Search

Background: Polyunsaturated fatty acids play a crucial role in neuronal function, and the modification of these compounds in the brain could have an impact on neurodegenerative diseases such as Alzheimer's disease. Despite the fact that arachidonic acid is the second foremost polyunsaturated fatty acid besides docosahexaenoic acid, its role and the regulation of its transfer and mobilization in the brain are poorly known., Methods: Two groups of 39 adult male BALB/c mice were fed with an arachidonic acid-enriched diet or an oleic acid-enriched diet, respectively, for 12 weeks. After 10 weeks on the diet, mice received intracerebroventricular injections of either NaCl solution or amyloid-β peptide (Aβ) oligomers. Y-maze and Morris water maze tests were used to evaluate short- and long-term memory. At 12 weeks on the diet, mice were killed, and blood, liver, and brain samples were collected for lipid and protein analyses., Results: We found that the administration of an arachidonic acid-enriched diet for 12 weeks induced short-term memory impairment and increased deleterious effects of Aβ oligomers on learning abilities. These cognitive alterations were associated with modifications of expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, postsynaptic density protein 95, and glial fibrillary acidic protein in mouse cortex or hippocampus by the arachidonic acid-enriched diet and Aβ oligomer administration. This diet also led to an imbalance between the main ω-6 fatty acids and the ω-3 fatty acids in favor of the first one in erythrocytes and the liver as well as in the hippocampal and cortical brain structures. In the cortex, the dietary arachidonic acid also induced an increase of arachidonic acid-containing phospholipid species in phosphatidylserine class, whereas intracerebroventricular injections modified several arachidonic acid- and docosahexaenoic acid-containing species in the four phospholipid classes. Finally, we observed that dietary arachidonic acid decreased the expression of the neuronal form of acyl-coenzyme A synthetase 4 in the hippocampus and increased the cytosolic phospholipase A 2 activation level in the cortices of the mice., Conclusions: Dietary arachidonic acid could amplify Aβ oligomer neurotoxicity. Its consumption could constitute a risk factor for Alzheimer's disease in humans and should be taken into account in future preventive strategies. Its deleterious effect on cognitive capacity could be linked to the balance between arachidonic acid-mobilizing enzymes.

Physiologically, renal medullary cells are surrounded by a hyperosmolar interstitium. However, different pathological situations can induce abrupt changes in environmental osmolality, causing cell stress. Therefore, renal cells must adapt to survive in this new condition. We previously demonstrated that, among the mechanisms involved in osmoprotection, renal cells upregulate triglyceride biosynthesis (which helps preserve glycerophospholipid synthesis and membrane homeostasis) and cyclooxygenase‐2 (which generates prostaglandins from arachidonic acid) to maintain lipid metabolism in renal tissue. Herein, we evaluated whether hyperosmolality modulates phospholipase A2 (PLA2) activity, leading to arachidonic acid release from membrane glycerophospholipid, and investigated its possible role in hyperosmolality‐induced triglyceride synthesis and accumulation. We found that hyperosmolality induced PLA2 expression and activity in Madin‐Darby canine kidney cells. Cytosolic PLA2 (cPLA2) inhibition, but not secreted or calcium‐independent PLA2 (sPLA2 or iPLA2, respectively), prevented triglyceride synthesis and reduced cell survival. Inhibition of prostaglandin synthesis with indomethacin not only failed to prevent hyperosmolality‐induced triglyceride synthesis but also exacerbated it. Similar results were observed with the peroxisomal proliferator activated receptor gamma (PPARγ) agonist rosiglitazone. Furthermore, hyperosmolality increased free intracellular arachidonic acid levels, which were even higher when prostaglandin synthesis was inhibited by indomethacin. Blocking PPARγ with GW‐9662 prevented the effects of both indomethacin and rosiglitazone on triglyceride synthesis and even reduced hyperosmolality‐induced triglyceride synthesis, suggesting that arachidonic acid may stimulate triglyceride synthesis through PPARγ activation. These results highlight the role of cPLA2 in osmoprotection, since it is essential to provide arachidonic acid, which is involved in PPARγ‐regulated triglyceride synthesis, thus guaranteeing cell survival. [ABSTRACT FROM AUTHOR]

The SARS-CoV-2 virus that is the cause of coronavirus disease 2019 (COVID-19) affects not only peripheral organs such as the lungs and blood vessels, but also the central nervous system (CNS)—as seen by effects on smell, taste, seizures, stroke, neuropathological findings and possibly, loss of control of respiration resulting in silent hypoxemia. COVID-19 induces an inflammatory response and, in severe cases, a cytokine storm that can damage the CNS. Antimalarials have unique properties that distinguish them from other anti-inflammatory drugs. (A) They are very lipophilic, which enhances their ability to cross the blood-brain barrier (BBB). Hence, they have the potential to act not only in the periphery but also in the CNS, and could be a useful addition to our limited armamentarium against the SARS-CoV-2 virus. (B) They are non-selective inhibitors of phospholipase A2 isoforms, including cytosolic phospholipase A2 (cPLA2). The latter is not only activated by cytokines but itself generates arachidonic acid, which is metabolized by cyclooxygenase (COX) to pro-inflammatory eicosanoids. Free radicals are produced in this process, which can lead to oxidative damage to the CNS. There are at least 4 ways that antimalarials could be useful in combating COVID-19. (1) They inhibit PLA2. (2) They are basic molecules capable of affecting the pH of lysosomes and inhibiting the activity of lysosomal enzymes. (3) They may affect the expression and Fe2+/H+ symporter activity of iron transporters such as divalent metal transporter 1 (DMT1), hence reducing iron accumulation in tissues and iron-catalysed free radical formation. (4) They could affect viral replication. The latter may be related to their effect on inhibition of PLA2 isoforms. Inhibition of cPLA2 impairs an early step of coronavirus replication in cell culture. In addition, a secretory PLA2 (sPLA2) isoform, PLA2G2D, has been shown to be essential for the lethality of SARS-CoV in mice. It is important to take note of what ongoing clinical trials on chloroquine and hydroxychloroquine can eventually tell us about the use of antimalarials and other anti-inflammatory agents, not only for the treatment of COVID-19, but also for neurovascular disorders such as stroke and vascular dementia. [ABSTRACT FROM AUTHOR]

During radiotherapy, an inflammatory response might be induced by activating various enzymes involved in membrane lipid metabolism. The eicosanoid pathway associated with cytosolic phospholipase A2 (cPLA2), cyclooxygenases (COXs), and lipoxygenases (LOXs) can be induced by radiation, and many lipid metabolites might contribute to cancer-associated inflammation, cell proliferation, and cell survival in cancer. The lipid metabolites are also involved in the establishment of the tumor-associated microenvironment through promotion of angiogenesis and formation of vascular network. These biological activities of lipid metabolites are responsible for malignant progression with the acquisition of radioresistance, leading to unsatisfactory outcome of cancer radiotherapy. Many efforts have been made to identify the mechanisms associated with bioactive lipid metabolites and radiation signaling that lead to radioresistance and to develop potent radiosensitizers to improve therapeutic efficacy. Beneficial outcomes would be achieved by targeting the enzymes, such as cPLA2, COXs, and LOXs, responsible for arachidonic acid metabolism and cancer-associated inflammation during cancer radiotherapy. The current study demonstrated a brief review for the radioresistant effects of bioactive lipid metabolites and their enzymes in cancer and the radiosensitizing effects of inhibitors for the enzymes on cancer therapy. [ABSTRACT FROM AUTHOR]

Background: Polyunsaturated fatty acids play a crucial role in neuronal function, and the modification of these compounds in the brain could have an impact on neurodegenerative diseases such as Alzheimer's disease. Despite the fact that arachidonic acid is the second foremost polyunsaturated fatty acid besides docosahexaenoic acid, its role and the regulation of its transfer and mobilization in the brain are poorly known. Methods: Two groups of 39 adult male BALB/c mice were fed with an arachidonic acid-enriched diet or an oleic acid-enriched diet, respectively, for 12 weeks. After 10 weeks on the diet, mice received intracerebroventricular injections of either NaCl solution or amyloid-β peptide (Aβ) oligomers. Y-maze and Morris water maze tests were used to evaluate short- and long-term memory. At 12 weeks on the diet, mice were killed, and blood, liver, and brain samples were collected for lipid and protein analyses. Results: We found that the administration of an arachidonic acid-enriched diet for 12 weeks induced short-term memory impairment and increased deleterious effects of Aβ oligomers on learning abilities. These cognitive alterations were associated with modifications of expression of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, postsynaptic density protein 95, and glial fibrillary acidic protein in mouse cortex or hippocampus by the arachidonic acid-enriched diet and Aβ oligomer administration. This diet also led to an imbalance between the main ω-6 fatty acids and the ω-3 fatty acids in favor of the first one in erythrocytes and the liver as well as in the hippocampal and cortical brain structures. In the cortex, the dietary arachidonic acid also induced an increase of arachidonic acid-containing phospholipid species in phosphatidylserine class, whereas intracerebroventricular injections modified several arachidonic acid- and docosahexaenoic acid-containing species in the four phospholipid classes. Finally, we observed that dietary arachidonic acid decreased the expression of the neuronal form of acyl-coenzyme A synthetase 4 in the hippocampus and increased the cytosolic phospholipase A2 activation level in the cortices of the mice. Conclusions: Dietary arachidonic acid could amplify Aβ oligomer neurotoxicity. Its consumption could constitute a risk factor for Alzheimer's disease in humans and should be taken into account in future preventive strategies. Its deleterious effect on cognitive capacity could be linked to the balance between arachidonic acid-mobilizing enzymes. [ABSTRACT FROM AUTHOR]

Studies of the response of RAW264.7 cells (RAW) to lipopolysaccharide (LPS) were carried out to determine why these cells do not demonstrate the prostaglandin (PG)-dependent autocrine regulation of tumor necrosis factor-α (TNF-α) secretion observed in primary resident peritoneal macrophages (RPMs). The major cyclooxygenase (COX) product of LPS-stimulated RAW was PGD2, with lesser amounts of PGE2. LPS-treated RAW produced PGs more slowly and reached their maximal PG synthetic rate later than did LPS-treated RPMs, as a result of lower constitutive COX-1 expression and a slower rate of COX-2 induction. Cytosolic phospholipase A2 and levels of free arachidonic acid were similar in RAW and RPMs. In contrast to RPMs, LPS-treated RAW produced high quantities of TNF-α, which were not altered in the presence of COX inhibitors. This failure of endogenous PGs to suppress TNF-α secretion was explained by the absence of the prostaglandin D2 receptor and the low levels of PGE2 produced during the first 2 h of the LPS response.These studies demonstrate that autocrine regulation of TNF-α secretion in response to LPS is greatly facilitated by a COX-1-mediated rapid accumulation of PGs as well by a correspondence between the PGs produced and the receptors expressed by the cells.

Berberine (BBR) has been suggested as a potential candidate anticancer agent due to its high anticancer activity and multiple mechanisms. In the present study, the inhibitory effect of BBR on hepatocellular carcinoma (HCC) via the suppression of the arachidonic acid (AA) metabolic pathway was investigated. BBR was demonstrated to reduce the viabilities of H22, HepG2 and Bel-7404 cells, in a dose- and time-dependent manner, and increase the number of apoptotic cells. BBR induced the translocation of apoptosis-inducing factor between the mitochondria and the nucleus, and had no effects on the protein expression levels of caspase-3 or -9. In addition, BBR significantly suppressed the protein expression levels of cytosolic phospholipase A2 (cPLA2) and cyclooxygenase (COX)-2 and elevated the content ratio of AA to prostaglandin E2 (PGE2). Furthermore, BBR reduced the volume and weight of tumors in a H22 transplanted tumor model in mice. The results of the present study demonstrated that elevation in the ratio of AA to PGE2 via suppression of the protein expression of cPLA2 and COX-2 in the AA metabolic pathway is involved in the inhibitory effect of BBR in HCC. [ABSTRACT FROM AUTHOR]

Abstract: Cytosolic phospholipase A2α (cPLA2α) affects membrane fluidity and permeability by catalyzing the hydrolysis of membrane phospholipids. We hypothesize that cPLA2α deficiency induces rigidity and architectural changes in cell membranes, especially in large cortical neurons. These membrane changes are discernible using light and electron microscopy. Through careful comparison with wild-type counterparts, we observed significant morphological changes in cortical neurons of cPLA2α knockout mice. These changes included the following: (1) increased numbers of nucleoli and enlarged nuclei, (2) narrower spaces between the inner and outer nuclear membranes, (3) reduced numbers of nuclear pores and altered nuclear pore structure, and (4) morphological changes in synaptic clefts. These results further suggest that cPLA2α and its cleaved arachidonic acids play important roles in cortical neuronal maturation and in normal neurochemical processes. [Copyright &y& Elsevier]

Abstract: Azithromycin is a macrolide antibiotic with well-described anti-inflammatory properties which can be attributed, at least partially, to its action on macrophages. We have previously shown, with 18 different macrolide molecules, that IL-6 and PGE2 inhibition correlates with macrolide accumulation, as well as with their binding to phospholipids in J774A.1 cells. The present study was performed in order to substantiate the hypothesis that biological membranes are a target for macrolide anti-inflammatory activity. By analyzing the effect of azithromycin on overall eicosanoid production, we found that in LPS-stimulated J774A.1 cells, azithromycin, like indomethacin, inhibited the synthesis of all eicosanoids produced downstream of COX. Upstream of COX, azithromycin inhibited arachidonic acid release in the same way as a cPLA2 inhibitor, while indomethacin had no effect. Further comparison revealed that in LPS-stimulated J774A.1 cells, the cPLA2 inhibitor showed the same profile of inhibition as azithromycin in inhibiting PGE2, IL-6, IL-12p40 and arachidonic acid release. Therefore, we propose that the anti-inflammatory activity of azithromycin in this model may be due to interactions with cPLA2, causing inadequate translocation of the enzyme or disturbing physical interactions with its substrates. [Copyright &y& Elsevier]

Abstract: Eicosanoids are potent lipid mediators derived from phospholipase (PL)-released arachidonic acid (AA) coupled to subsequent metabolism by cyclooxygenase (COX)-1/2 or lipoxygenases (LO) which are involved in a variety of homeostatic biological functions and inflammation. We have investigated three lupeolic acids (LA) from the gum resin of Boswellia carterii for their ability to interfere with eicosanoid biosynthesis in human blood cells. A novel, yet unknown C(28)-hydroxylated LA, that is, 3α-acetoxy-28-hydroxylup-20(29)-en-4β-oic acid (Ac-OH-LA) was found to inhibit the biosynthesis of COX-, 5-LO- and 12-LO-derived eicosanoids from endogenous AA in activated platelets, neutrophils, and monocytes from human blood with consistent IC50 values of 2.3–6.9μM. In contrast, two other LAs lacking the C(28)-OH moiety were essentially inactive in this respect. Inhibition of eicosanoids by Ac-OH-LA correlated with reduced release of AA in intact cells. When AA was exogenously provided as substrate for cellular eicosanoid biosynthesis the inhibitory effects of Ac-OH-LA were essentially reversed, even though some inhibition of 5-LO and COX-1 product formation still remained. Finally, by means of a cell-free phospholipid hydrolysis assay using human recombinant cytosolic PLA2α, we show that Ac-OH-LA may directly interfere with cPLA2α activity (IC50 =3.6μM). Together, we identified a novel, naturally occuring C(28)-hydroxylated LA which acts as efficient inhibitor of cPLA2α and consequently suppresses eicosanoid biosynthesis in intact cells. [Copyright &y& Elsevier]

Abstract: Prostaglandin E2 (PGE2) is an arachidonic acid (AA)-derived signaling molecule that can influence host immune responses to infection or vaccination. In this study, we investigated PGE2 production in vitro by cells infected with the poxvirus vaccine strain, modified vaccinia Ankara virus (MVA). Human THP-1 cells, murine bone marrow-derived dendritic cells, and murine C3HA fibroblasts all accumulated PGE2 to high levels in culture supernatants upon infection with MVA. We also demonstrated that MVA induced the release of AA from infected cells, and this was, most unusually, independent of host cytosolic phospholipase A2 activity. The accumulation of AA and PGE2 was dependent on viral gene expression, but independent of canonical NF-κB signaling via p65/RelA. The production of PGE2 required host cyclooxygenase-2 (COX-2) activity, and COX-2 protein accumulated during MVA infection. The results of this study provide insight into a novel aspect of MVA biology that may affect the efficacy of MVA-based vaccines. [Copyright &y& Elsevier]

Background & Aims: Cytosolic phospholipase A2α (cPLA2α) is a rate-limiting key enzyme controlling the release of arachidonic acid (AA) substrate for the synthesis of prostaglandins and leukotrienes. This study was designed to explore the role of hepatocyte cPLA2α in Fas-mediated liver injury, in vivo. Methods: Transgenic mice with targeted expression of cPLA2α under control of the albumin-promoter enhancer and wild-type mice were injected intraperitoneally with anti-Fas antibody Jo2 or lipopolysaccharide plus d-galactosamine and monitored for liver injury and survival at various time points. Results: The cPLA2α Tg mice resist Fas-induced liver failure, as reflected by the lower serum transaminase levels, fewer apoptotic hepatocytes, reduced caspase activation, and reduced PARP cleavage when compared to the matched wild type mice. Inhibition of cPLA2α by its pharmacological inhibitor, pyrrolidine, enhanced Jo2-induced liver injury in both cPLA2α Tg and wild type mice. Hepatic overexpression of cPLA2α increases the expression of EGFR in the liver and the EGFR inhibitor, AG1478, exacerbated Jo2-mediated liver injury. The cPLA2α transgenic mice develop more prominent liver tissue damage than wild-type mice after LPS/d-galactosamine injection. Conclusions: Hepatocyte cPLA2α protects against Fas-induced liver injury and this effect is mediated at least in part through the upregulation of EGFR. [ABSTRACT FROM AUTHOR]

Abstract: The olfactory bulbectomized rat is an animal model of depression with a number of neurochemical, neuroendocrinological and behavioural features resembling human depression. Arachidonic acid (AA) is a second messenger released from the neuronal membrane phospholipids following the stimulation of the receptors coupled with G-proteins to the cytosolic phospholipase A (cPLA2) signalling pathway. The signalling of several neurotransmitter systems which are deregulated in OBX rats (serotonergic, dopaminergic, cholinergic, and glutamatergic) converges on the cPLA2 signalling pathway. The aim of the present study was to assess the incorporation coefficient k* [ml/g/s] of AA in a large number of brain regions in the OBX rat, as a parameter reflecting AA turnover. Male Sprague–Dawley rats (160–200g) were randomly assigned into an intact Sprague–Dawley (SPD) group, a Sham-operated (SHAM) group or an olfactory bulbectomized (OBX) group (n =5 per group). Two weeks following the surgeries (SHAM and OBX rats) or without any prior intervention (SPD rats), the k* was measured using [1-14C] AA autoradiography. Two-way ANOVA followed by the Newman–Keuls test revealed the following: (1) significantly increased AA turnover in the OBX rats, relative to the SHAM rats, in 17 out of 27 assessed brain regions; and (2) no significant differences in AA turnover between the SHAM and the SPD rats. The results suggest the upregulation of one or more neurotransmitter systems or receptors acting through the PLA2 signalling pathway, or the components of the cPLA2 signalling system itself. Taken together with the previous measurements, one can conclude that this elevation is likely related to upregulation in the brain serotonergic system because of the elevated 5-HT synthesis of the OBX rats. [Copyright &y& Elsevier]

Abstract: Glioma is characterized by an active production of proangiogenic molecules. We observed that conditioned medium (CM) from C6 glioma significantly enhanced proliferation and migration of immortalized rat brain GP8.3 endothelial cells (ECs) and primary bovine brain microvascular ECs. The glioma CM effect was significantly reduced by cytosolic (cPLA2) and Ca++-independent (iPLA2) phospholipase A2, cyclooxygenase-2, and protein kinase inhibitors. In GP8.3 ECs, cPLA2 and iPLA2 enzyme activities and phosphorylation of cPLA2, significantly stimulated after 24h CM co-incubation, were attenuated by PLA2, PI3-K, MEK-1, and ERK1/2 inhibitors. By confocal microscopy, in glioma CM-stimulated ECs, enhancement of fluorescence signals for phospho-cPLA2, phospho-ERK1/2, phospho-PKCα, COX-2, and iPLA2 was in parallel observed. Electroporation of anti-iPLA2 and cPLA2 antibodies and siRNAs directed against iPLA2 and cPLA2 significantly inhibited cell proliferation and migration. Incubation of CM- or VEGF peptide-stimulated ECs with antibodies against VEGF or VEGFR-1/-2 receptors strongly reduced mitotic rate, cell migration, and phospho-cPLA2 and iPLA2 protein levels. The findings suggest that PLA2 activities are involved in stimulating EC migration and proliferation in the presence of glioma CM and that cPLA2 is positively regulated upstream by PI3-K, PKCα, and ERK1/2 signal cascades. Our work provides new insights in understanding EC metabolism and signaling during tumor angiogenesis. [ABSTRACT FROM AUTHOR]

Celecoxib is a selective inhibitor of cyclooxygenase-2 (COX-2). It may reduce the risk of cancer formation by affecting the metabolism of arachidonic acid (AA), which has been implicated in the development of cancer. Accordingly, this study was designed to determine the effects of celecoxib on the AA pathway in mouse hepatoma H22 cells. Celecoxib was found to inhibit the proliferation of H22 cells in a dose- and time-dependent manner. Low doses (50 and 100 µmol·L-1) of celecoxib caused an increase in the expression of cytosolic phospholipase A2 (cPLA2), but did not affect the expression of COX-2 in terms of the mRNA and protein levels. Surprisingly, the amount of AA was elevated and the level of prostaglandin E2 (PGE2) was unaltered in the culture supernatant. At higher celecoxib doses (200 and 400 µmol·L-1), the mRNA and protein of both COX-2 and cPLA2 were inhibited. The concentration of AA was increased, and PGE2 level was depressed in H22 cells. The ratio of AA to PGE2 was increased in a dose-dependent manner. Our findings suggest that the imbalance between AA and PGE2, characterized by increased AA at a low dosage and decreased PGE2 at a high dosage of celecoxib, was an important indicator of cytotoxicity of celecoxib on H22 cells. Le célécoxib est un inhibiteur sélectif de la cyclooxygénase-2 (COX-2). Il peut réduire le risque de formation de cancer en modifiant le métabolisme de l’acide arachidonique (AA) impliqué dans le développement du cancer. Conséquemment, cette étude a été conçue pour déterminer les effets du célécoxib sur la voie de l’AA dans les cellules d’hépatome de souris H22. Dans cette étude, le célécoxib a inhibé la prolifération des cellules H22 de façon dépendante de la concentration et du temps. De faibles doses (50 µmol·L-1 et 100 µmol·L-1) de célécoxib ont causé une augmentation de l’expression de la phospholipase A2 cytosolique (cPLA2), mais n’ont pas affecté l’expression de l’ARNm et de la protéine COX-2. De façon surprenante, la quantité d’AA était élevée et le niveau de prostaglandine E2 (PGE2) du milieu de culture n’était pas modifié. À plus fortes doses de célécoxib (200 µmol·L-1 et 400 µmol·L-1), l’expression des protéinesCOX-2 et cPLA2 et de leur ARNm était inhibée. La concentration d’AA était augmentée et le niveau de PGE2 des cellules H22 était diminué. Le ratio AA/PGE2 était augmenté de façon dépendante de la concentration. Nos résultats suggèrent qu’un déséquilibre entre les quantités d’AA et de PGE2 caractérisé par une augmentation d’AA à faible dose et une diminution de PGE2 à forte dose de célécoxib, constituait un indicateur important de la cytotoxicité du célécoxib sur les cellules H22. [ABSTRACT FROM AUTHOR]

Abstract: Cytosolic phospholipase A2α (cPLA2α, Group IVA phospholipase A2) is a central mediator of arachidonate release from cellular phospholipids for the biosynthesis of eicosanoids. cPLA2α translocates to intracellular membranes including the Golgi in response to a rise in intracellular calcium level. The enzyme’s calcium-dependent phospholipid-binding C2 domain provides the targeting specificity for cPLA2α translocation to the Golgi. However, other features of cPLA2α regulation are incompletely understood such as the role of phosphorylation of serine residues in the catalytic domain and the function of basic residues in the cPLA2α C2 and catalytic domains that are proposed to interact with anionic phospholipids in the membrane to which cPLA2α is targeted. Increasing evidence strongly suggests that cPLA2α plays a role in regulating Golgi structure, tubule formation and intra-Golgi transport. For example, recent data suggests that cPLA2α regulates the transport of tight junction and adherens junction proteins through the Golgi to cell–cell contacts in confluent endothelial cells. However, there are now examples where data based on knockdown using siRNA or pharmacological inhibition of enzymatic activity of cPLA2α affects fundamental cellular processes yet these phenotypes are not observed in cells from cPLA2α deficient mice. These results suggest that in some cases there may be compensation for the lack of cPLA2α. Thus, there is continued need for studies employing highly specific cPLA2α antagonists in addition to genetic deletion of cPLA2α in mice. [Copyright &y& Elsevier]

Arachidonic acid (AA) signaling is upregulated in the caudate-putamen and frontal cortex of unilaterally 6-hydroxydopamine (6-OHDA) lesioned rats, a model for asymmetrical Parkinson disease. AA signaling can be coupled to D2-like receptor initiated AA hydrolysis from phospholipids by cytosolic phospholipase A2 (cPLA2) and subsequent metabolism by cyclooxygenase (COX)-2. In unilaterally 6-OHDA- and sham-lesioned rats, we measured brain expression of cPLA2, other PLA2 enzymes, and COX-2. Activity and protein levels of cPLA2 were significantly higher as was COX-2-protein in caudate-putamen, frontal cortex and remaining brain on the lesioned compared to intact side of the 6-OHDA lesioned rats, and compared to sham brain. Secretory sPLA2 and Ca2+-independent iPLA2 expression did not differ between sides or groups. Thus, the tonically increased ipsilateral AA signal in the lesioned rat corresponds to upregulated cPLA2 and COX-2 expression within the AA metabolic cascade, which may contribute to symptoms and pathology in Parkinson disease. [ABSTRACT FROM AUTHOR]

In mammalian cells, cytosolic phospholipase A2 (cPLA2) displays a variety of activities through the release of arachidonic acid in response to cellular stimuli. In this study, we characterize the putative cPLA2-like protein, AoPlaA, in the filamentous fungus Aspergillus oryzae. When AoPlaA-EGFP was expressed in A. oryzae, it localized to the tubular structures that was costained by the marker dye for the mitochondria. A biochemical fractionation experiment showed that AoPlaA was present in the mitochondria-enriched fraction. The presence of an N-terminal cleavable mitochondrial targeting signal in AoPlaA was demonstrated by N-terminal amino acid sequence analysis, and by showing that chimeric proteins consisting of N-terminal 65 or 50 amino acids of AoPlaA fused to enhanced green fluorescent protein (EGFP) localized to the mitochondria. Submitochondrial fractionation of AoPlaA expressed in Saccharomyces cerevisiae demonstrated that AoPlaA localizes to the intramembrane space of the mitochondria. Taken together, the results presented here demonstrate a novel, submitochondrial localization of the cPLA2-like protein in the filamentous fungi. [ABSTRACT FROM AUTHOR]

Juxtaglomerular cells or granular cells synthesize, store, and secrete renin needed for the renin-angiotensin-aldosterone (RAS) to fine tune the maintenance of blood pressure and blood volume. The baroreceptor function of these granular cells may function as a nuclear mechanotransducer to sense and transmit the extracellular physical forces to their chromatin to regulate renin gene expression and secretion. Cell nucleus serves as a sensor and responds to the changes in pressure and stretch that triggers calcium release and cPLA2 (cytosolic phospholipase A2) activation that, in turn, releases arachidonic acid (AA) (and possibly, other unsaturated fatty acids) from the cell membrane lipid pool. AA influences the cytoskeletal system and is involved and regulates exocytosis. AA is the precursor of a variety of products that have a regulatory role in the formation and action of RAS, inflammation, and immune response. These results imply that AA is involved in nuclear mechanotransduction mechanism in the baroreceptor function of granular cells and in the regulation of synthesis and secretion of renin. [ABSTRACT FROM AUTHOR]

Abstract: The phospholipase A2 (PLA2)-prostanoid cascade is involved in cannabinoid receptor-mediated neuronal functions. We investigated the signaling mechanism for the release of arachidonic acid by cannabinoids, 2-arachidonoyl glycerol (2-AG) and HU210, in rat PC12 cells and in primary cultured cells from the mouse cerebellum. The effect of selective inhibitors for signaling pathways and/or enzymes (α type cytosolic PLA2 (cPLA2α), G protein, Src kinases, phospholipase C, protein kinase C) was assessed. Methods included translocation of the chimeric protein GFP-cPLA2α, the activities of Src family kinases, Ca2+-dependent fluorescence and cyclic AMP accumulation. Treatment with 2-AG and HU210 at greater concentrations than 3 μM caused the release of arachidonic acid, and the response was inhibited by AM251 (an antagonist of cannabinoid CB1 receptor) and by pyrrophenone (a selective inhibitor of cPLA2α) in PC12 cells. The cannabinoid treatment caused the intracellular translocation of cPLA2α and an increase in the intracellular Ca2+ level. Treatment with HU210 caused tyrosine phosphorylation of Src and Fyn, and increased their kinase activities. Pretreatment with inhibitors of tyrosine kinases or phospholipase C abolished the cannabinoids-induced release of arachidonic acid and Ca2+ response, and protein kinase C inhibitor reduced the release of arachidonic acid. 2-AG caused the release of arachidonic acid from cultured cells of the mouse cerebellum via similar mechanisms. These data reveal that cannabinoids activated cPLA2α in a Src-phospholipase C-protein kinase C-dependent manner probably via cannabinoid CB1 receptor and/or CB1-like receptor in neuronal cells. [Copyright &y& Elsevier]

Phospholipase A2 (PLA2) enzymes encompass a superfamily of at least 13 extracellular and intracellular esterases that hydrolyze the sn-2 fatty acyl bonds of phospholipids to yield fatty acids and lysophospholipids. The purpose of this study was to characterize which phospholipase paralog regulates NMDA receptor-mediated arachidonic acid (AA) release. Using mixed cortical cell cultures containing both neurons and astrocytes, we found that [3H]-AA released into the extracellular medium following NMDA receptor stimulation (100 μM) increased with time and was completely prevented by the addition of the NMDA receptor antagonist MK-801 (10 μM) or by removal of extracellular Ca2+. Neither diacylglycerol lipase inhibition (RHC-80267; 10 μM) nor selective inhibition of Ca2+-independent PLA2 [bromoenol lactone (BEL); 10 μM] alone had an effect on NMDA receptor-stimulated release of [3H]-AA. Release was prevented by methyl arachidonyl fluorophosphonate (MAFP) (5 μM) and AACOCF3 (1 μM), inhibitors of both cytosolic PLA2 (cPLA2) and Ca2+-independent PLA2 isozymes. This inhibition effectively translated to block of NMDA-induced prostaglandin (PG) production. An inhibitor of p38MAPK, SB 203580 (7.5 μM), also significantly reduced NMDA-induced PG production providing suggestive evidence for the role of cPLA2α. Its involvement in release was confirmed using cultures derived from mice deficient in cPLA2α, which failed to produce PGs in response to NMDA receptor stimulation. Interestingly, neither MAFP, AACOCF3 nor cultures derived from cPLA2α null mutant animals showed any protection against NMDA-mediated neurotoxicity, indicating that inhibition of this enzyme may not be a viable protective strategy in disorders of the cortex involving over-activation of the NMDA receptor. [ABSTRACT FROM AUTHOR]

Abstract: Although CpG containing DNA is an important regulator of innate immune responses via toll-like receptor 9 (TLR9), excessive activation of this receptor is detrimental to the host. Here, we show that cytosolic phospholipase A2 (cPLA2) activation is important for TLR9-mediated inducible nitric oxide synthase (iNOS) expression. Activation of TLR9 signaling by CpG induces iNOS expression and NO production. Inhibition of TLR9 blocked the iNOS expression and NO production. The CpG also stimulates cPLA2-hydrolyzed arachidonic acid (AA) release. Inhibition of cPLA2 activity by inhibitor attenuated the iNOS expression by CpG response. Additionally, knockdown of cPLA2 protein by miRNA also suppressed the CpG-induced iNOS expression. Furthermore, the CpG rapidly phosphorylates three MAPKs and Akt. A potent inhibitor for p38 MAPK or Akt blocked the CpG-induced AA release and iNOS expression. These results suggest that TLR9 activation stimulates cPLA2 activity via p38 or Akt pathways and mediates iNOS expression. [Copyright &y& Elsevier]

Leukotrienes (LT) exert stimulatory effects on myelopoiesis, beside their inflammatory and immunomodulating effects. Here, we have studied the expression and activity of the enzymes involved in the synthesis of leukotriene B4 (LTB4) in acute myeloid leukemia (AML) cells (16 clones) and G-CSF mobilized peripheral blood CD34+ cells. CD34+ cells from patients with non-myeloid malignancies expressed cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase activating protein (FLAP), and leukotriene A4 (LTA4) hydrolase but not 5-lipoxygenase (5-LO). The enzyme cPLA2 was abundantly expressed in AML cells and the activity of the enzyme was high in certain AML clones. The expression of 5-LO, FLAP, and LTA4 hydrolase in AML clones was in general lower than in healthy donor polymorphonuclear leukocytes (PMNL). The calcium ionophore A23187-induced release of [14C] arachidonic acid (AA) in AML cells was low, compared with PMNL, and did not correlate with the expression of cPLA2 protein. Biosynthesis of LTB4, upon calcium ionophore A23187 activation, was only observed in five of the investigated AML clones and only three of the most differentiated clones produced similar amounts of LTB4 as PMNL. The capacity of various cell clones to produce LTs could neither be explained by the difference in [1 − 14C] AA release nor 5-LO expression. Taken together, these results indicate that LT synthesis is under development during early myelopoiesis and the capacity to produce LTs is gained upon maturation. High expression of cPLA2 in AML suggests a putative role of this enzyme in the pathophysiology of this disease. [ABSTRACT FROM AUTHOR]

Addition of bacterial lipopolysaccharides (LPS) and interferon-γ (IFN-γ) to rat astrocytes in primary culture promotes an early release of arachidonic acid (ARA) associated with an immediate inhibition of neuronal nitric oxide synthase (nNOS). Preventing the release of constitutive nitric oxide (NO) is indeed critical for activation of the nuclear factor kappa B, and for the expression of inducible nitric oxide synthase responsible for the formation of large amounts of NO. LPS/IFN-γ also promotes an early release of superoxide, via activation of NADPH oxidase, but the generation of peroxynitrite (ONOO−) is prevented by the different timing of superoxide (minutes) and NO (hours) formation. Upstream inhibition of the ARA-dependent nNOS inhibitory signaling, however, caused the parallel release of superoxide and constitutive NO, thereby leading to formation of ONOO− levels triggering loss of ATP and mitochondrial membrane potential followed by the mitochondrial release of cytochrome c, activation of caspase 3 and morphological evidence of apoptosis. Nanomolar levels of exogenous ARA prevented all these events via inhibition of early ONOO− formation. Thus, the ARA-dependent nNOS inhibition observed in astrocytes exposed to pro-inflammatory stimuli, as LPS/IFN-γ, is critical for both the expression of nuclear factor kappa B-dependent genes and for survival. [ABSTRACT FROM AUTHOR]

Cytosolic phospholipase A2 (cPLA2) group IVα is a critical enzyme involved in the liberation of arachidonic acid from cellular membranes. cPLA2−/− mice have reduced allergen-induced bronchoconstriction and bronchial hyperresponsiveness. The goal of this study was to investigate polymorphisms of the (CA)n and (T)n microsatellites and surrounding regions in the cPLA 2α gene promoter. We analysed the cPLA 2 promoter regions containing (CA)n and (T)n repeats in 87 patients with severe asthma and in 48 control subjects by bidirectional sequencing. Functional studies were performed utilizing reporter genes derived from subjects with varying numbers of these repeats, and on constructs with a series of deletions. We found that the (CA)n and (T)n regions are polymorphic and that constructs with CA or T repeats or CA and T repeats deleted revealed, respectively, a 41·8 ± 7%, 22·3 ± 5% and 100 ± 20% increase in reporter gene activity. A lower number of CA or T repeats caused higher cPLA 2 promoter luciferase activity. The group of shorter alleles of the (CA)n microsatellite region ( n = 12–18) ( Pcor = 0·00006), and the group of shorter alleles of (T)n repeats region ( n = 17–38) ( Pcor = 0·0039) occurred significantly more often in patients with severe asthma. We also found novel SNPs in positions −292 C > G, −185 A > C, −180 T > C and −165 A > C. Two of them were associated with the severe asthma phenotype: −180T allele ( Pcor = 0·03996) and −185 A allele ( Pcor = 0·03966). These results demonstrate that (CA)n and (T)n repeats may have an influence on cPLA2 transcription which might play a role in severe asthma pathogenesis. [ABSTRACT FROM AUTHOR]

Excessive N-methyl-d-aspartate (NMDA) signaling is thought to contribute to bipolar disorder symptoms. Lithium and carbamazepine, effective against bipolar mania, are reported in rats to reduce brain transcription of an arachidonic acid selective calcium-dependent cytosolic phospholipase A2 (cPLA2), as well as expression of one of its transcription factors, activator protein (AP)-2. In this study, we determined if chronic administration of NMDA (25 mg/kg i.p.) to rats would increase brain cPLA2 and AP-2 expression, as these antimanic drugs are known to down-regulate excessive NMDA signaling. Administration of a daily subconvulsive dose of NMDA to rats for 21 days decreased frontal cortex NMDA receptor (NR)-1 and NR-3A subunits and increased cPLA2 activity, phosphorylation, protein, and mRNA levels. The activity and protein levels of secretory phospholipase A2 or calcium-independent phospholipase A2 were not changed significantly. Chronic NMDA also increased the DNA-binding activity of AP-2 and the protein levels of its α and β subunits. These changes were absent following acute (3 h earlier) NMDA administration. The changes, opposite to those found following chronic lithium or carbamazepine, are consistent with up-regulated arachidonic acid release due to excessive NR signaling and may be a contributing factor to bipolar mania. [ABSTRACT FROM AUTHOR]

Abstract: Limited evidence suggests that brain cytosolic phospholipase A2 (cPLA2), which selectively releases arachidonic acid (AA) from membrane phospholipids, and cyclooxygenase-2 (COX-2), the rate-limiting enzyme for AA metabolism to prostanoids, change as a function of normal aging. In this study, we examined the protein levels of cPLA2 and COX-2 enzymes in hippocampus, frontal pole and cerebellum from young (2–5 years old), middle-aged (8–11 years old) and old (23 years old) male and female Rhesus monkeys. In the cerebellum, cPLA2 protein level was higher in the young brain as compared to levels seen at both middle-aged and old. Similarly, in the frontal pole, the young brain showed a higher level of COX-2 protein as compared to the levels seen at both older ages. For both, once an animal reached 8–11 years of age the levels appeared to remain relatively constant over the next decade. Immunohistochemistry of COX-2 protein within the brain demonstrated no significant change in the localization to neurons within the frontal pole. Qualitatively, a greater number of neurons were positively stained for COX-2 in the young brain than in the aged brain. Based on the previous reports of localization of cPLA2 and COX-2 at post-synaptic sites in neurons results from the current study suggest that the elevated protein levels of the two enzymes seen in the younger brain is related to the greater potential for synaptic plasticity across multiple neurons as a function of age and that cPLA2 and COX-2 may be considered as post-synaptic markers. [Copyright &y& Elsevier]

Abstract: Peroxynitrite, a highly reactive nitrogen species, promotes in U937 cells (a promonocytic cell line) a mitochondrial permeability transition (MPT)-dependent necrosis. An initial event triggered by peroxynitrite (i.e., inhibition of complex III of the mitochondrial respiratory chain) is responsible for the time-dependent formation of H2O2, essential for the occurrence of cell death. Otherwise non-toxic concentrations of peroxynitrite nevertheless commit cells to MPT-dependent necrosis, which is however prevented by a cytoprotective signaling driven by arachidonic acid (AA) released by the cytosolic PLA2 isoform. Interestingly, the mechanism whereby delayed formation of H2O2 promotes toxicity in cells exposed to intrinsically toxic concentrations of peroxynitrite is independent of the accumulation of additional damage. Cell death is in fact mediated by inhibition of the AA-dependent cytoprotective signaling. Exogenous AA, however, prevented toxicity also under these conditions. An additional point to be made is that the major findings obtained using U937 cells were reproduced in different cell types belonging to the monocyte/macrophage lineage. Hence, within the context of the inflammatory response, monocytes and macrophages may cope with peroxynitrite by using AA, a signaling molecule largely available at the inflammatory sites. [Copyright &y& Elsevier]

Recently we found that 1-methyldodecanoylindole-2-carboxylic acid (1) and 1-[2-(4-carboxyphenoxy)ethyl]-3-dodecanoylindole-2-carboxylic acid (4) were inhibitors of the cytosolic phospholipase A 2 α (cPLA 2 α)-mediated arachidonic acid release in calcium ionophore A23187-stimulated human platelets with IC 50 -values of 4.8 μM (1) and 0.86 μM (4). We have now replaced the 3-acyl residue of these compounds by alkylated sulfinyl-, sulfony-, sulfinamoyl-, sulfamoyl-, carbonylamino-, or carbonylaminomethyl-substituents. Structure–activity relationship studies revealed that the pronounced cellular activity of 4 strongly depends on the presence of the 3-acyl moiety. Surprisingly, when testing 4 and its derivatives in an assay with the isolated cPLA 2 , none of these compounds showed an inhibitory potency at 10 μM indicating that they do not inhibit cPLA 2 α in the cells by a direct interaction with the active site of the enzyme. [ABSTRACT FROM AUTHOR]

Abstract: Here, we show that actin polymerisation inhibitors such as latrunculin B (LB), and to a minor extent also cytochalasin D (Cyt D), enhance the release of arachidonic acid (AA) as well as nuclear translocation of 5-lipoxygenase (5-LO) and 5-LO product synthesis in human polymorphonuclear leukocytes (PMNL), challenged with thapsigargin (TG) or N-formyl-methionyl-leucyl-phenylalanine. The concentration-dependent effects of LB (EC50 ≈200 nM) declined with prolonged preincubation (>3 min) prior TG and were barely detectable when PMNL were stimulated with Ca2+-ionophores. Investigation of the stimulatory mechanisms revealed that LB (or Cyt D) elicits Ca2+ mobilisation and potentiates stimulus-induced elevation of intracellular Ca2+, regardless of the nature of the stimulus. LB caused rapid but only moderate activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK)2. The selective Src family kinase inhibitors PP2 and SU6656 blocked LB- or Cyt D-mediated Ca2+ mobilisation and suppressed the upregulatory effects on AA release and 5-LO product synthesis, without affecting AA metabolism evoked by ionophore alone. We conclude that in PMNL, inhibitors of actin polymerisation cause enhancement of intracellular Ca2+ levels through Src family kinase signaling, thereby facilitating stimulus-induced release of AA and 5-LO product formation. [Copyright &y& Elsevier]

The authors have previously demonstrated that the oxidant t-buOOH stimulates phospholipase A 2 (PLA 2 ) activity in bovine pulmonary artery endothelial cells (S. Chakraborti et al. American Journal of Physiology, 257, L430–L437, 1989). Herein, the authors sought to investigate the mechanism by which t-buOOH stimulates PLA 2 activity and the role of protein kinase C (PKC) in this scenario. Treatment of bovine pulmonary artery endothelial cells with t-buOOH stimulated an aprotinin-sensitive protease activity, PKC activity, and PLA 2 activity in the cell membrane. Pretreatment with intracellular Ca 2+ chelator (BAPTA-AM), PKCα inhibitor (Go6976), cPLA 2 inhibitor (AACOCF 3 ), and pertussis toxin prevented t-buOOH–stimulated PLA 2 activity. Immunoblot studies with aprotinin, cPLA 2 , PKCα, and Giα antibodies revealed their presence in the endothelial membrane. Immunoblot studies of the cell membrane isolated from t-buOOH–stimulated cells with cPLA 2 and PKCα antibodies elicited an apparent increase in their immunoreactive protein profiles along with an additional 47-kDa immunoreactive fragment in the membrane. t-buOOH caused Giα phosphorylation in the membrane and pretreatment with Go6976 prevented the phosphorylation. Overall, these results suggest that t-buOOH stimulates an aprotinin-sensitive protease activity that proteolytically activates PKCα and that subsequently phosphorylates a pertussis toxin–sensitive protein, resulting in the stimulation of cPLA 2 activity in the cell membrane. [ABSTRACT FROM AUTHOR]

Abstract: A short-term growth of U937 cells in serum-free medium causes a prompt, mitochondrial permeability transition (MPT)-dependent necrotic response after exposure to an otherwise non-toxic concentration of peroxynitrite. This event is mediated by inhibition of extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation, essential for the cytosolic phospholipase A2-dependent arachidonic acid (AA) release evoked by peroxynitrite. Reduced availability of the lipid messenger would therefore limit the efficiency of the AA-dependent survival signalling and cause an MPT-based necrosis. Since peroxynitrite further reduces the extent of ERK1/2 phosphorylation, regardless of whether cells had been grown in serum-free or -containing medium, it appears that basal ERK1/2 phosphorylation is a critical determinant for the survival response of U937 cells to a non-toxic, but nevertheless MPT-committing, concentration of peroxynitrite. [Copyright &y& Elsevier]

Abstract: Exposure to an otherwise non-toxic concentration of peroxynitrite (ONOO−) promotes toxicity in U937 cells supplemented with pharmacological inhibitors of protein kinase C (PKC). This effect is not associated with enhanced formation of H2O2 and is in fact causally linked to inhibition of the cytoprotective signalling driven by endogenous arachidonic acid (AA). The outcome of various approaches using PKC or phospholipase A2 inhibitors, cytosolic phospholipase A2 or PKCα antisense-oligonucleotide-transfected cells and supplementation with exogenous AA or tetradecanoylphorbol acetate, as well as PKC down-regulated cells, indicated that ONOO− promotes AA-dependent cytosol to membrane translocation of PKCα, an event critical for the cytoprotective signalling under investigation. Evidence for a similar mechanism regulating survival of human monocytes exposed to ONOO− is also presented. These results, along with our previous work on this topic, contribute to the definition of the mechanism whereby monocytes survive to ONOO− in inflamed tissues. [Copyright &y& Elsevier]

Abstract: We have previously shown that, in bovine retina pericytes, amyloid β(1–42) and its truncated form containing amino acids 25–35, after 24 h treatment, stimulate arachidonic acid (AA) release and phosphatidylcholine hydrolysis, by activation of both cytosolic (cPLA2) and Ca2+-independent (iPLA2) phospholipase A2. A putative role for MAP kinases in this process emerged. Here we studied the role of the MAP-kinase family as well as both cPLA2 and iPLA2 mRNA expression by a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in the same sublethal model of amyloid-β (Aβ) damage to pericytes in vitro. Aβ(25–35) peptide evoked AA release as well as stimulated phosphorylation of ERK1/2, p38 MAPKs and cPLA2, but not c-Jun N-terminal kinase (JNK/SAPK). PD98059, an inhibitor of ERK-activating kinase MEK-1, and SB203580, an inhibitor of p38 protein kinase, abolished the stimulation of AA release and MAPK activities. In cells stimulated by Aβ(25–35) peptide, Western blotting and confocal microscopy analyses confirmed either an increase in the phosphorylated form of ERKs and p38 or their nuclear translocation. A complete inhibition of MAPK activation and AA release was also observed when pericytes were treated with GF109203X, a general PKC inhibitor, indicating the important role of both PKC and the two MAPKs in mediating the Aβ peptide response. Compared with samples untreated or treated with reverse Aβ(35–25) peptide, pretreatment with 50 μM Aβ(25–35) for 24 h significantly increased the level of constitutively expressed iPLA2 mRNA by 25%, which seems to depend on the activation of kinases. By contrast, the level of cPLA2 mRNA remained unchanged. Together, these data link either the stimulation of PKC-ERK-p38 cascades or PLA2 activity by Aβ peptide to prooxidant mechanism induced by amyloid, which may initially stimulate the cell reaction as well as metabolic repair, such as during inflammation. [Copyright &y& Elsevier]

Abstract: Hexaclorobenzene (HCB), one of the most persistent environmental pollutants, can cause a wide range of toxic effects including cancer in animals, and hepatotoxicity and porphyria both in humans and animals. In the present study, liver microsomal cytochrome P450 (CYP)-dependent arachidonic acid (AA) metabolism, hepatic PGE production, and cytosolic phospholipase A2 (cPLA2) activity were investigated in an experimental model of porphyria cutanea tarda induced by HCB. Female Wistar rats were treated with a single daily dose of HCB (100 mg kg−1 body weight) for 5 days and were sacrificed 3, 10, 17, and 52 days after the last dose. HCB treatment induced the accumulation of hepatic porhyrins from day 17 and increased the activities of liver ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), and aminopyrine N-demethylase (APND) from day 3 after the last dose. Liver microsomes from control and HCB-treated rats generated, in the presence of NADPH, hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatrienoic acids (EETs), 11,12-Di HETE, and ω-OH/ω-1-OH AA. HCB treatment caused an increase in total NADPH CYP-dependent AA metabolism, with a higher response at 3 days after the last HCB dose than at the other time points studied. In addition, HCB treatment markedly enhanced PGE production and release in liver slices. This HCB effect was time dependent and reached its highest level after 10 days. At this time cPLA2 activity was shown to be increased. Unexpectedly, HCB produced a significant decrease in cPLA2 activity on the 17th and 52nd day. Our results demonstrated for the first time that HCB induces both the cyclooxygenase and CYP-dependent AA metabolism. The effects of HCB on AA metabolism were previous to the onset of a marked porphyria and might contribute to different aspects of HCB-induced liver toxicity such as alterations of membrane fluidity and membrane-bound protein function. Observations also suggested that a possible role of cPLA2 in the early increase of AA metabolism cannot be excluded. However, the existence of other pathway(s) for metabolizable AA generation different from cPLA2 activation is also proposed. [Copyright &y& Elsevier]

Cyclooxygenase-2 (COX-2) is a key molecule in the inflammatory pathway in amyotrophic lateral sclerosis (ALS). Cytosolic phospholipase A (cPLA2) is an important enzyme providing substrate for cyclooxygenases. We therefore examined cPLA2 expression in human ALS and mutant Cu/Zn superoxide dismutase (SOD1) transgenic mice and its relation to COX-2. Immunohistochemistry and real-time RT-PCR revealed elevated cPLA2 protein and its mRNA levels in the lumbar spinal cord of mutant SOD1 mice. COX-2 immunoreactivity was increased in lumbar spinal cord sections from both familial ALS (FALS) and sporadic ALS (SALS) as compared to controls, and cPLA2 immunoreactivity was increased in a patient with FALS. Oral administration of the non-selective cyclooxygenase (COX) inhibitor, sulindac, extended the survival (by 10%) of G93A SOD1 mice as compared to littermate controls. Sulindac, as well as the selective COX-2 inhibitors, rofecoxib and celecoxib reduced cPLA2 immunoreactivity in the lumbar spinal cord of G93A transgenic mice. Sulindac treatment preserved motor neurons, and reduced microglial activation and astrocytosis, in the spinal cord of G93A SOD1 transgenic mice. These results suggest that cPLA2 plays an important role in supplying arachidonic acid to the COX-2 driven inflammatory pathway in ALS associated with SOD1 mutations. [ABSTRACT FROM AUTHOR]

We previously demonstrated that brain cyclooxygenase (COX)-2 mRNA and protein levels, and prostaglandin E2 (PGE2) level, are down-regulated in cytosolic phospholipase A2 (cPLA2) -deficient mice. To further investigate the interaction between upstream and downstream enzymes involved in brain prostaglandin synthesis, we examined expression and activity of COX-1, of different PLA2 enzymes and of prostaglandin E synthase (PGES) enzymes in COX-2–/– mice. We found that the PGE2 level was decreased by 51.5% in the COX-2–/– mice brains, indicating a significant role of COX-2 in brain formation of PGE2. However, when we supplied exogenous arachidonic acid (AA) to brain homogenates, COX activity was increased in the COX-2–/– mice, suggesting a compensatory activation of COX-1 and an intracellular compartmentalization of the COX isozymes. Consistent with COX-1 increased activity, brain expression of COX-1 protein and mRNA also was increased. Activity and expression of cPLA2 and secretory PLA2 (sPLA2) enzymes, supplying AA to COX, were significantly increased. Also, the PGE2 biosynthetic pathway downstream from COX-2 was affected in the COX-2–/– mice, as decreased expression of microsomal prostaglandin E synthase-2 (mPGES-2), but not mPGES-1 or cytosolic PGES, was observed. Overall, the data suggest that compensatory mechanisms exist in COX-2–/– mice and that mPGES-2 is functionally coupled with COX-2. [ABSTRACT FROM AUTHOR]

Treatment of bovine pulmonary artery endothelial cells with the calcium ionophore, A23187, stimulates the cell membrane associated protease activity, phospholipase A2 (PLA2) activity, and arachidonic acid (AA) release from the cells. Pretreatment of the cells with arachidonyl-trifluomethylketone (AACOCF3), a cPLA2 inhibitor, but not bromoenollactone (BEL), a iPLA2 inhibitor, prevents A23187 stimulated PLA2 activity and AA release without producing an appreciable alteration of the protease activity. Pretreatment of the cells with aprotinin, an ambient protease inhibitor, prevents the increase in the protease activity and cPLA2 activity in the membrane and AA release from the cells caused by both low and high doses of A23187, and also inhibits protein kinase C (PKC) activity caused by high doses of A23187. Immunoblot study of the endothelial cell membrane isolated from A23187 (10 μM)-treated cells with polyclonal PKCα antibody elicited an increase in the 80 kDa immunoreactive protein band along with an additional 47 kDa immunoreactive fragment. Pretreatment of the cells with aprotinin abolished the 47 kDa immunoreactive fragment in the immunoblot. Immunoblot study of the endothelial membrane with polyclonal cPLA2 antibody revealed that treatment of the cells with A23187 dose-dependently increases cPLA2 immunoreactive protein profile in the membrane. It therefore appears from the present study that treatment of the cells with a low dose of A23187 (1 μM) causes a small increase in an aprotinin-sensitive protease activity and that stimulates cPLA2 activity in the cell membrane without an involvement of PKC. By contrast, treatment of the cells with a high dose of 10 μM of A23187 causes optimum increase in the protease activity and that plays an important role in activating PKCα, which subsequently stimulates cPLA2 activity in the cell membrane. Although pretreatment of the cells with pertussis toxin caused ADP ribosylation of a 41 kDa protein in the cell membrane, it did not inhibit the cPLA2 activity and AA release caused by both low and high doses of A23187. [Copyright &y& Elsevier]

U937, THP-1, and J774 cells or human monocytes and macrophages display similar levels of sensitivity to peroxynitrite and exposure to an otherwise non-toxic concentration of the oxidant in the presence of a phospholipase A2 inhibitor was invariably associated with the onset of mitochondrial permeability transition (MPT)-dependent toxicity. These events were prevented by exogenous arachidonic acid (AA). In general, the protective concentrations of AA were greater in those cell types releasing more AA. Thus, non-toxic concentrations of peroxynitrite commit cells belonging to the monocyte/macrophage lineage to MPT-dependent toxicity that is however prevented by endogenous AA. [Copyright &y& Elsevier]

The present study was aimed at examining the mechanism whereby an otherwise non-toxic concentration of peroxynitrite promotes a rapid necrotic response in U937 cells in which cytosolic phospholipase A2 is pharmacologically inhibited or genetically depleted. We found that loss of viable cells is appreciable 15 min after addition of peroxynitrite, does not further increase at 30 min and is mediated by mitochondrial permeability transition (MPT). Both MPT and toxicity were prevented by exogenous arachidonic acid (AA). Various experimental approaches produced results consistent with the notion that the AA-dependent protective mechanism takes place 10–15 min after addition of peroxynitrite. The observation that the extent of DNA strand scission induced by peroxynitrite did not vary under conditions of different AA availability suggests that this event is either upstream to mitochondrial dysfunction or irrelevant for cytotoxicity. Collectively, these data indicate that a non-toxic concentration of peroxynitrite commits U937 cells to MTP-dependent necrosis that is however prevented by endogenous AA. Thus, mitochondria are a likely target of the cytoprotective signalling triggered by AA. [Copyright &y& Elsevier]

Cytosolic phospholipase A2 (cPLA2) plays an important role in the stimulus-dependent hydrolysis of sn-2 ester bond from membrane phospholipids, releasing arachidonic acid (AA), which along with its metabolites is involved in a number of regulatory functions. The present study examined the effect of methylmercury (MeHg; 0, 2.5, 5.0 μM) on cPLA2 activation in primary hippocampal neurons by assessing the release of 3H-AA. A significant increase in AA release was observed in cultures treated with 5 μM MeHg (10, 30, 60 and 120 min). This effect was due to neuronal cPLA2 activation, since it was completely abolished by arachidonyl trifluoromethyl ketone (AACOCF3), a specific inhibitor of cPLA2. Additional studies confirmed, by means of western blot analysis, that MeHg (5.0 and 10 μM; 16 h) potently increases neuronal cPLA2 protein expression. These results suggest that cPLA2-stimulated hydrolysis and release of AA are potential mediators of MeHg-induced neurotoxicity. [Copyright &y& Elsevier]

Cytosolic phospholipase A2 (cPLA2) stimulates the hydrolysis of sn-2 ester bond in membrane phospholipids releasing arachidonic acid (AA) and lysophospholipids. The present study examined the effect of methylmercury (MeHg) on cPLA2 activation and AA release in primary cultures of neonatal rat cerebral astrocytes. Astrocytes were preloaded overnight at 37 °C with 3H-AA to metabolically label phospholipids. The effect of MeHg on the activation of cPLA2 was measured by the release of 3H-AA from astrocytes over 120 min. MeHg (5 μM) caused a significant increase in AA release at 10, 30, 60, and 120 min, whereas 2.5 μM MeHg significantly increased AA release only at 120 min. MeHg-induced increase in 3H-AA release was due to cPLA2 activation, since arachidonyl trifluoromethyl ketone (AACOCF3), a selective inhibitor of cPLA2, completely abolished MeHg’s effect. Consistent with these observations, MeHg (5.0 and 10.0 μM) increased cPLA2 mRNA (6 h) and cPLA2 protein expression (5.0 and 10.0 μM; 24 h). The time-course of these effects suggests an immediate direct or indirect effect of MeHg on cPLA2 activation and 3H-AA release as well as a long-term effect involving the induction of cPLA2. Thin layer chromatographic analysis of 3H-AA-labeled phospholipids showed that MeHg-stimulated astrocyte 3H-AA release was not due to increased incorporation of 3H-AA into the putative substrates of cPLA2. These results invoke cPLA2 as a putative target for MeHg toxicity, and support the notion that cPLA2-stimulated hydrolysis and release of AA play a critical role in MeHg-induced neurotoxicity. [Copyright &y& Elsevier]

Cyclooxgenase (COX) and phospholipase A2 (PLA2) are crucial rate-limiting enzymes involved in the conversion of arachidonic acid to prostaglandin H2, the precursor of various compounds including prostaglandins (PGs), prostacyclin, and thromboxanes in the process of PGs'' synthesis. Recent studies have shown increased levels of COX2 in adjacent cirrhotic tissue of hepatocellular carcinoma. The relationship between the expression of COX2 or cytosolicPLA2 (cPLA2) and liver fibrosis has not been described previously. We used 45 formalin-fixed, paraffin-embedded liver tissue samples obtained by needle biopsies from patients with chronic hepatitis, consisting of 7 cases of F0, 10 cases of F1, 10 cases of F2, 9 cases of F3 and 9 cases of liver cirrhosis (LC) according to the New Inuyama Classification of the staging of liver fibrosis. The expression of COX2 and cPLA2 was investigated by immunohistochemistry, western blotting and image analysis. The positive signals for COX2 and cPLA2 were observed in the cytoplasm of hepatocytes. The signal intensity of COX2 increased significantly with the progression of liver fibrosis (P<0.001) and no significant difference was observed in the relative amount of cPLA2 from group F0 to group LC. According to the New Inuyama Classification of hepatitis activity grading, 45 samples were classified as group A1 (23 cases), group A2 (19 cases) and group A3 (3 cases). No significant differences were found in the relative amount of COX2 and cPLA2 between group A1 and group A2–3. Significant correlation was observed between the relative amount of COX2 and hyaluronan (P<0.01). Our findings suggested that COX2 may be involved in liver fibrogenesis. [Copyright &y& Elsevier]

Background: The 85-kDa cytosolic phospholipase A2 (cPLA2) mediates arachidonic acid (AA) release in MDCK cells. Although calcium and mitogen-activated protein kinases regulate cPLA2, the correlation of cPLA2 translocation and phosphorylation with MAPK activation and AA release is unclear. Results: MEK1 inhibition by U0126 inhibited AA release in response to ATP and ionomycin. This directly correlated with inhibition of ERK activation but not with phosphorylation of cPLA2 on Ser505, which was only partially inhibited by ERK inhibition. Inhibition of AA release by U0126 was still observed when stoichiometric phosphorylation of cPLA2 on Ser505 was maintained by activating p38 with anisomycin. Translocation kinetics of wild-type cPLA2 and cPLA2 containing S505A or S727A mutations to Golgi were similar in response to ATP and ionomycin and were not affected by U0126. Conclusions: These results suggest that the ability of cPLA2 to hydrolyze membrane phospholipid is reduced by inhibition of the MEK1/ERK pathway and that the reduction in activity is independent of cPLA2 phosphorylation and translocation to membrane. The results also demonstrate that cPLA2 mutated at the phosphorylation sites Ser505 and Ser727 translocated with similar kinetic as wild-type cPLA2. [ABSTRACT FROM AUTHOR]

We have reported that cadmium (Cd) stimulates bone resorption via prostaglandin E2 (PGE2), which is mainly produced in osteoblasts. Prostaglandin (PGs) is regulated by arachidonic acid (AA) release by phospholipase A2 (PLA2) and its conversion to PGs by cyclooxygenase (COX). In the present study, we investigated the possibility that Cd-induced PGE2 synthesis was mediated through PLA2 or COX or both using primary mouse osteoblastic cells in serum-free medium. Cd at 1 microM and above stimulated 14C-AA release from 14C-AA-prelabeled osteoblastic cells. PLA2 activity of cytosolic fraction in Cd-treated cells preferentially hydrolyzed AA at the Sn2 position of phospholipids and was inhibited by arachidonyltrifluoromethyl ketone (AACOCF3), an inhibitor of cytosolic PLA2 (cPLA2). Cd at 1 microM and above increased cPLA2 activity and the level of constitutive cPLA2 mRNA. Secretory PLA2 mRNA was not detected. On the other hand, Cd at 1 microM and above stimulated PGE2 production and its production was inhibited by an inhibitor of COX-2 (NS-398). Cd at 1 microM and above markedly stimulated COX-2 mRNA expression and slightly increased the level of COX-1 mRNA. An inhibitor of COX-1 (varelylsalicylic acid) did not affect Cd-induced PGE2 production. In addition, Cd-induced PGE2 synthesis was inhibited by AA-COCF3, On the other hand, IL-1 alpha, an inducer of COX-2, did not stimulated PGE2 production in present culture system. When IL-1 alpha- or Cd-treated cells were incubated with AA for 10 minutes, IL-1 alpha-treated cells as well as Cd-treated ones caused an increase in PGE2 production. This suggests that the mechanism of Cd-induced PGE2 production is different from that of IL-1 alpha, which may require an activation of cPLA2. From these results, it was found that Cd by itself stimulated PGE2 production by two successive steps that Cd increased cPLA2 activity and then COX-2 induction. [ABSTRACT FROM AUTHOR]