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The study was aimed at investigating in vivo and in vitro the involvement of the cGMP/cGMP-dependent protein kinase (PKG) signaling pathway in MPP+-induced cytosolic phospholipase A2 (cPLA2) activation of dopaminergic neurons. MPP+ activated neuronal nitric oxide synthase (NOS)/soluble guanylyl cyclase/cGMP pathway in mouse midbrain and striatum, and in pheochromocytoma cell line 12 cells, and caused an upward shift in [Ca2+]i level in the latter. The activation was accompanied by increases in total and phosphorylated cPLA2, and increased arachidonic acid release. Effects of selective inhibitors [2-oxo-1,1,1-trifluoro-6,9-12,15-heneicosatetraene (AACOCF3), (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)2h-pyran-2-one (BEL)] indicated the main impact of cPLA2 on arachidonic acid release in pheochromocytoma cell line 12 cells. Treatment of the cells with the protein kinase inhibitors GF102610x, UO126, and KT5823, and with the nitric oxide synthase (NOS) inhibitor NNLA revealed the involvement of protein kinase C (PKC) and extracellular signal-regulated kinases 1 and 2 (ERK 1/2), with the possible key role of PKG, in cPLA2 phosphorylation at Ser505. Inhibitors of cPLA2 and PKG increased viability and reduced MPP+-induced apoptosis of the cells. Our results indicate that the neuronal NOS/cGMP/PKG pathway stimulates cPLA2 phosphorylation at Ser505 by activating PKC and ERK1/2, and suggest that up-regulation of this pathway in experimental models of Parkinson’s disease may mediate dopaminergic neuron degeneration and death through activation of cPLA2. [ABSTRACT FROM AUTHOR]

Abstract: Up-regulation of cytosolic phospholipase A2 (cPLA2) by cigarette smoke extract (CSE) may play a critical role in airway inflammatory diseases. However, the mechanisms underlying CSE-induced cPLA2 expression in human tracheal smooth muscle cells (HTSMCs) remain unknown. CSE induced cPLA2 protein and mRNA expression, and ROS generation was attenuated by pretreatment with a reactive oxygen species (ROS) scavenger (N-acetylcysteine), or inhibitors of NADPH oxidase (diphenyleneiodonium chloride, apocynin) and transfection with p47 phox siRNA, suggesting that CSE-induced cPLA2 expression was mediated through NADPH oxidase activation and ROS production in HTSMCs. Furthermore, CSE-induced cPLA2 expression was attenuated by pretreatment with the inhibitors of MEK1/2 (U0126), p38 MAPK (SB202190), and JNK (SP600125), which were further confirmed by transfection with siRNAs of JNK1, p42, and p38 to down-regulate the expression of respective proteins and reduce cPLA2 expression. Induction of cPLA2 by CSE was attenuated by selective inhibitors of NF-κB (helenalin) and AP-1 (curcumin). Moreover, promoter assays revealed that increases of cPLA2, NF-κB, and AP-1 luciferase activities stimulated by CSE were attenuated by these inhibitors. These results suggest that in HTSMCs, CSE induced NADPH oxidase activation leading to phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK. These reactions induced nuclear transcription NF-κB and AP-1 activities which were essential for CSE-induced cPLA2 gene expression. [Copyright &y& Elsevier]

Abstract: Both A23187 and formyl-Met-Leu-Phe (fMLP) induced the release of arachidonic acid and the production of thromboxane B2 and leukotriene B4 from rat neutrophils that were inhibited by acetylshikonin in a concentration-dependent manner. Acetylshikonin blocked exogenous arachidonic acid-induced leukotriene B4 and thromboxane B2 production in neutrophils and inhibited the enzymatic activity of ram seminal vesicles cyclooxygenase and human recombinant 5-lipoxygenase, whereas it had no effect on cytosolic phospholipase A2 activity, in cell-free systems. 3-Morpholinosydnonimine- and 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13-HpODE)-mediated dihydrorhodamine 123 oxidation (to assess the lipid peroxide and peroxynitrite scavenging activity) was reduced by acetylshikonin. The membrane recruitment of cytosolic phospholipase A2 was inhibited, but the phosphorylation of cytosolic phospholipase A2 was enhanced, by acetylshikonin in the A23187-induced response. Acetylshikonin alone stimulated extracellular signal regulated kinase (ERK) phosphorylation and enhanced this response in cells stimulated with A23187 and fMLP. The phosphorylation of ERKs and cytosolic phospholipase A2 was attenuated by U0126, a mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor. Acetylshikonin facilitated both A23187- and fMLP-mediated translocation of 5-lipoxygenase to the membrane. Acetylshikonin attenuated both fMLP- and ionomycin-mediated [Ca2+]i elevation. These results indicate that the inhibition of eicosanoid production by acetylshikonin is due to the attenuation of cytosolic phospholipase A2 membrane recruitment via the decrease in [Ca2+]i and to the blockade of cyclooxygenase and 5-lipoxygenase activity. [Copyright &y& Elsevier]

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]

Small intestinal ulcers are frequent complications of therapy with nonsteroidal anti-inflammatory drugs (NSAIDs). We present here a genetic deficiency of eicosanoid biosynthesis that illuminates the mechanism of NSAID-induced ulcers of the small intestine. Eicosanoids and metabolites were measured by isotope dilution with mass spectrometry. cDNA was obtained by reverse transcription and sequenced following amplification with RT-PCR. We investigated the cause of chronic recurrent small intestinal ulcers, small bowel perforations, and gastrointestinal blood loss in a 45-year-old man who was not taking any cyclooxygenase inhibitor. Prostaglandin metabolites in urine were significantly depressed. Serum thromboxane B2 (TxB2) production was 4.6% of normal controls ( P < 0.006), and serum 12-HETE was 1.3% of controls ( P < 0.005). Optical platelet aggregation with simultaneous monitoring of ATP release demonstrated absent granule secretion in response to ADP and a blunted aggregation response to ADP and collagen, but normal response to arachidonic acid (AA). LTB4 biosynthesis by ionophore-activated leukocytes was only 3% of controls, and urinary LTE4 was undetectable. These findings suggested deficient AA release from membrane phospholipids by cytosolic phospholipase A2-α (cPLA2-α), which regulates cyclooxygenase- and lipoxygenase-mediated eicosanoid production by catalyzing the release of their substrate, AA. Sequencing of cPLA2-α cDNA demonstrated two heterozygous nonsynonymous single-base-pair mutations: Ser111Pro (S111P) and Arg485His (R485H), as well as a known single nucleotide polymorphism (SNP), Lys651Arg (K651R). Characterization of this cPLA2-α deficiency provides support for the importance of prostaglandins in protecting small intestinal integrity and indicates that loss of prostaglandin biosynthesis is sufficient to produce small intestinal ulcers. [ABSTRACT FROM AUTHOR]

Background: We have recently reported that tumor necrosis factor (TNF)-α plays an important role in the development of a late anaphylactic reaction, but the downstream pathway beyond TNF-α remains unclear. Objective: It was the aim of this study to examine whether TNF-α induces late-phase anaphylaxis via the activation of cytosolic phospholipase A2 (cPLA2). Methods: Using a murine model of active systemic anaphylaxis to penicillin V, the induction of the late phase of anaphylaxis was quantified by measuring the increase in hematocrit value as well as the plasma level of platelet-activating factor in TNF-α knockout mice. Phosphorylation of mitogen-activated protein kinases (MAPKs) and cPLA2 was measured by immunoprecipitation. cPLA2 activity was assessed by using 1-stearoyl-2-[1-14C] arachidonyl-sn-glycero-3-phosphocholine as the substrate. Results: Phosphorylation and enzymatic activity of cPLA2, and phosphorylation of the 3 known MAPKs, i.e. p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH2-terminal kinase, were markedly increased in a TNF-α-dependent way in the lungs of mice undergoing anaphylaxis. A specific cPLA2 inhibitor significantly attenuated the late anaphylactic symptoms. Either p38 or an ERK inhibitor significantly attenuated not only cPLA2 phosphorylation and activity, but also the late-phase anaphylaxis. Conclusion: TNF-α-induces cPLA2 activation through the pathway involving p38 MAPK and ERK activation and appears to be the key mechanism leading to the development of late-phase anaphylaxis. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Abruquinone A, a natural isoflavanquinone, suppressed A23187- and formyl-Met-Leu-Phe (fMLP)-induced production of thromboxane B2 and leukotriene B4 from rat neutrophils. This compound failed to inhibit the enzymatic activity of ram seminal vesicles cyclooxygenase (COX) and human recombinant 5-lipoxygenase (5-LO) in cell-free systems. Abruquinone A diminished the arachidonic acid release from [3H]arachidonic acid-loaded neutrophils stimulated with either fMLP or A23187, whereas it had no inhibitory effect on the cytosolic phospholipase A2 (cPLA2) activity of neutrophil cytosolic fraction. Based on the Western blot analysis, the nuclear membrane recruitment of cPLA2 and 5-LO was inhibited by abruquinone A in A23187- as well as in fMLP-stimulated cells. Moreover, the phosphorylation of both cPLA2 and extracellular signal regulated kinases (ERKs) induced by fMLP and A23187 was attenuated by abruquinone A in a parallel concentration-dependent manner. Abruquinone A attenuated both fMLP- and ionomycin-mediated [Ca2+]i elevation in a concentration range that inhibited the recruitment of cPLA2 to nuclear membrane. These results indicate that the blockade of leukotriene B4 production by abruquinone A implicates the attenuation of 5-LO membrane translocation. Inhibition of thromboxane B2 production by abruquinone A is due to the attenuation of cPLA2 membrane recruitment and/or cPLA2 phosphorylation through the blockade of [Ca2+]i elevation and ERK activation, respectively. [Copyright &y& Elsevier]

Cytosolic phospholipase A2 (cPLA2) is a key enzyme that mediates arachidonic acid metabolism, which causes cerebral ischemia-induced oxidative injury, blood–brain barrier (BBB) dysfunction, and edema. Recent reports have shown that p38 mitogen-activated protein kinase (MAPK) is related to phosphorylation and activation of cPLA2 and release of arachidonic acid. However, involvement of the p38 MAPK pathway in cPLA2 activation and of reactive oxygen species in expression of p38 MAPK/cPLA2 after ischemia–reperfusion injury in the brain remains unclear. To address these issues, we used a model of transient focal cerebral ischemia (tFCI) in rats. Western blot analysis showed a significant increase in expression of phospho-p38 MAPK and phospho-cPLA2 in rat brain cortex after tFCI. Activity assays showed that both p38 MAPK and cPLA2 activation markedly increased 1 day after reperfusion. Intraventricular administration of SB203580 significantly suppressed activation and phosphorylation of cPLA2 and attenuated BBB extravasation and subsequent edema. Moreover, overexpression of copper/zinc-superoxide dismutase remarkably diminished activation and phosphorylation of both p38 MAPK and cPLA2 after reperfusion. These findings suggest that the p38 MAPK/cPLA2 pathway may promote BBB disruption with secondary vasogenic edema and that superoxide anions can stimulate this pathway after ischemia–reperfusion injury.Journal of Cerebral Blood Flow & Metabolism (2008) 28, 1686–1696; doi:10.1038/jcbfm.2008.60; published online 11 June 2008 [ABSTRACT FROM AUTHOR]

Radiosensitivity of various normal tissues is largely dependent on radiation-triggered signal transduction pathways. Radiation simultaneously initiates distinct signaling from both DNA damage and cell membrane. Specifically, DNA strand breaks initiate cell-cycle delay, strand-break repair or programmed cell death, whereas membrane-derived signaling through phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) enhances cell viability. Here, activation of cytosolic phospholipase A2 (cPLA2) and production of the lipid second-messenger lysophosphatidylcholine were identified as initial events (within 2 min) required for radiation-induced activation of Akt and ERK1/2 in vascular endothelial cells. Inhibition of cPLA2 significantly enhanced radiation-induced cytotoxicity due to an increased number of multinucleated giant cells and cell cycle-independent accumulation of cyclin B1 within 24–48 h of irradiation. Delayed programmed cell death was detected at 72–96 h after treatment. Endothelial functions were also affected by inhibition of cPLA2 during irradiation resulting in attenuated cell migration and tubule formation. The role of cPLA2 in the regulation of radiation-induced activation of Akt and ERK1/2 and cell viability was confirmed using human umbilical vein endothelial cells transfected with shRNA for cPLA2α and cultured embryonic fibroblasts from cPLA2α−/− mice. In summary, an immediate radiation-induced cPLA2-dependent signaling was identified that regulates cell viability and, therefore, represents one of the key regulators of radioresistance of vascular endothelial cells.Cell Death and Differentiation (2008) 15, 1641–1653; doi:10.1038/cdd.2008.93; published online 20 June 2008 [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]

Toxoplasma gondii-derived heat shock protein 70 ( T.g.HSP70) was proven to induce IFN-γ-dependent lethal anaphylactic reaction in T. gondii-infected mice through an alternative PAF-mediated pathway, but not the classical immunoglobulin (Ig)E-dependent pathway. Although marked IFN-γ production was observed by CD11b+, CD11c+, CD4+ and CD8+ splenocytes, CD11b+ and CD11c+ cells were shown to be the key effecter cells which generated pro-inflammatory lipid such as PAF and caused T.g.HSP70-induced anaphylactic reaction. In the present study, we found that the T.g.HSP70-induced anaphylactic reaction was not observed in TLR 4-deficient (−/−) mice, whereas it was observed in WT and TLR2−/− mice. The mRNA expression of PAF-AH, the main enzyme for PAF degradation, increased in T. gondii-infected WT and TLR2−/− but not in TLR4−/− mice after T.g.HSP70 injection. Furthermore, phosphorylation of cPLA2, which is the key enzyme for pro-inflammatory lipid generation, was detected in CD11b+ splenocytes of WT and TLR2−/− mice but not in TLR4−/− mice. Subsequently, cPLA2 activation was suppressed by inhibiting the TLR4-directed p38 and p44/42 MAPK pathways. However, T.g.HSP70-induced anaphylactic reaction was observed in TRIF−/− mice, but not in MyD88−/− mice. These findings indicate the cPLA2 activated-PAF production via TLR4/MyD88-dependent, but not TRIF-dependent, signaling pathway in T.g.HSP70-induced anaphylactic reaction in T. gondii-infected mice. [ABSTRACT FROM AUTHOR]

Abstract: When exposed to enteric pathogens intestinal epithelial cells produce several cytokines and other proinflammatory mediators. To date there is no evidence that the ether-lipid platelet-activating factor (PAF) is one of these mediators. Our results revealed a significant increase in PAF production by human colonic tissue 4 h after infection by enterohemorrhagic Escherichia coli (EHEC) or Salmonella enteritidis. PAF is produced in the gut by cells of the immune system in response to bacterial infection. To determine whether the epithelial cells of colonic mucosa might also modulate PAF levels, we carried out PAF quantification and analysis of the enzymes involved in PAF synthesis in 5-day-old (undifferentiated) or 28-day-old (differentiated) Caco-2 cell cultures. Infection of undifferentiated Caco-2 cells with either bacterium had no effect on PAF levels, whereas in differentiated cells, infection by S. enteritidis increased PAF levels. Following infection by S. enteritidis, there were no changes in the activity of dithiothreitol-insensitive choline phosphotransferase. However, the enzymes of the remodeling pathway cytosolic phospholipase A2, which catalyzes the formation of the PAF precursor lysoPAF, and lysoPAF acetyltransferase, are activated in the infected epithelial cells. This response is Ca2+-dependent. [Copyright &y& Elsevier]

Abstract: We examined the adhesion and proliferation of immortalized endothelial cells GP8.39 (ECs) onto polyethyleneterephtalate (PET) and polyhydroxymethylsiloxane (PHMS) thin films, functionalized by UV-O3 treatment and/or protein immobilization. The modified surface topography showed partial oxidation for both polymers, a slight increase in wettability and monopolar basic character for PET, and a hydrophilic bipolar acid–base behaviour for PHMS. UV-O3 treatment did not induce significant roughness changes (under 1 nm) as shown by atomic force spectroscopy measurements (AFM). The EC adhesion and spreading onto untreated and modified surfaces were investigated both before and after immobilization of collagen (CA) and fibronectin (FN) adlayers. AFM analyses showed an open-weave protein layer on both untreated polymers which became a tight-woven net after UV-O3 irradiation of underlying films. On day 5 after seeding, cell count analyses on irradiated PET surfaces, CA/FN-coated or not, showed EC adhesion and proliferation significantly greater than those on untreated polymers, indicating that UV-O3 irradiation promoted fast endothelialization. A less pronounced EC spreading behaviour on treated PHMS was observed. In ECs grown on irradiated and CA- or FN-coated PET, the levels of phospho-protein kinase Cα (p-PKCα, phospho-ERK1/2, and phospho-cytosolic phospholipase A2 (p-cPLA2), all enzymes taken as signaling markers of cell adhesion and proliferation, decreased in comparison to those in CA- or FN-coated untreated PET. In contrast, in ECs grown on UV-O3-treated PHMS, Western blot analyses showed increased levels of p-PKCα, p-ERK1/2 and p-cPLA2 in comparison with cells grown onto untreated polymer. The growth response of ECs to the substrates was related to the changes of polarity properties of UV-O3-treated polymer films, from hydrophobic/neutral towards hydrophilic/charged layers, and the signaling pathway remodelling to the cell proliferation degree. [Copyright &y& Elsevier]

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]

Activation of P2X3 and P2X2/3 receptors (P2X3R/P2X2/3R), ionotropic ATP receptor subtypes, in primary sensory neurons is involved in neuropathic pain, a debilitating chronic pain that occurs after peripheral nerve injury. However, the underlying mechanisms remain unknown. We investigated the role of cytosolic phospholipase A2 (cPLA2) as a downstream molecule that mediates the P2X3R/P2X2/3R-dependent neuropathic pain. We found that applying ATP to cultured dorsal root ganglion (DRG) neurons increased the level of Ser505-phosphorylated cPLA2 and caused translocation of Ser505-phosphorylated cPLA2 to the plasma membrane. The ATP-induced cPLA2 activation was inhibited by a selective antagonist of P2X3R/P2X2/3R and by a selective inhibitor of cPLA2. In the DRG in vivo, the number of cPLA2-activated neurons was strikingly increased after peripheral nerve injury but not after peripheral inflammation produced by complete Freund’s adjuvant. Pharmacological blockade of P2X3R/P2X2/3R reversed the nerve injury-induced cPLA2 activation in DRG neurons. Moreover, administering the cPLA2 inhibitor near the DRG suppressed nerve injury-induced tactile allodynia, a hallmark of neuropathic pain. Our results suggest that P2X3R/P2X2/3R-dependent cPLA2 activity in primary sensory neurons is a key event in neuropathic pain and that cPLA2 might be a potential target for treating neuropathic pain. [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: NADPH oxidase Nox2 is involved in the production of superoxide by rheumatoid synovial cells, constitutively and after pro-inflammatory cytokine treatment. The aims of the study were to evaluate the capacity of these cells to produce the superoxide anion in response to arachidonic acid (AA), and to study the involvement of cytosolic phospholipase A2 (cPLA2) in the cytokine regulation of Nox2. Superoxide production was quantified in synovial cells obtained from six patients with rheumatoid arthritis (RA) and six with osteoarthritis (OA), stimulated with (i) AA, and (ii) PLA2 inhibitors prior to IL-1β or TNF-α treatment. Total cellular AA concentrations and PLA2 activity were measured; effects of cytokines and NADPH oxidase inhibitors on the AA-activatable proton channel opening were also studied. Our results demonstrated that AA enhanced superoxide production in RA and OA cells; this production was significantly inhibited by iodonium diphenyl and apocynin. cPLA2 inhibitors inhibited both IL-1β and TNF-α-induced superoxide production in RA and OA cells. Basal PLA2 activity was significantly more important in RA cells than in OA cells; PLA2 activity was increased in IL-1β and TNF-α pre-treated RA cells, and cPLA2 inhibitors inhibited this activity. Opening of the AA-activatable proton channel was amplified when RA cells were pre-treated with both IL-1β and TNF-α, and iodonium diphenyl and apocynin inhibited these cytokine effects. We concluded that AA is an important cofactor for synovial NADPH oxidase activity. Despite their direct effects on p47-phox phosphorylation, cytokines can also regulate the Nox2 activity though the AA-activatable associated H+ channel. [Copyright &y& Elsevier]

Background/Aims: Thromboxane A2 (TXA2) has been suggested to play a significant role in the development of portal hypertension in fibrosis, and Kupffer cell (KC) derived TXA2 has been shown to mediate the hyperresponsiveness of the portal circulation to the vasoconstrictive actions of endothelin-1 (ET-1) during endotoxemia. The aim of this study was to determine whether the double stresses of prefibrotic changes and endotoxemia additively activate KC to increase release of TXA2 in response to ET-1, resulting in elevated portal resistance. Methods: One week Bile duct ligation (BDL) rats and sham-operated controls were subjected to isolated liver perfusions following LPS or saline for 6h. In a separate experiment, KC were isolated from BDL or sham rats and incubated with LPS or saline for 6h before the ET-1 treatment. Results: The double stresses of early fibrosis and LPS resulted in a greater sustained increase in portal pressure in response to ET-1 in BDL rats, and this increase correlated well with the much enhanced release of TXA2 in the perfusate. Media from the cultured KC showed significantly greater TXA2 release in response to ET-1 in BDL group than those in sham group, and LPS exacerbated this effect. Protein levels of cytosolic phospholipase A2 (cPLA2), cyclooxygenase-2, and thromboxane synthase were also significantly elevated in KC from BDL rats. ET-1 produced a marked increase in cPLA2 activation as measured by the phosphorylation of cPLA2 in KC of both BDL and sham groups. LPS greatly exacerbated the activation of cPLA2. Conclusions: The data suggest that the double stresses additively activate KC with an upregulation of the key enzymes in the TXA2 biosynthesis and release increased amount of TXA2 via the augmented activation of cPLA2 in response to ET-1, which leads to the increased portal resistance and ultimately hepatic microcirculatory dysfunction. [Copyright &y& Elsevier]

We assessed 1) whether pretreatment before ischemia with pioglitazone (Pio) limits infarct size (IS) and whether this protective effect is due to nitric oxide synthase (NOS) and/or prostaglandin production, as has been shown for atorvastatin (ATV); and 2) whether Pio and ATV have synergistic effects on myocardial protection. Sprague-Dawley rats received oral ATV (10 mg·kg-1·day-1), Pio (10 mg·kg-1·day-1), their combination (Pio + ATV), or water alone for 3 days. Additional rats received Pio (10 mg·kg-1·day-1) for 3 days and intravenous SC-58125 [a cyclooxygenase-2 (COX-2) inhibitor] or SC-560 (a COX-1 inhibitor) 15 min before ischemia. Rats underwent 30 min of myocardial ischemia and 4 h of reperfusion, or hearts were harvested for analysis. IS in the Pio and in the ATV groups was significantly smaller than in the sham-treated group. IS in the Pio + ATV group was smaller than in all other groups (P < 0.001 vs. each group). The protective effect of Pio was abrogated by SC-58125 but not by SC-560. Pio, ATV, and Pio + ATV increased the expression and activity of cytosolic phospholipase A2 (cPLA2) and COX-2. ATV increased phosphorylated-Akt, phosphorylated-endothelial NOS (P-eNOS), inducible NOS, and COX-2 levels. In contrast, Pio caused an insignificant increase in myocardial levels of phosphorylated-Akt but did not change P-eNOS and iNOS expression. In conclusion, the IS-limiting effects of Pio and ATV involve COX-2. However, the upstream steps differ. ATV induced eNOS phosphorylation and iNOS. cPLA2, and COX-2 expression, whereas Pio induced mainly the expression and activity of cPLA2. The effects of Pio and ATV were additive. [ABSTRACT FROM AUTHOR]

Abstract: Disulfiram (an alcohol-aversive drug) and related compounds are known to provoke several side effects involving behavioral and neurological complications. N,N-diethyldithiocarbamate (DDC) is considered as one of the main toxic species of disulfiram and acts as an inhibitor of superoxide dismutase. Since arachidonic acid (AA) formation is regulated by reactive oxygen species (ROS) and related to toxicity in neuronal cells, we investigated the effects of DDC on AA release and expression of the α type of cytosolic phospholipase A2 (cPLA2α) in PC12 cells. Treatment with 80–120 μM DDC that causes a moderate increase in ROS levels without cell toxicity stimulated cPLA2α mRNA and its protein expression. The expression was mediated by extracellular-signal-regulated kinase (ERK1/2), one of the mitogen-activated protein kinases. Treatment with N G nitro-l-arginine methyl ester (an inhibitor of nitric oxide synthase, 1 mM) and oxy-hemoglobin (a scavenger of nitric oxide, 2 mg/mL) abolished the DDC-induced responses (ERK1/2 phosphorylation and cPLA2α expression). We also showed DDC-induced up-regulation of the mRNA expression of lipocortin 1, an inhibitor of PLA2. Furthermore, DDC treatment of the cells enhanced Ca2+-ionophore-induced AA release in 30 min, although the effect was limited. Changes in AA metabolism in DDC-treated cells may have a potential role in mediating neurotoxic actions of disulfiram. In this study, we show the first to demonstrate the up-regulation of cPLA2α expression by DDC treatment in neuronal cells. [Copyright &y& Elsevier]

Abstract: Mortality from prostate cancer is a result of progression of cancer cells to become androgen-refractory and metastatic. Eicosanoid products of the cyclooxygenase (COX) and lipoxygenase (LOX) pathways are important mediators of the proliferation of prostate cancer cells in culture and regulate tumour vascularisation and metastasis in animal models. Pharmacological agents that block either COX or LOX products effectively reduce the size of prostate cancer xenografts. Recently, phospholipase A2 (PLA2) enzymes, which regulate the provision of arachidonic acid to both COX- and LOX-derived eicosanoids, are found to also regulate the growth of prostate cancer cells and tumours, with one enzyme, secreted PLA2-IIA, being increased in prostate cancer tissues. Annexin A1 and A2, known inhibitors of cytosolic phospholipase A2-α activity, are absent in prostate cancer tissues. We propose that PLA2 enzyme function is dysregulated by aberrant up regulation of secreted enzymes and downregulation of endogenous inhibitors of cytosolic phospholipase A2 activity in prostate cancer and that this dysregulation contributes to the pathogenesis of prostate cancer. Thus, in addition to COX and LOX enzymes, PLA2 enzymes represent important targets for the treatment of prostate cancer. [Copyright &y& Elsevier]

Abstract: Cytosolic phospholipase A2 (cPLA2) plays a pivotal role in mediating agonist-induced arachidonic acid (AA) release for prostaglandins (PG) synthesis induced by bacterial lipopolysaccharide (LPS) and cytokines. However, the intracellular signaling pathways mediating LPS-induced cPLA2 expression and PGE2 synthesis in canine tracheal smooth muscle cells (TSMCs) remains unknown. LPS-induced expression of cPLA2 and release of PGE2 was attenuated by inhibitors of tyrosine kinase (genistein), phosphatidylcholine-phospholipase C (D609), phosphatidylinositol-phospholipase C (U73122), PKC (GF109203X and staurosporine), removal of Ca2+ by BAPTA/AM plus EDTA, MEK1/2 (PD98059), p38 (SB202190), JNK (SP600125), and phosphatidylinositol 3-kinase (PI3-K; LY294002 and wortmannin). The involvement of MPAKs in LPS-induced responses was further confirmed by transfection of TSMCs with dominant negative mutants of ERK2 and p38. LPS-induced cPLA2 expression and PGE2 synthesis was inhibited by a selective NF-κB inhibitor (helenalin) and transfection with dominant negative mutants of NF-κB inducing kinase (NIK), IκB kinase (IKK)-α, and IKK-β, consistent with that LPS-stimulated both IκB-α degradation and NF-κB translocation into nucleus in these cells. LPS-stimulated cPLA2 phosphorylation was inhibited by PD98059, GF109203X, and staurosporine, indicating the regulation by p42/p44 MAPK and PKC. Moreover, LPS-induced up-regulation of cPLA2 and COX-2 linked to PGE2 synthesis was inhibited by AACOCF3 (a selective cPLA2 inhibitor), implying the involvement of cPLA2 in these responses. These findings suggest that phosphorylation and expression of cPLA2 correlates with the release of PGE2 from LPS-challenged TSMCs, at least in part, mediated through MAPKs and NF-κB signaling pathways. LPS-mediated responses were modulated by PLC, Ca2+, PKC, tyrosine kinase, and PI3-K in TSMCs. [Copyright &y& Elsevier]

Abstract: Recent data have revealed that soluble oligomeric amyloid-β peptide (Aβ) may be the proximate effectors of neuronal injuries and death in Alzheimer''s disease (AD) by unknown mechanisms. Consistently, we recently demonstrated the critical role of a redox-sensitive cytosolic calcium-dependent phospholipase A2 (cPLA2)-arachidonic acid (AA) pathway in Aβ oligomer-induced cell death. According to the involvement of oxidative stress and polyunsaturated fatty acids like AA in the regulation of sphingomyelinase (SMase) activity, the present study underlines the role of SMases in soluble Aβ-induced apoptosis. Soluble Aβ oligomers induced the activation of both neutral and acidic SMases, as demonstrated by the direct measurement of their enzymatic activities, by the inhibitory effects of both specific neutral and acidic SMase inhibitors, and by gene knockdown using antisense oligonucleotides. Furthermore, soluble Aβ-mediated activation of SMases and subsequent cell death were found to be inhibited by antioxidant molecules and a cPLA2-specific inhibitor or antisense oligonucleotide. We also demonstrate that sphingosine-1-phosphate is a potent neuroprotective factor against soluble Aβ oligomer-induced cell death and apoptosis by inhibiting soluble Aβ-induced activation of acidic sphingomyelinase. These results suggest that Aβ oligomers induce neuronal death by activating neutral and acidic SMases in a redox-sensitive cPLA2-AA pathway. [Copyright &y& Elsevier]

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]

Angiotensin II (ANG II) promotes neointimal growth in the balloon-injured rat carotid artery. However, the mechanism by which ANG II stimulates neointimal growth during vascular injury is not known. In cultured vascular smooth muscle cells, ANG II activates Akt through cytosolic phospholipase A2 (cPLA2)-dependent phospholipase D2 (PLD2). This study was conducted to determine whether ANG II-induced neointimal thickening is mediated via cPLA2- and PLD2-activated Akt in balloon-injured rat carotid arteries. ANG II-stimulated neointimal growth was inhibited by exposure of the injured carotid arteries to an adenovirus containing a dominant negative Akt mutant (intima-to-media ratio from 3.01 ± 0.31 to 1.44 ± 0.14, P < 0.01) or a retrovirus containing cPLA2 small interfering RNA (siRNA; intima-to-media ratio from 3.01 ± 0.31 to 1.16 ± 0.36, P < 0.001) or PLD2 siRNA (intima-to-media ratio from 3.01 ± 0.31 to 1.33 ± 0.11, P < 0.001). The effect of cPLA2 and PLD2 siRNA to reduce the ANG II-induced increase in neointimal thickening was associated with reduced expression of cPLA2 and PLD2 as determined by immunohistochemical analysis in injured carotid arteries. Western blot analysis showed that Akt phosphorylation that was increased by ANG II was inhibited in injured carotid arteries 2 days after exposure to cPLA2 or PLD2 siRNA or in injured arteries isolated after exposure to these agents for 30 mm and then placed in tissue culture media for 24 h in the presence of these agents. These data suggest that the ANG II-induced neointimal growth is mediated by the activation of Akt through a mechanism dependent on cPLA2 and PLD2 activation in balloon-injured rat carotid arteries. [ABSTRACT FROM AUTHOR]

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]

Background: Late-phase airway hyperresponsiveness (AHR) in asthma is considered the event leading to persistent inflammation in the lungs, but the molecular mechanisms involved in this process are poorly understood. Objective: To examine the role of TNF-α in the development of a late AHR and airway inflammation in asthma. Methods: We established a murine model of asthma with not only biphasic AHR to methacholine but also airway eosinophilia. The effect of TNF-α blockade was determined by using anti–TNF-α antibody and TNF-α knockout mice. Cytosolic phospholipase A2 (cPLA2) mRNA expression and activity were assessed by using RT-PCR and 1-stearoyl-2-[1-14C] arachidonyl-sn-glycero-3-phosphocholine as the substrate, respectively. Results: TNF-α blockade resulted in significant inhibition of the late AHR without affecting the early AHR, and reduction in airway eosinophilia and inflammation. cPLA2 activity was increased in asthmatic lungs in a TNF-α–dependent way, and cPLA2 inhibitor blocked late AHR and airway eosinophilia. TNF-α also stimulated the synthesis of cPLA2 metabolites such as leukotriene B4 and platelet-activating factor in the airway. Specific inhibitors of cPLA2 metabolites inhibited the late AHR and airway eosinophilia. Conclusions: TNF-α is the proximal key cytokine capable of developing late-phase AHR and subsequent airway inflammation through expression/activation of cPLA2. [Copyright &y& Elsevier]

Abstract: Experimental evidence suggests that leptin operates on the tissues, including skeletal muscle, also by modulating gene expression. Using electrophoretic mobility shift assays, we have shown that physiological doses of leptin promptly increase the binding of C2C12 cell nuclear extracts to peroxisome proliferator-activated receptor (PPAR) response elements in oligonucleotide probes and that all three PPAR isoforms participate in DNA-binding complexes. We pre-treated C2C12 cells with AACOCF3, a specific inhibitor of cytosolic phospholipase A2 (cPLA2), an enzyme that supplies ligands to PPARs, and found that it abrogates leptin-induced PPAR DNA-binding activity. Leptin treatment significantly increased cPLA2 activity, evaluated as the release of [3H]arachidonic acid from pre-labelled C2C12 cells, as well as phosphorylation. Further, using MEK1 inhibitor PD-98059 we showed that leptin activates cPLA2 through ERK induction. These results support a direct effect of leptin on skeletal muscle cells, and suggest that the hormone may modulate muscle transcription also by precocious activation of PPARs through ERK–cPLA2 pathway. [Copyright &y& Elsevier]

Abstract: Glial activation and inflammation following brain injury may initiate and maintain the process of neurodegeneration. Both glia and neurons synthesize proinflammatory mediators such as interleukin 1 beta (IL-1β), cytosolic phospholipase A2 (cPLA2), cyclooxygenase-2 (COX-2), and prostaglandins. The molecular mechanisms by which IL-1β regulates inflammatory genes such as cPLA2 and COX-2 in glial and neuronal cells are poorly understood. We have studied IL-1β-mediated gene regulation in an established glial and neuronal human cell lines. We report that IL-1β induced cPLA2 and COX-2 mRNA and protein expression and subsequent prostaglandin E2 (PGE2) release in a time-dependent manner in H4 neuroglioma cells. Both SB203580 and PD98059 [p38 and p42/44 mitogen-activated protein kinase (MAPKs) inhibitors, respectively] reduced IL-1β-induced PGE2 production, while only SB203580 reduced both cPLA2 and COX-2 expression. Similarly, in SKNSH neuroblastoma cells, both SB203580 and PD98059 reduced IL-1β-induced PGE2 release, with no detectable COX-2 and cPLA2 protein expression in these cells. Our results indicate that the signaling mechanisms of p38 and p42/44 MAPKs play a role in IL-1β-mediated PGE2 release in both of these cell lines, with differences upstream at the level of cPLA2/COX-2 expression. IL-1β-induced cPLA2 and COX-2 gene expression is modulated through the p38 MAPK pathway in both neuroglioma and neuroblastoma cells. Understanding the signaling mechanisms involved in IL-1β-mediated inflammatory processes in both glia and neuronal cells may provide potential targets for therapeutic intervention for neurological disorders. [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]

We investigated changes in cytosolic phospholipase A2 (cPLA2) and calcium-independent PLA2 (iPLA2) activities in bovine retina capillary pericytes after stimulation with 50 μM amyloid-β (Aβ) (1–42) and its (25–35) fragment, over 24 h (mild, sublethal model of cell damage). In the presence of Aβ peptides, we found that cPLA2 activity was increased and translocated from the cytosolic fraction to the membrane system, particularly in the nuclear region. Reversed-sequence Aβ(35–25) peptide did not stimulate or induce cPLA2 translocation. Exposure to both Aβ peptides had no significant effect on cPLA2 protein content as tested by Western immunoblot analysis. The addition of Aβs to quiescent pericytes was followed by phosphorylation of cPLA2 and arachidonic acid release. Treatment with inhibitors (AACOCF3, staurosporine and cycloheximide) resulted in a sharp decrease in basal and stimulated cPLA2 activity. Inactivating effects of bromoenol lactone (BEL), inhibitor of iPLA2, demonstrated that the stimulation of total PLA2 activity by Aβs was mediated by both PLA2 enzymes. Taken together with our previous observations that both Aβ peptides may induce hydrolysis of phosphatidylcholine, the present results provide evidence that this process is cooperatively mediated by cPLA2 activation/translocation and iPLA2 activation. The effect is very likely triggered by a mild prooxidant mechanism which was not able to divert the cell to degeneration. The data confirm the hypothesis that pericytes could be a target of potential vascular damage and reactivity during processes involving amyloid accumulation. [Copyright &y& Elsevier]

Leptin is a hormone secreted by adipocytes in correlation with total body fat mass. In addition to regulating energy homeostasis, leptin modulates immune functions such as macrophage phagocytosis and cytokine synthesis. Previously, we reported defective leukotriene synthesis in macrophages from leptin-deficient mice that could be restored with exogenous leptin. In the present study, we utilized macrophages from normal rodents to explore the mechanism by which leptin could enhance cellular leukotriene synthesis. Leptin pretreatment of either rat alveolar or murine peritoneal macrophages for 16 h dose dependently increased the synthesis of leukotriene B4 and cysteinyl leukotrienes in response to calcium ionophore or the particulate zymosan. Leptin also enhanced calcium ionophore-stimulated release of free arachidonic acid. Calcium-dependent and -independent arachidonoylselective phospholipase activities in macrophage lysates were likewise increased following leptin treatment. Immunoblot analysis of leptin-treated cells revealed that group IVC iPLA2 (cPLA2γ) protein expression increased ∼80%. These data demonstrate for the first time that phospholipase A2 activity and cPLA2γ protein levels in alveolar macrophages represent targets for upregulation by leptin and provide previously unrecognized mechanisms by which this hormone can promote inflammatory responses. [ABSTRACT FROM AUTHOR]

Exposure to ultraviolet B (UVB) irradiation induces acute skin inflammation such as erythema (sunburn) and edema, and prostaglandin (PG)E2 in the epidermis plays an important role as its prominent mediator. In the present study, we investigated the effect of nobiletin (5,6,7,8,3′,4′-hexamethoxy flavone) from Citrus depressa, on the production of PGE2 in UVB-irradiated human keratinocytes. When keratinocytes were irradiated with 60 mJ of UVB/cm2, the production and gene expression of cyclooxygenase (COX)-2, but not COX-1, were augmented along with an increase in PGE2 levels. The augmented COX-2 production was transcriptionally suppressed by nobiletin. In addition, neither the release of [14C]arachidonic acid from membrane phospholipids nor the gene expression of cytosolic phospholipase A2 (cPLA2) was altered in UVB-irradiated human keratinocytes. However, nobiletin was found to inhibit the release of [14C]arachidonic acid by decreasing the Ca2+-dependent activity of cPLA2. Furthermore, topical treatment of nobiletin on the skin of the back prevented the UVB-induced increase of transepidermal water loss and hyperplasia of the epidermis in hairless mice. Therefore, these results suggest that nobiletin inhibits the UVB-induced production of PGE2 not only by suppressing the expression of COX-2 but also by decreasing the activity of cPLA2 in human keratinocytes. Furthermore, nobiletin may be useful as a novel sunscreen reagent to be applied for protection against photoinflammation and photoaging. [Copyright &y& Elsevier]

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]

A non-radioactive spectrometric assay for the evaluation of inhibitors of cytosolic phospholipase A2 (cPLA2) is described. The enzyme was isolated from human platelets applying anion exchange chromatography. Sonicated covesicles consisting of 0.2 mM 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine and 0.1 mM 1,2-dioleoyl-sn-glycerol were used as enzyme substrate. The cPLA2 activity was determined by measuring the arachidonic acid released by the enzyme with reversed-phase HPLC and UV detection at 200 nm after cleaning up the samples by solid phase extraction. Two known cPLA2 inhibitors were used to validate the test assay. [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]

We have demonstrated that magnolol suppressed thromboxane B2 (TXB2) and leukotriene B4 (LTB4) formation in A23187-stimulated rat neutrophils. Maximum inhibition was obtained with about 10 μM magnolol. Magnolol was more effective in the inhibition of cyclooxygenase (COX) activity than in the inhibition of 5-lipoxygenase (5-LO) activity as assessed by means of enzyme activity determination in vitro and COX and 5-LO metabolic capacity analyses in vivo. Magnolol alone stimulated cytosolic phospholipase A2 (cPLA2) phosphorylation and the translocation of 5-LO and cPLA2 to the membrane, and evoked arachidonic acid (AA) release. Recruitment of both 5-LO and cPLA2 to the membranes was suppressed by EGTA. Arachidonyl trifluoromethyl ketone (AACOCF3), a PLA2 inhibitor, bromoenol lactone (BEL), a Ca2+-independent PLA2 (iPLA2) inhibitor, and EGTA suppressed the magnolol-induced AA release. However, none of the follows affected magnolol-induced AA-release: 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580), a p38 mitogen-activated protein kinase (MAPK) inhibitor, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), a MAPK kinase (MEK) inhibitor, or 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF109203X), a protein kinase C (PKC) inhibitor. In addition, magnolol at 30 μM did not stimulate the p38 MAPK and extracellular signal-regulated kinase 2 (ERK2) enzyme activities. These results indicated that magnolol inhibits the formation of prostaglandins and leukotrienes in A23187-stimulated rat neutrophils, probably through a direct blockade of COX and 5-LO activities. The stimulatory effects of magnolol at high concentration on the membrane association of 5-LO and cPLA2 are attributable to the elevation of [Ca2+]i, and on the AA release is likely via activation of cPLA2 and iPLA2. [Copyright &y& Elsevier]