Naegleria fowleri amoebae express a membrane-associated calcium-independent phospholipase A(2)

Naegleria fowleri, a free-living amoeba, is the causative agent of primary amoebic meningoencephalitis. Previous reports have demonstrated that N. fowleri expresses one or more forms of phospholipase A(2) (PLA(2)) and that a secreted form of this enzyme is involved in pathogenesis. However, the molecular nature of these phospholipases remains largely unknown. This study was initiated to determine whether N. fowleri expresses analogs of the well-characterized PLA(2)s that are expressed by mammalian macrophages. Amoeba cell homogenates contain a PLA(2) activity that hydrolyzes the substrate that is preferred by the 85 kDa calcium-dependent cytosolic PLA(2), cPLA(2). However, unlike the cPLA(2) enzyme in macrophages, this activity is largely calcium-independent, is constitutively associated with membranes and shows only a modest preference for phospholipids that contain arachidonate. The amoeba PLA(2) activity is sensitive to inhibitors that block the activities of cPLA(2)-alpha and the 80 kDa calcium-independent PLA(2), iPLA(2), that are expressed by mammalian cells. One of these compounds, methylarachidonyl fluorophosphonate, partially inhibits the constitutive release of [(3)H]arachidonic acid from pre-labeled amoebae. Together, these data suggest that N. fowleri expresses a constitutively active calcium-independent PLA(2) that may play a role in the basal phospholipid metabolism of these cells.     

Mapping the phospholipid-binding surface and translocation determinants of the C2 domain from cytosolic phospholipase A2.

Cytosolic phospholipase A2 (cPLA2) plays a key role in the generation of arachidonic acid, a precursor of potent inflammatory mediators. Intact cPLA2 is known to translocate in a calcium-dependent manner from the cytosol to the nuclear envelope and endoplasmic reticulum. We show here that the C2 domain of cPLA2 alone is sufficient for this calcium-dependent translocation in living cells. We have identified sets of exposed hydrophobic residues in loops known as calcium-binding region (CBR) 1 and CBR3, which surround the C2 domain calcium-binding sites, whose mutation dramatically decreased phospholipid binding in vitro without significantly affecting calcium binding. Mutation of a residue that binds calcium ions (D43N) also eliminated phospholipid binding. The same mutations that prevent phospholipid binding of the isolated C2 domain in vitro abolished the calcium-dependent translocation of cPLA2 to internal membranes in vivo, suggesting that the membrane targeting is driven largely by direct interactions with the phospholipid bilayer. Using fluorescence quenching by spin-labeled phospholipids for a series of mutants containing a single tryptophan residue at various positions in the cPLA2 C2 domain, we show that two of the calcium-binding loops, CBR1 and CBR3, penetrate in a calcium-dependent manner into the hydrophobic core of the phospholipid bilayer, establishing an anchor for docking the domain onto the membrane.     

Stimulation-dependent recruitment of cytosolic phospholipase A2-alpha to EA.hy.926 endothelial cell membranes leads to calcium-independent association.

Cytosolic phospholipase A2-alpha (cPLA2-alpha) is a calcium-activated enzyme involved in agonist-induced arachidonic acid release. In endothelial cells, free arachidonic acid is predominantly converted into prostacyclin, a potent vasodilator and inhibitor of platelet activation. As the rate-limiting step in prostacyclin production is the generation of free arachidonic acid by cPLA2-alpha, this enzyme has become an attractive pharmacological target and the focus of many studies. Following stimulation with calcium-mobilizing agonists, cPLA2-alpha translocates to intracellular phospholipid membranes via its C2 domain. In this study, the calcium-induced association of cPLA2-alpha with EA.hy.926 endothelial cell membranes was investigated. Subcellular fractionation and immunofluorescence studies showed that following stimulation with histamine, thrombin or the calcium ionophore A23187, cPLA2-alpha relocated to intracellular membranes. Treatment of A23187-stimulated cells with EGTA or BAPTA-AM demonstrated that a substantial pool of cPLA2-alpha remained associated with membrane fractions in a calcium-independent manner. Furthermore, immunofluorescence microscopy studies revealed that cells stimulated for periods of greater than 10 min showed a high proportion of calcium-independent membrane-associated cPLA2-alpha. Calcium-independent membrane association of cPLA2-alpha was not due to hydrophobic or cytoskeletal interactions. Finally, the recombinant C2 domain of cPLA2-alpha exhibited calcium-independent membrane binding to membranes isolated from A23187-stimulated cells but not those isolated from nonstimulated cells. These findings suggest that novel mechanisms involving accessory proteins at the target membrane play a role in the regulation of cPLA2-alpha. Such regulatory associations could enable the cell to discriminate between the varying levels of cytosolic calcium induced by different stimuli.     

The caveolin scaffolding domain modifies 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor binding properties by inhibiting phospholipase A2 activity

Activation of the enzyme phospholipase (PLA 2) has been proposed to be part of the molecular mechanism involved in the alteration of 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) glutamate receptor responsiveness during long term changes in synaptic plasticity (long term potentiation). This study assesses the effect of the caveolin-1 scaffolding domain (CSD) on the activity of the regulatory enzyme PLA2. Caveolin-1 is a 22-kDa cholesterol-binding membrane protein known to inhibit the activity of most of its interacting partners. Our results show that the calcium-dependent cytosolic form of PLA2 (cPLA2) and caveolin-1 co-localized in mouse primary hippocampal neuron cultures and that they were co-immunoprecipitated from mouse hippocampal homogenates. A peptide corresponding to the scaffolding domain of caveolin-1 (Cav-(82-101)) dramatically inhibited cPLA2 activity in purified hippocampal synaptoneurosomes. Activation of endogenous PLA2 activity with KCl or melittin increased the binding of [3H]AMPA to its receptor. This effect was almost completely abolished by the addition of the CSD peptide to these preparations. Moreover, we demonstrated that the inhibitory action of the CSD peptide on AMPA receptor binding properties is specific (because a scrambled version of this peptide failed to have any effect) and that it is mediated by an inhibition of PLA2 enzymatic activity (because the CSD peptide failed to have an effect in membrane preparations lacking endogenous PLA2 activity). These results raised the possibility that caveolin-1, via the inhibition of cPLA2 enzymatic activity, may interfere with synaptic facilitation and long term potentiation formation in the hippocampus.     

Identification of the expressed form of human cytosolic phospholipase A2beta (cPLA2beta): cPLA2beta3 is a novel variant localized to mitochondria and early endosomes

In this study, we identify the principal splice variant of human cytosolic phospholipase A(2)beta (cPLA(2)beta) (also known as Group IVB cPLA(2)) present in cells. In human lung, spleen, and ovary and in a lung epithelial cell line (BEAS-2B), cPLA(2)beta is expressed as a 100-kDa protein, not the 114-kDa form originally predicted. Using RNA interference, the 100-kDa protein in BEAS-2B cells was confirmed to be cPLA(2)beta. BEAS-2B cells contain three different RNA splice variants of cPLA(2)beta (beta1, beta2, and beta3). cPLA(2)beta1 is identical to the previously cloned cPLA(2)beta, predicted to encode a 114-kDa protein. However, cPLA(2)beta2 and cPLA(2)beta3 splice variants are smaller and contain internal deletions in the catalytic domain. The 100-kDa cPLA(2)beta in BEAS-2B cells is the translated product of cPLA(2)beta3. cPLA(2)beta3 exhibits calcium-dependent PLA(2) activity against palmitoyl-arachidonyl-phosphatidylethanolamine and low level lysophospholipase activity but no activity against phosphatidylcholine. Unlike Group IVA cPLA(2)alpha, cPLA(2)beta3 is constitutively bound to membrane in unstimulated cells, localizing to mitochondria and early endosomes. cPLA(2)beta3 is widely expressed in tissues, suggesting that it has a generalized function at these unique sites.     

Cytosolic phospholipase A2-mediated regulation of phospholipase D2 in leukocyte cell lines.

Phospholipase D (PLD) has been implicated in a variety of cellular processes, including inflammation, secretion, and respiratory burst. Two distinct PLD isoforms, designated PLD1 and PLD2, have been cloned; however, the regulatory mechanism for each PLD isoform is not clear. In our present study we investigated how PLD2 activity is regulated in mouse lymphocytic leukemia L1210 cells, which mainly contain PLD2, and in PLD2 -transfected COS-7 cells. Intriguingly, A23187, a calcium ionophore that induces calcium influx, potently stimulates PLD activity in these two cell lines, suggesting that Ca2+ might be implicated in the regulation of the PLD2 activity. In addition to the A23187-induced PLD2 activation, A23187 also increases PLA2-mediated arachidonic acid release, and the A23187-stimulated PLD2 and PLA2 activities could be blocked by pretreatment of the cells with cytosolic calcium-dependent PLA2 (cPLA2) inhibitors, such as arachidonyl trifluoromethyl ketone and methyl arachidonyl fluorophosphonate in these two cell lines. Moreover, the A23187-induced PLD2 and PLA2 activities could be inhibited by cotransfection with antisense cPLA2 oligonucleotide. These results suggest a role for cPLA2 in the regulation of PLD2 activity in vivo. The inhibitory effect of arachidonyl trifluoromethyl ketone on the A23187-induced PLD2 activity could be recovered by addition of exogenous lysophosphatidylcholine. This study is the first to demonstrate that PLD2 activity is up-regulated by Ca2+ influx and that cPLA2 may play a key role in the Ca2+-dependent regulation of PLD2 through generation of lysophosphatidylcholine.     

Inhibition of cytosolic phospholipase A2 by annexin I. Specific interaction model and mapping of the interaction site

Annexins (ANXs) display regulatory functions in diverse cellular processes, including inflammation, immune suppression, and membrane fusion. However, the exact biological functions of ANXs still remain obscure. Inhibition of phospholipase A(2) (PLA(2)) by ANX-I, a 346-amino acid protein, has been observed in studies with various forms of PLA(2). "Substrate depletion" and "specific interaction" have been proposed for the mechanism of PLA(2) inhibition by ANX-I. Previously, we proposed a specific interaction model for inhibition of a 100-kDa porcine spleen cytosolic form of PLA(2) (cPLA(2)) by ANX-I (Kim, K. M., Kim, D. K., Park, Y. M., and Na, D. S. (1994) FEBS Lett. 343, 251-255). Herein, we present an analysis of the inhibition mechanism of cPLA(2) by ANX-I in detail using ANX-I and its deletion mutants. Deletion mutants were produced in Escherichia coli, and inhibition of cPLA(2) activity was determined. The deletion mutant ANX-I-(1-274), containing the N terminus to amino acid 274, exhibited no cPLA(2) inhibitory activity, whereas the deletion mutant ANX-I-(275-346), containing amino acid 275 to the C terminus, retained full activity. The protein-protein interaction between cPLA(2) and ANX-I was examined using the deletion mutants by immunoprecipitation and mammalian two-hybrid methods. Full-length ANX-I and ANX-I-(275-346) interacted with the calcium-dependent lipid-binding domain of cPLA(2). ANX-I-(1-274) did not interact with cPLA(2). Immunoprecipitation of A549 cell lysate with anti-ANX-I antibody resulted in coprecipitation of cPLA(2). These results are consistent with the specific interaction mechanism rather than the substrate depletion model. ANX-I may function as a negative regulator of cPLA(2) in cellular signal transduction.     

Activation of cytosolic phospholipase A2 and fatty acid transacylase is essential but not sufficient for thrombin-induced smooth muscle cell proliferation

Thrombin is a potent stimulant of smooth muscle cell (SMC) proliferation in inflammatory conditions, leading to pathological thickening of vascular walls in atherosclerosis and airway remodeling in asthma. Cell proliferation requires the formation and remodeling of cell membrane phospholipids (PLs), involving the activation of PL-metabolizing enzymes. Yet, the role of specific PL-metabolizing enzymes in SMC proliferation has hardly been studied. To bridge this gap, in the present study, we investigated the role of key enzymes involved in PL metabolism, the PL-hydrolyzing enzyme phospholipase A2 (PLA2) and the PL-synthesizing enzyme lysophosphatidic acid-fatty acid transacylase (LPAAT), in thrombin-induced proliferation of bovine aortic SMCs (BASMCs). Concomitantly with the induction of BASMC proliferation, thrombin activated cytosolic PLA2 (cPLA2-alpha), expressed by selective release of arachidonic acid and mRNA expression, as well as LPAAT, expressed by nonselective incorporation of fatty acid and mRNA expression. Specific inhibitors of these enzymes, arachidonyl-trifluoromethyl-ketone for cPLA2 and thimerosal for LPAAT, suppressed their activities, concomitantly with suppression of BASMC proliferation, suggesting a mandatory requirement for cPLA2 and LPAAT activation in thrombin-induced SMC proliferation. Thrombin acts through the protease-activated receptor (PAR-1), and, accordingly, we found that thrombin-induced BASMC proliferation was suppressed by the PAR-1 inhibitor SCH-79797. However, the PAR-1 inhibitor did not prevent thrombin-induced mRNA expression of cPLA2 and LPAAT, implying that the activation of cPLA2 and LPAAT is essential but not sufficient for thrombin-induced proliferation of BASMCs.     

Roles of C-terminal processing, and involvement in transacylation reaction of human group IVC phospholipase A2 (cPLA2gamma)

The phospholipase A2s (PLA2s) are a diverse group of enzymes that hydrolyze the sn-2 fatty acid from phospholipids and play a role in a wide range of physiological functions. A 61-kDa calcium-independent PLA2, termed cPLA2gamma, was identified as an ortholog of cPLA2alpha with approximately 30% overall sequence identity. cPLA2gamma contains a potential prenylation motif at its C terminus, and is known to have PLA2 and lysophospholipase activities, but its physiological roles have not been clarified. In the present study, we expressed various forms of recombinant cPLA2gamma, including non-prenylated and non-cleaved forms, in order to investigate the effects of C-terminal processing. We examined the expression of the wild type and non-prenylated (SCLA) forms of cPLA2gamma, and found that the SCLA form was expressed normally and retained almost full activity. Expression of the prenylated and non-cleaved form of cPLA2gamma using yeast mutants lacking prenyl protein proteases AFC1 (a-factor-converting enzyme) and RCE1 (Ras-converting enzyme) revealed decreased expression in the mutant strain compared to that in the wild type yeast, suggesting that complete C-terminal processing is important for the functional expression of cPLA2gamma. In addition, cPLA2gamma was found to have coenzyme A (CoA)-independent transacylation and lysophospholipid (LPL) dismutase (LPLase/transacylase) activities, suggesting that it may be involved in fatty acid remodeling of phospholipids and the clearance of toxic lysophospholipids in cells.     

Cytosolic phospholipase A2-alpha is necessary for platelet-activating factor biosynthesis, efficient neutrophil-mediated bacterial killing, and the innate immune response to pulmonary infection: cPLA2-alpha does not regulate neutrophil NADPH oxidase activity

The role of a cytosolic phospholipase A(2)-alpha (cPLA(2)-alpha) in neutrophil arachidonic acid release, platelet-activating factor (PAF) biosynthesis, NADPH oxidase activation, and bacterial killing in vitro, and the innate immune response to bacterial infection in vivo was examined. cPLA(2)-alpha activity was blocked with the specific cPLA(2)-alpha inhibitor, Pyrrolidine-1 (human cells), or by cPLA(2) -alpha gene disruption (mice). cPLA(2)-alpha inhibition or gene disruption led to complete suppression of neutrophil arachidonate release and PAF biosynthesis but had no effect on neutrophil NADPH oxidase activation, FcgammaII/III or CD11b surface expression, primary or secondary granule secretion, or phagocytosis of Escherichia coli in vitro. In contrast, cPLA(2)-alpha inhibition or gene disruption diminished neutrophil-mediated E. coli killing in vitro, which was partially rescued by exogenous arachidonic acid or PAF but not leukotriene B(4). Following intratracheal inoculation with live E. coli in vivo, pulmonary PAF biosynthesis, inflammatory cell infiltration, and clearance of E. coli were attenuated in cPLA(2)-alpha(-/-) mice compared with wild type littermates. These studies identify a novel role for cPLA(2)-alpha in the regulation of neutrophil-mediated bacterial killing and the innate immune response to bacterial infection.     

A novel arachidonic acid-selective cytosolic PLA2 contains a Ca(2+)-dependent translocation domain with homology to PKC and GAP

We report the cloning and expression of a cDNA encoding a high molecular weight (85.2 kd) cytosolic phospholipase A2 (cPLA2) that has no detectable sequence homology with the secreted forms of PLA2. We show that cPLA2 selectively cleaves arachidonic acid from natural membrane vesicles and demonstrate that cPLA2 translocates to membrane vesicles in response to physiologically relevant changes in free calcium. Moreover, we demonstrate that an amino-terminal 140 amino acid fragment of cPLA2 translocates to natural membrane vesicles in a Ca(2+)-dependent fashion. Interestingly, we note that this 140 amino acid domain of cPLA2 contains a 45 amino acid region with homology to PKC, p65, GAP, and PLC. We suggest that this homology delineates a Ca(2+)-dependent phospholipid-binding motif, providing a mechanism for the second messenger Ca2+ to translocate and activate cytosolic proteins.     

C2 domains from different Ca2+ signaling pathways display functional and mechanistic diversity

The ubiquitous C2 domain is a conserved Ca2+ triggered membrane-docking module that targets numerous signaling proteins to membrane surfaces where they regulate diverse processes critical for cell signaling. In this study, we quantitatively compared the equilibrium and kinetic parameters of C2 domains isolated from three functionally distinct signaling proteins: cytosolic phospholipase A2-alpha (cPLA2-alpha), protein kinase C-beta (PKC-beta), and synaptotagmin-IA (Syt-IA). The results show that equilibrium C2 domain docking to mixed phosphatidylcholine and phosphatidylserine membranes occurs at micromolar Ca2+ concentrations for the cPLA2-alpha C2 domain, but requires 3- and 10-fold higher Ca2+ concentrations for the PKC-beta and Syt-IA C2 domains ([Ca2+](1/2) = 4.7, 16, 48 microM, respectively). The Ca2+ triggered membrane docking reaction proceeds in at least two steps: rapid Ca2+ binding followed by slow membrane association. The greater Ca2+ sensitivity of the cPLA2-alpha domain results from its higher intrinsic Ca2+ affinity in the first step compared to the other domains. Assembly and disassembly of the ternary complex in response to rapid Ca2+ addition and removal, respectively, require greater than 400 ms for the cPLA2-alpha domain, compared to 13 ms for the PKC-beta domain and only 6 ms for the Syt-IA domain. Docking of the cPLA2-alpha domain to zwitterionic lipids is triggered by the binding of two Ca2+ ions and is stabilized via hydrophobic interactions, whereas docking of either the PKC-beta or the Syt-IA domain to anionic lipids is triggered by at least three Ca2+ ions and is maintained by electrostatic interactions. Thus, despite their sequence and architectural similarity, C2 domains are functionally specialized modules exhibiting equilibrium and kinetic parameters optimized for distinct Ca2+ signaling applications. This specialization is provided by the carefully tuned structural and electrostatic parameters of their Ca2+ and membrane-binding loops, which yield distinct patterns of Ca2+ coordination and contrasting mechanisms of membrane docking.     

Identification of a cellular protein that functionally interacts with the C2 domain of cytosolic phospholipase A(2)alpha

Cytosolic phospholipase A(2) (cPLA(2)) alpha plays critical roles in lipid mediator synthesis. We performed far-Western analysis and identified a 60-kDa protein (P60) that interacted with cPLA(2)alpha in a Ca(2+)-dependent manner. Peptide microsequencing revealed that purified P60 was identical to vimentin, a major component of the intermediate filament. The interaction occurred between the C2 domain of cPLA(2)alpha and the head domain of vimentin. Immunofluorescence microscopic analysis demonstrated that cPLA(2)alpha and vimentin colocalized around the perinuclear area in cPLA(2)alpha-overexpressing human embryonic kidney 293 cells following A23187 stimulation. Forcible expression of vimentin in vimentin-deficient SW13 cells augmented A23187-induced arachidonate release. Moreover, overexpression of the vimentin head domain in rat fibroblastic 3Y1 cells exerted a dominant inhibitory effect on arachidonate metabolism, significantly reducing A23187-induced arachidonate release and attendant prostanoid generation. These results suggest that vimentin is an adaptor for cPLA(2)alpha to function properly during the eicosanoid-biosynthetic process.     

Observations of rotation within the F(o)F(1)-ATP synthase: deciding between rotation of the F(o)c subunit ring and artifact

Annexin-I (ANX-I) is a 37-kDa protein with a calcium-dependent phospholipid-binding property. Previously we have observed the inhibition of cytosolic phospholipase A2 (cPLA2) by ANX-I in the studies using purified recombinant ANX-I, and proposed a specific interaction model for the mechanism of cPLA2 inhibition by ANX-I [Kim et al. (1994) FEBS Lett. 343, 251-255]. Here we have studied the role of ANX-I in the cPLA2 signaling pathway by transient transfection assay. The stimulation of Rat2 fibroblast cells with phorbol 12-myristate 13-acetate (PMA) induced the c-fos serum response element (SRE). The SRE stimulation by PMA was dramatically reduced by (1) pretreatment with a cPLA2-specific inhibitor, arachidonyltrifluoromethyl ketone, or (2) co-transfection with antisense cPLA2 oligonucleotide, indicating that the SRE activation was through cPLA2 activation. Co-transfection with an ANX-I expression vector also reduced the SRE stimulation by PMA, suggesting the inhibition of cPLA2 by ANX-I. The active domain of ANX-I was mapped using various deletion mutants. ANX-I(1-113) and ANX-I(34-346) were fully active, whereas ANX-I(114-346) abolished the activity. Therefore the activity was in the amino acid 34 to 113 region, which corresponds to the conserved domain I of ANX-I.     

The role of cytosolic phospholipase A2-alfa in regulation of phagocytic functions

Phospholipase A2(s) (PLA2(s)) are a family of enzymes that is present in a variety of mammalian and nonmammalian sources. Phagocytic cells contain cytosolic PLA2 (cPLA2) as well as several types of secreted PLA2, all of which have the potential to produce proinflammatory lipid mediators. The role of the predominant form of cPLA2 present in neutrophils is cPLA2alpha was studied by many groups. By modulating its expression in a variety of phagocytes it was found that it plays a major role in formation of eicosanoids. In addition, it was reported that cPLA2alpha also regulates the NADPH oxidase activation. The specificity of its effect on the NADPH oxidase is evident by results demonstrating that the differentiation process as well as other phagocytic functions are normal in cPLA2alpha-deficient PLB cell model. The novel dual subcellular localization of cPLA2alpha in different compartments, in the plasma membranes and in the nucleus, provides a molecular mechanism for the participation of cPLA2alpha in different processes (stimulation of NADPH oxidase and formation of eicosanoids) in the same cells.     

Role of phosphatidylinositol 4,5-bisphosphate in the activation of cytosolic phospholipase A2-alpha

Cytosolic phospholipase A(2)-alpha (cPLA(2)) plays an important role in the release of arachidonic acid and in cell injury. Activation of cPLA(2) is dependent on a rise in cytosolic Ca(2+) concentration, membrane association via the Ca(2+)-dependent lipid binding (CaLB) domain, and phosphorylation. This study addresses the activation of cPLA(2) via potential association with membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)), including the role of a "pleckstrin homology (PH)-like" region of cPLA(2) (amino acids 263-354). In cells incubated with complement, phorbol myristate acetate+the Ca(2+) ionophore, A23187, or epidermal growth factor+A23187, expression of the PH domain of phospholipase C-delta1 (which sequesters membrane PIP(2)) attenuated cPLA(2) activity. Stimulated cPLA(2) activity was also attenuated by the expression of cPLA(2) 135-366, or cPLA(2) 2-366, and expression of a PIP(2)-specific 5'-phosphatase. However, in a yeast-based assay that tests the ability of proteins to bind to membrane lipids, including PIP(2), with high affinity, only cPLA(2) 1-200 (CaLB domain) was able to interact with membrane lipids, whereas cPLA(2)s 135-366, 2-366, 201-648, and 1-648 were unable to do so. Therefore, cPLA(2) activity can be modulated by sequestration or depletion of cellular PIP(2), although the interaction of cPLA(2) with membrane PIP(2) appears to be indirect, or of weak affinity.     

Inhibition of phospholipase A2 reduces neurite outgrowth and neuronal viability

Phospholipase A2 (PLA(2)) has been implicated in neurodevelopmental processes and in the early development of the nervous system. We investigated the effects of the inhibition of calcium-dependent and calcium-independent subtypes of cytosolic PLA2 (cPLA2 and iPLA2) on the development and viability of primary cultures of cortical and hippocampal neurons. PLA2 in these cultures was continuously inhibited with methylarachidonyl-fluorophosphonate (MAFP), an irreversible inhibitor of cPLA2 and iPLA2, or with bromoenol lactone (BEL), an irreversible selective iPLA2 inhibitor. The effect of PLA2 inhibitors on the development of neuronal cultures was ascertained by total cell count and morphological characterisation. Neuronal viability was quantified with MTT assays. Inhibition of PLA2 resulted in reduction of neuritogenesis and neuronal viability, disrupting neuronal homeostasis and leading to neuronal death. We conclude that the functional integrity of both calcium-dependent and calcium-independent cytosolic PLA2 is necessary for the in vitro development of cortical and hippocampal neurons.     

Developmental maturation of primate placental trophoblast: placental cytosolic and secretory phospholipase A2 expression after estrogen suppression of baboons

We have demonstrated that the baboon placenta expressed the mRNAs and proteins for secretory and cytosolic phospholipase A2 (PLA2) enzymes and that cPLA2 expression increased with advancing gestation in association with the increase in placental estrogen production. To determine whether estrogen regulates placental PLA2 expression, as it does other aspects of syncytiotrophoblast functional differentiation, we compared sPLA2 and cPLA2 mRNA levels in placentas obtained on day 165 of gestation (term = day 184) from baboons that were untreated or treated during the second half of gestation with the aromatase inhibitor CGS 20267 or CGS 20267 and estradiol. Maternal saphenous and uterine vein estradiol levels were reduced (P < 0.05) by approximately 95% by treatment with CGS 20267 and restored by concomitant administration of CGS 20267 and estrogen. However, sPLA2 and cPLA2 mRNA levels expressed as a ratio of beta-actin were similar in whole villous placenta from baboons that were untreated or treated with CGS 20267 or CGS 20267 plus estrogen. PLA2 expression in an enriched fraction of nontrophoblast cells of the baboon placenta was also not altered by CGS 20267 treatment. Collectively these findings indicate that placental cPLA2 and sPLA2 expression is not estrogen-dependent. Because estrogen has been shown to regulate other aspects of placental steroidogenesis, we suggest that the regulatory role of estrogen on syncytiotrophoblast functional maturation is specific.     

Cytosolic phospholipase A2-alpha and cyclooxygenase-2 localize to intracellular membranes of EA.hy.926 endothelial cells that are distinct from the endoplasmic reticulum and the Golgi apparatus

Cytosolic phospholipase A2-alpha (cPLA2-alpha) is a calcium-activated enzyme that plays an important role in agonist-induced arachidonic acid release. In endothelial cells, free arachidonic acid can be converted subsequently into prostacyclin, a potent vasodilator and inhibitor of platelet activation, through the action of cyclooxygenase (COX) enzymes. Here we study the relocation of cPLA2-alpha in human EA.hy.926 endothelial cells following stimulation with the calcium-mobilizing agonist, A23187. Relocation of cPLA2-alpha was seen to be highly cell specific, and in EA.hy.926 cells occurred primarily to intracellular structures resembling the endoplasmic reticulum (ER) and Golgi. In addition, relocation to both the inner and outer surfaces of the nuclear membrane was observed. Colocalization studies with markers for these subcellular organelles, however, showed colocalization of cPLA2-alpha with nuclear membrane markers but not with ER or Golgi markers, suggesting that the relocation of cPLA2-alpha occurs to sites that are separate from these organelles. Colocalization with annexin V was also observed at the nuclear envelope, however, little overlap with staining patterns for the potential cPLA2-alpha interacting proteins, annexin I, vimentin, p11 or actin, was seen in this cell type. In contrast, cPLA2-alpha was seen to partially colocalize specifically with the COX-2 isoform at the ER-resembling structures, but not with COX-1. These studies suggest that cPLA2-alpha and COX-2 may function together at a distinct and novel compartment for eicosanoid signalling.     

The calcium binding loops of the cytosolic phospholipase A2 C2 domain specify targeting to Golgi and ER in live cells

Translocation of cytosolic phospholipase A2 (cPLA2) to Golgi and ER in response to intracellular calcium mobilization is regulated by its calcium-dependent lipid-binding, or C2, domain. Although well studied in vitro, the biochemical characteristics of the cPLA2C2 domain offer no predictive value in determining its intracellular targeting. To understand the molecular basis for cPLA2C2 targeting in vivo, the intracellular targets of the synaptotagmin 1 C2A (Syt1C2A) and protein kinase Calpha C2 (PKCalphaC2) domains were identified in Madin-Darby canine kidney cells and compared with that of hybrid C2 domains containing the calcium binding loops from cPLA2C2 on Syt1C2A and PKCalphaC2 domain backbones. In response to an intracellular calcium increase, PKCalphaC2 targeted plasma membrane regions rich in phosphatidylinositol-4,5-bisphosphate, and Syt1C2A displayed a biphasic targeting pattern, first targeting phosphatidylinositol-4,5-bisphosphate-rich regions in the plasma membrane and then the trans-Golgi network. In contrast, the Syt1C2A/cPLA2C2 and PKCalphaC2/cPLA2C2 hybrids targeted Golgi/ER and colocalized with cPLA2C2. The electrostatic properties of these hybrids suggested that the membrane binding mechanism was similar to cPLA2C2, but not PKCalphaC2 or Syt1C2A. These results suggest that primarily calcium binding loops 1 and 3 encode structural information specifying Golgi/ER targeting of cPLA2C2 and the hybrid domains.