phospholipase A(2) (PLA(2)) inhibitor, attenuated the activity of hVPLA(2); trifluoromethylketone, an inhibitor of cytosolic group IVA PLA(2) (gIVA-PLA(2)), had no inhibitory effect on hVPLA(2)-mediated adhesion. Activation of beta(2) integrin-dependent adhesion by hVPLA(2) did not cause ERK1/2 activation and was independent of gIVA-PLA(2) phosphorylation. In other studies, eosinophils cocultured with epithelial cells were stimulated with FMLP/cytochalasin B (FMLP/B) and/or endothelin-1 (ET-1) before LTC(4) assay. FMLP/B alone caused release of LTC(4) from eosinophils, which was augmented by coculture with epithelial cells activated with ET-1. Addition of MCL-3G1 to cocultured cells caused approximately 50% inhibition of LTC(4) secretion elicited by ET-1, which was blocked further by trifluoromethylketone. Our data indicate that hVPLA(2) causes focal clustering of CD11b and beta(2) integrin adhesion by a novel mechanism that is independent of arachidonic acid synthesis and gIVA-PLA(2) activation. We also demonstrate that gVPLA(2), endogenously secreted from activated epithelial cells, promotes secretion of LTC(4) in cocultured eosinophils.     

Group IVA phospholipase A2 is necessary for the biogenesis of lipid droplets

Lipid droplets (LD) are organelles present in all cell types, consisting of a hydrophobic core of triacylglycerols and cholesteryl esters, surrounded by a monolayer of phospholipids and cholesterol. This work shows that LD biogenesis induced by serum, by long-chain fatty acids, or the combination of both in CHO-K1 cells was prevented by phospholipase A(2) inhibitors with a pharmacological profile consistent with the implication of group IVA cytosolic phospholipase A(2) (cPLA(2)alpha). Knocking down cPLA(2)alpha expression with short interfering RNA was similar to pharmacological inhibition in terms of enzyme activity and LD biogenesis. A Chinese hamster ovary cell clone stably expressing an enhanced green fluorescent protein-cPLA(2)alpha fusion protein (EGFP-cPLA(2)) displayed higher LD occurrence under basal conditions and upon LD induction. Induction of LD took place with concurrent phosphorylation of cPLA(2)alpha at Ser(505). Transfection of a S505A mutant cPLA(2)alpha showed that phosphorylation at Ser(505) is key for enzyme activity and LD formation. cPLA(2)alpha contribution to LD biogenesis was not because of the generation of arachidonic acid, nor was it related to neutral lipid synthesis. cPLA(2)alpha inhibition in cells induced to form LD resulted in the appearance of tubulo-vesicular profiles of the smooth endoplasmic reticulum, compatible with a role of cPLA(2)alpha in the formation of nascent LD from the endoplasmic reticulum.     

2-Oxoamide inhibitors of cytosolic group IVA phospholipase A2 with reduced lipophilicity

Cytosolic GIVA phospholipase A₂ (GIVA cPLA₂) initiates the eicosanoid pathway of inflammation and thus inhibitors of this enzyme constitute novel potential agents for the treatment of inflammatory diseases. Traditionally, GIVA cPLA₂ inhibitors have suffered systemically from high lipophilicity. We have developed a variety of long chain 2-oxoamides as inhibitors of GIVA PLA₂. Among them, AX048 was found to produce a potent analgesic effect. We have now reduced the lipophilicity of AX048 by replacing the long aliphatic chain with a chain containing an ether linked aromatic ring with in vitro inhibitory activities similar to AX048.     

Group IVA phospholipase A2 regulates testosterone biosynthesis by murine Leydig cells and is required for timely sexual maturation

In the present paper, we report that PLA2G4A (Group IVA phospholipase A2) is important in the development and function of rodent testes. Interstitial cells of rat testes had high PLA2 (phospholipase A2) activity that was very sensitive to the PLA2G4A-preferential inhibitor AACOCF3 (arachidonyl trifluoromethyl ketone). PLA2G4A protein was expressed primarily in the interstitial cells of wild-type mouse testes throughout maturation. Although Pla2g4a knockout (Pla2g4a-/-) male mice are fertile, their sexual maturation was delayed, as indicated by cauda epididymal sperm count and seminal vesicle development. Delayed function of Pla2g4a-/- mice testes was associated with histological abnormalities including disorganized architecture, swollen appearance and fewer interstitial cells. Basal secretion of testosterone was attenuated significantly and steroidogenic response to hCG (human chorionic gonadotropin) treatment was reduced in Pla2g4a-/- mice compared with their Pla2g4a+/+ littermates during the sexual maturation period. Chemical inhibition of PLA2G4A activity by AACOCF3 or pyrrophenone significantly reduced hCG-stimulated testosterone production in cultured rat interstitial cells. AACOCF3 inhibited forskolin- and cAMP analogue-stimulated testosterone production. These results provide the first evidence that PLA2G4A plays a role in male testes physiology and development. These results may have implications for the potential clinical use of PLA2G4A inhibitors.     

Mechanism of regulation of group IVA phospholipase A2 activity by Ser727 phosphorylation

Although group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) has been reported to be phosphorylated at multiple Ser residues, the mechanisms by which phosphorylation at different sites regulates cPLA(2)alpha activities are not fully understood. To explore the possibility that phosphorylation of Ser(727) modulates cellular protein-protein interactions, we measured the effect of Ser(727) mutations on the interaction of cPLA(2)alpha with a reported cPLA(2)alpha-binding protein, p11. In vitro activity assays and membrane binding measurements by surface plasmon resonance analysis showed that a heterotetramer (A2t) of p11 and annexin A2, but not p11 or annexin A2 alone, directly binds cPLA(2)alpha via Ser(727), which keeps the enzyme from binding the membrane and catalyzing the phospholipid hydrolysis. Phosphorylation of Ser(727) disrupts this inhibitory cPLA(2)alpha-A2t interaction, thereby activating cPLA(2)alpha. Subcellular translocation and activity measurements in HEK293 cells cotransfected with cPLA(2)alpha and p11 also showed that p11, in the form of A2t, inhibits cPLA(2)alpha by the same mechanism and that phosphorylation of Ser(727) activates cPLA(2)alpha by interfering with the inhibitory cPLA(2)alpha-A2t interaction. Collectively, these studies provide new insight into the regulatory mechanism of cPLA(2)alpha through Ser(727) phosphorylation.     

Cytosolic phospholipase A2 Group IValpha but not secreted phospholipase A2 Group IIA, V, or X induces interleukin-8 and cyclooxygenase-2 gene and protein expression through peroxisome proliferator-activated receptors gamma 1 and 2 in human lung cells

It has been reported that interleukin-8 (IL-8) and cyclooxygenase-2 (COX-2) expression is regulated by peroxisome proliferator-activated receptor (PPAR)-gamma synthetic ligands. We have shown previously that cytosolic phospholipase A2 (cPLA2) is able to activate gene expression through PPAR-gamma response elements (Pawliczak, R., Han, C., Huang, X. L., Demetris, A. J., Shelhamer, J. H., and Wu, T. (2002) J. Biol. Chem. 277, 33153-33163). In this study we investigated the influence of cPLA2 and secreted phospholipase A2 (sPLA2) Group IIA, Group V, and Group X on IL-8 and COX-2 expression in human lung epithelial cells (A549 cells). We also studied the results of cPLA2 activation by epidermal growth factor (EGF) and calcium ionophore (A23187) on IL-8 and COX-2 reporter gene activity, mRNA level, and protein synthesis. cPLA2 overexpression and activation increased both IL-8 and COX-2 reporter gene activity. Overexpression and activation of Group IIA, Group V, or Group X sPLA2s did not increase IL-8 and COX-2 reporter gene activity. Methyl arachidonyl fluorophosphate, a cPLA2 inhibitor, inhibited the effect of A23187 and of EGF on both IL-8 and COX-2 reporter gene activity, steady state levels of IL-8 and COX-2 mRNA, and IL-8 and COX-2 protein expression. Small inhibitory RNAs directed against PPAR-gamma1 and -gamma2 blunted the effect of A23187 and of EGF on IL-8 and COX-2 protein expression. Moreover small inhibitory RNAs directed against cPLA2 decreased the effect of A23187 and EGF on IL-8 and COX-2 protein expression. These results demonstrate that cPLA2 has an influence on IL-8 and COX 2 gene and protein expression at least in part through PPAR-gamma.     

Activation of cytosolic phospholipase A2 in permeabilized human neutrophils

Neutrophils (PMN) contain two types of phospholipase A₂ (PLA₂), a 14 kDa ‘secretory’ Type II PLA₂ (sPLA₂) and an 85 kDa ‘cytosolic’ PLA₂ (cPLA₂), that differ in a number of key characteristics: (1) cPLA₂ prefers arachidonate (AA) as a substrate but hydrolyzes all phospholipids; sPLA₂ is not AA specific but prefers ethanolamine containing phosphoacylglycerols. (2) cPLA₂ is active at nM calcium (Ca²⁺) concentrations; sPLA₂ requires μM Ca²⁺ levels. (3) cPLA₂ activity is regulated by phosphorylation; sPLA₂ lacks phosphorylation sites. (4) cPLA₂ is insensitive to reduction; sPLA₂ is inactivated by agents that reduce disulfide bonds. We utilized PMN permeabilized with Staphylococcus aureus α-toxin to determine whether one or both forms of PLA₂ were activated in porated cells under conditions designed to differentiate between the two enzymes. PMN were labeled with [³H]AA to measure release from phosphatidylcholine and phosphatidylinositol; gas chromatography-mass spectrometry was utilized to determine total AA release (mainly from phosphatidylethanolamine) and to asses oleate and linoleate mass. A combination of 500 nM Ca²⁺, a guanine nucleotide, and stimulation with n-formyl-met-leu-phe (FMLP) were necessary to induce maximal AA release in permeabilized PMN measured by either method; AA was preferentially released. [³H]AA and AA mass release occurred in parallel over time. A hydrolyzable form of ATP was necessary for maximum AA release and staurosporin inhibited PLA₂ activation. Dithiothreitol treatment had little affect on [³H]AA release and metabolism but inhibited AA mass release. Assay of cell supernatants after cofactor addition did not detect sPLA₂ activity and the cytosolic buffer utilized did not support activity of recombinant sPLA₂. These results strongly suggested that cPLA₂ was the enzyme activated in the permeabilized cell model and this is the first report which unambiguously demonstrates AA release in response to activation of a specific type of PLA₂ in PMN.     

Overexpression of cytosolic group IVA phospholipase A2 protects cells from Ca2+-dependent death

The calcium ionophore ionomycin induces apoptosis-like events in the human embryonic kidney cell line at early times. Plasma membrane blebbing, mitochondrial depolarization, externalization of phosphatidylserine, and nuclear permeability changes can all be observed within 15 min of treatment. However, there is no activation of caspases or chromatin condensation. Expression of a fusion protein containing the enhanced green fluorescent protein (EGFP) and human cytosolic Group IVA phospholipase A(2)alpha (EGFP-cPLA(2)alpha) in these cells prevents ionomycin-induced phosphatidylserine externalization and death. Cells expressing the cPLA(2)alpha mutant D43N, which does not bind calcium, retain their susceptibility to ionomycin-induced cell death. Both nonexpressing and EGFP-D43N-cPLA(2)alpha-expressing human embryonic kidney cells can be spared from ionomycin-induced cell death by pretreating them with exogenous arachidonic acid. Moreover, during calcium overload, mitochondrial depolarization is significantly lower in the EGFP-cPLA(2)alpha-expressing cells than in cells expressing normal amounts of cPLA(2)alpha. These results suggest that early cell death events promoted by an overload of calcium can be prevented by the presence of high levels of arachidonic acid.     

Anionic lipids activate group IVA cytosolic phospholipase A2 via distinct and separate mechanisms

Previously, ceramide-1-phosphate (C1P) and phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] were demonstrated to be potent and specific activators of group IVA cytosolic phospholipase A2 (cPLA2alpha). In this study, we hypothesized that these anionic lipids functionally activated the enzyme by distinctly different mechanisms. Indeed, surface plasmon resonance and surface dilution kinetics demonstrated that C1P was a more potent effector than PI(4,5)P2 in decreasing the dissociation constant of the cPLA2alpha-phosphatidylcholine (PC) interaction and increasing the residence time of the enzyme on the vesicles/micelles. PI(4,5)P2, in contrast to C1P, decreased the Michaelis-Menten constant, increasing the catalytic efficiency of the enzyme. Furthermore, PI(4,5)P2 activated cPLA2alpha with a stoichiometry of 1:1 versus C1P at 2.4:1. Lastly, PI(4,5)P2, but not C1P, increased the penetration ability of cPLA2alpha into PC-rich membranes. Therefore, this study demonstrates two distinct mechanisms for the activation of cPLA2alpha by anionic lipids. First, C1P activates cPLA2alpha by increasing the residence time of the enzyme on membranes. Second, PI(4,5)P2 activates the enzyme by increasing catalytic efficiency through increased membrane penetration.     

Group IVA phospholipase A2 is associated with the storage of lipids in adipose tissue and liver

Prostaglandin (PG) E(2) is considered to participate in the storage of fat in adipocytes and hepatocytes, but roles of group IVA phospholipase A(2) (PLA(2)), a key PLA(2) isozyme in the arachidonic acid cascade, remain unclear. The present study examined the possible involvement of the enzyme using group IVA PLA(2)-deficient mice (C57BL/6 background, 22 weeks of age) fed a normal diet (5.3% fat). The ratio of epididymal fat pad weight to body weight was significantly reduced in group IVA PLA(2)-deficient mice compared to wild-type mice. Histological analysis revealed that in group IVA PLA(2)-deficient mice, the adipocytes were smaller, and hepatocytes bearing cytoplasmic vacuolation were scarce. Hepatic triglyceride content and the serum levels of PGE(2) in the deficient mice were also lower. However, there was no difference in the serum levels of insulin, glucose, non-esterified free fatty acid, or total cholesterol between the deficient and wild-type mice. Our findings suggest that group IVA PLA(2) is involved in the storage of lipids in the adipose tissue and liver and in determining circulating PGE(2) levels.     

Retinol induces platelet aggregation via activation of phospholipase A2

All-trans-retinol induced aggregation of rabbit platelets, and this effect could be inhibited by a cyclooxygenase inhibitor and a thromboxane A2 (TXA2) receptor antagonist, indicating an essential role for endogenously produced TXA2. We found a two-phase arachidonic acid release in retinol-stimulated platelets. The first phase was induced by the action of retinol alone and not inhibited by TXA2 receptor antagonist. The second phase was induced via synergistic action of retinol and initially generated small amount of TXA2, and was inhibited by the antagonist. Moreover, we discussed that the arachidonic acid release may be mediated by the action of phospholipase A2.     

Differential inside-out activation of beta2-integrins by leukotriene B4 and fMLP in human neutrophils

We have investigated how LTB4, an endogenous chemoattractant encountered early in the inflammatory process, and fMLP, a bacteria-derived chemotactic peptide emanating from the site of infection, mediate inside-out regulation of the beta2-integrin. The role of the two chemoattractants on beta2-integrin avidity was investigated by measuring their effect on beta2-integrin clustering and surface mobility, whereas their effect on beta2-integrin affinity was measured by the expression of a high affinity epitope, a ligand-binding domain on beta2-integrins, and by integrin binding to s-ICAM. We find that the two chemoattractants modulate the beta2-integrin differently. LTB4 induces an increase in integrin clustering and surface mobility, but only a modest increase in integrin affinity. fMLP evokes a large increase in beta2-integrin affinity as well as in clustering and mobility. Lipoxin, which acts as a stop signal for the functions mediated by pro-inflammatory agents, was used as a tool for further examining the inside-out mechanisms. While LTB4-induced integrin clustering and mobility were inhibited by lipoxin, only a minor inhibition of fMLP-induced beta2-integrin avidity and no inhibition of integrin affinity were detected. The different modes of the inside-out regulation of beta2-integrins suggest that distinct mechanisms are involved in the beta2-integrin modulation induced by various chemoattractants.     

Effect of tryptophan insertions on the properties of the human group IIA phospholipase A2: mutagenesis produces an enzyme with characteristics similar to those of the human group V phospholipase A2

An important characteristic of the human group IIA secreted phospholipase A(2) (IIA PLA(2)) is the extremely low activity of this enzyme with phosphatidylcholine (PC) vesicles, mammalian cell membranes, and serum lipoproteins. This characteristic is reflected in the lack of ability of this enzyme to bind productively to zwitterionic interfaces. Part of the molecular basis for this lack of activity is an absence of tryptophan, a residue with a known preference for residing in the interfacial region of zwitterionic phospholipid bilayers. In this paper we have replaced the eight residues that make up the hydrophobic collar on the interfacial binding surface of the enzyme with tryptophan. The catalytic and interfacial binding properties of these mutants have been investigated, particularly those properties associated with binding to and hydrolysis of zwitterionic interfaces. Only the insertion of a tryptophan at position 3 or 31 produces mutants that significantly enhance the activity of the human IIA enzyme against zwitterionic interfaces and intact cell membranes. Importantly, the ability of the enzyme mutants to hydrolyze PC-rich interfaces such as the outer plasma membrane of mammalian cells was paralleled by enhanced interfacial binding to zwitterionic interfaces. The corresponding double tryptophan mutant (V3,31W) displays a specific activity on PC vesicles comparable to that of the human group V sPLA2. This enhanced activity includes the ability to interact with human embryonic kidney HEK293 cells, previously reported for the group V enzyme [Kim, Y. J., Kim, K. P., Rhee, H. J., Das, S., Rafter, J. D., Oh, Y. S., and Cho, W. (2002) J. Biol. Chem. 277, 9358-9365].     

Involvement of cytosolic phospholipase A2 and secretory phospholipase A2 in arachidonic acid release from human neutrophils

The purpose of this study was to define the role of secretory phospholipase A2 (sPLA2), calcium-independent PLA2, and cytosolic PLA2 (cPLA2) in arachidonic acid (AA) release from fMLP-stimulated human neutrophils. While fMLP induced the release of extracellular sPLA2 activity and AA, 70% of sPLA2 activity remained associated with the cell. Treatment with the cell-impermeable sPLA2 inhibitors DTT or LY311-727, or the anti-sPLA2 Ab 3F10 all inactivated extracellular sPLA2 activity, but had minimal effect on neutrophil AA mass release. In contrast, coincubation of streptolysin-O toxin-permeabilized neutrophils with DTT, LY311-727, or 3F10 all decreased [3H8]AA release from [3H8]AA-labeled, fMLP-stimulated cells. Exposure to fMLP resulted in a decrease in the electrophoretic mobility of cPLA2, a finding consistent with cPLA2 phosphorylation, and stimulated the translocation of cPLA2 from cytosolic to microsomal and nuclear compartments. The role of cPLA2 was further evaluated with the cPLA2 inhibitor methyl arachidonyl fluorophosphonate, which attenuated cPLA2 activity in vitro and decreased fMLP-stimulated AA mass release by intact neutrophils, but had no effect on neutrophil sPLA2 activity. Inhibition of calcium-independent PLA2 with haloenol lactone suicide substrate had no effect on neutrophil cPLA2 activity or AA mass release. These results indicate a role for cPLA2 and an intracellular or cell-associated sPLA2 in the release of AA from fMLP-stimulated human neutrophils.