Leukotriene A synthase activity of purified mouse skin arachidonate 8-lipoxygenase expressed in Escherichia coli.
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Mouse skin 8-lipoxygenase was expressed in COS-7 cells by transient transfection of its cDNA in pEF-BOS carrying an elongation factor-1alpha promoter. When crude extract of the transfected COS-7 cells was incubated with arachidonic acid, 8-hydroxy-5,9,11, 14-eicosatetraenoic acid was produced as assessed by reverse- and straight-phase high performance liquid chromatographies. The recombinant enzyme also reacted on alpha-linolenic and docosahexaenoic acids at almost the same rate as that with arachidonic acid. Eicosapentaenoic and gamma-linolenic acids were also oxygenated at 43% and 56% reaction rates of arachidonic acid, respectively. In contrast, linoleic acid was a poor substrate for this enzyme. The 8-lipoxygenase reaction with these fatty acids proceeded almost linearly for 40 min. The 8-lipoxygenase was also expressed in an Escherichia coli system using pQE-32 carrying six histidine residues at N-terminal of the enzyme. The expressed enzyme was purified over 380-fold giving a specific activity of approximately 0.2 micromol/45 min per mg protein by nickel-nitrilotriacetate affinity chromatography. The enzymatic properties of the purified 8-lipoxygenase were essentially the same as those of the enzyme expressed in COS-7 cells. When the purified 8-lipoxygenase was incubated with 5-hydroperoxy-6,8,11, 14-eicosatetraenoic acid, two epimers of 6-trans-leukotriene B4, degradation products of unstable leukotriene A4, were observed upon high performance liquid chromatography. Thus, the 8-lipoxygenase catalyzed synthesis of leukotriene A4 from 5-hydroperoxy fatty acid. Reaction rate of the leukotriene A synthase was approximately 7% of arachidonate 8-lipoxygenation. In contrast to the linear time course of 8-lipoxygenase reaction with arachidonic acid, leukotriene A synthase activity leveled off within 10 min, indicating suicide inactivation. (+info)
Down-regulation of microglial cyclo-oxygenase-2 and inducible nitric oxide synthase expression by lipocortin 1.
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1. Activated microglial cells are believed to play an active role in most brain pathologies, during which they can contribute to host defence and repair but also to the establishment of tissue damage. These actions are largely mediated by microglial secretory products, among which are prostaglandins (PGs) and nitric oxide (NO). 2. The anti-inflammatory protein, lipocortin 1 (LC1) was reported to have neuroprotective action and to be induced by glucocorticoids in several brain structures, with a preferential expression in microglia. In this paper we tested whether the neuroprotective effect of LC1 could be explained by an inhibitory effect on microglial activation. 3. We have previously shown that bacterial endotoxin (LPS) strongly stimulates PGE2 and NO production in rat primary microglial cultures, by inducing the expression of the key enzymes cyclo-oxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), respectively. 4. Dexamethasone (DEX, 1-100 nM) and LC1-derived N-terminus peptide (peptide Ac2-26, 1-100 microg ml(-1)) dose-dependently inhibited the production of both PGE2 and NO from LPS-stimulated microglia. The inhibitory effects of DEX on NO and of the peptide on NO and PGE2 synthesis were partially abrogated by a specific antiserum, raised against the N-terminus of human LC1. The peptide Ac2-26 did not affect arachidonic acid release from control and LPS-stimulated microglial cultures. 5. Western blot experiments showed that the LPS-induced expression of COX-2 and iNOS was effectively down-regulated by DEX (100 nM) and peptide Ac2-26 (100 microg ml(-1)). 6. In conclusion, our findings support the hypothesis that LC1 may foster neuroprotection by limiting microglial activation, through autocrine and paracrine mechanisms. (+info)
Water maze performance is unaffected in artificially reared rats fed diets supplemented with arachidonic acid and docosahexaenoic acid.
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Four groups of male Long-Evans rats were reared artificially from postnatal d 5 to 18 by being fed through a gastrostomy tube with rat milk substitutes containing oils providing 10% linoleic acid and 1% alpha-linolenic acid (g/100 g fat); with the use of a 2 x 2 design, they were fed one of two levels of arachidonic acid (AA) and docosahexaenoic acid (DHA) (0.0 and 2.5 g/100 g of fatty acids). A fifth artificially reared group was fed a diet high in saturated fat, and a sixth group was reared by dams fed a standard AIN-93M diet. The pups were weaned onto modified AIN-93G diets, with a fat composition similar to that fed during the artificial rearing period. Behavioral testing was conducted between 6 and 9 wk of age; brain lipid composition was then assessed. Relative to the unsupplemented group (0.0 g/100 g AA and DHA), dietary supplementation resulted in a wide range of AA (84-103%) and particularly DHA (86-119%) levels in forebrain membrane phospholipids. AA supplementation increased AA levels and decreased DHA levels, and DHA supplementation increased DHA levels and decreased AA levels, with the magnitude of these effects dependent on the level of the other fatty acid. DHA levels were very low in the saturated fat group. The groups did not differ on the place or cued version of the Morris water-maze, but on a test of working memory, the saturated fat group was impaired relative to the suckled control group. Further correlational analyses in the artificially reared animals did not support a relationship between the wide range of DHA and AA levels in the forebrain and working-memory performance. (+info)
Identification of extremely reactive gamma-ketoaldehydes (isolevuglandins) as products of the isoprostane pathway and characterization of their lysyl protein adducts.
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Isoprostanes are prostaglandin-like compounds produced by non-enzymatic peroxidation of arachidonic acid. The cyclooxygenase-derived endoperoxide, prostaglandin H2, can undergo rearrangement to highly reactive gamma-ketoaldehyde secoprostanoids (levuglandin E2 and D2). We explored whether isoprostane endoperoxide intermediates also rearrange to levuglandin-like compounds (isolevuglandins). Formation of a series of isolevuglandins during oxidation of arachidonic acid in vitro was established utilizing a number of mass spectrometric analyses. However, these compounds could not be detected in free form in protein-containing biological systems, which we hypothesized was due to extremely rapid adduction to amines. This was supported by the finding that >60% of levuglandin E2 adducted to albumin within 20 s, whereas approximately 50% of 4-hydroxynonenal still remained unadducted after 1 h. By utilizing electrospray tandem mass spectrometry, we established that these compounds form oxidized pyrrole adducts (lactams and hydroxylactams) with lysine. Formation of isolevuglandin-lysine adducts on apolipoprotein B was readily detected during oxidation of low density lipoprotein following enzymatic digestion of the protein to single amino acids. These studies identify a novel series of extremely reactive products of the isoprostane pathway that rapidly form covalent adducts with lysine residues on proteins. This provides the basis to explore the formation of isolevuglandins in vivo to investigate the potential biological ramifications of their formation in settings of oxidant injury. (+info)
A non-capacitative pathway activated by arachidonic acid is the major Ca2+ entry mechanism in rat A7r5 smooth muscle cells stimulated with low concentrations of vasopressin.
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1. Depletion of the Ca2+ stores of A7r5 cells stimulated Ca2+, though not Sr2+, entry. Vasopressin (AVP) or platelet-derived growth factor (PDGF) stimulated Sr2+ entry. The cells therefore express a capacitative pathway activated by empty stores and a non-capacitative pathway stimulated by receptors; only the former is permeable to Mn2+ and only the latter to Sr2+. 2. Neither empty stores nor inositol 1,4,5-trisphosphate (InsP3) binding to its receptors are required for activation of the non-capacitative pathway, because microinjection of cells with heparin prevented PDGF-evoked Ca2+ mobilization but not Sr2+ entry. 3. Low concentrations of Gd3+ irreversibly blocked capacitative Ca2+ entry without affecting AVP-evoked Sr2+ entry. After inhibition of the capacitative pathway with Gd3+, AVP evoked a substantial increase in cytosolic [Ca2+], confirming that the non-capacitative pathway can evoke a significant increase in cytosolic [Ca2+]. 4. Arachidonic acid mimicked the effect of AVP on Sr2+ entry without stimulating Mn2+ entry; the Sr2+ entry was inhibited by 100 microM Gd3+, but not by 1 microM Gd3+ which completely inhibited capacitative Ca2+ entry. The effects of arachidonic acid did not require its metabolism. 5. AVP-evoked Sr2+ entry was unaffected by isotetrandrine, an inhibitor of G protein-coupled phospholipase A2. U73122, an inhibitor of phosphoinositidase C, inhibited AVP-evoked formation of inositol phosphates and Sr2+ entry. The effects of phorbol esters and Ro31-8220 (a protein kinase C inhibitor) established that protein kinase C did not mediate the effects of AVP on the non-capacitative pathway. An inhibitor of diacylglycerol lipase, RHC-80267, inhibited AVP-evoked Sr2+ entry without affecting capacitative Ca2+ entry or release of Ca2+ stores. 6. Selective inhibition of capacitative Ca2+ entry with Gd3+ revealed that the non-capacitative pathway is the major route for the Ca2+ entry evoked by low AVP concentrations. 7. We conclude that in A7r5 cells, the Ca2+ entry evoked by low concentrations of AVP is mediated largely by a non-capacitative pathway directly regulated by arachidonic acid produced by the sequential activities of phosphoinositidase C and diacylglycerol lipase. (+info)
Interleukin-1 stimulates Jun N-terminal/stress-activated protein kinase by an arachidonate-dependent mechanism in mesangial cells.
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BACKGROUND: We have studied interleukin-1 (IL-1)-stimulated signals and gene expression in mesangial cells (MCs) to identify molecular mechanisms of MC activation, a process characteristic of glomerular inflammation. The JNK1 pathway has been implicated in cell fate decisions, and IL-1 stimulates the Jun N-terminal/stress-activated protein kinases (JNK1/SAPK). However, early postreceptor mechanisms by which IL-1 activates these enzymes remain unclear. Free arachidonic acid (AA) activates several protein kinases, and because IL-1 rapidly stimulates phospholipase A2 (PLA2) activity release AA, IL-1-induced activation of JNK1/SAPK may be mediated by AA release. METHODS: MCs were grown from collagenase-treated glomeruli, and JNK/SAPK activity in MC lysates was determined using an immunocomplex kinase assay. RESULT: Treatment of MCs with IL-1 alpha induced a time-dependent increase in JNK1/SAPK kinase activity, assessed by phosphorylation of the activating transcription factor-2 (ATF-2). Using similar incubation conditions, IL-1 also increased [3H]AA release from MCs. Pretreatment of MCs with aristolochic acid, a PLA2 inhibitor, concordantly reduced IL-1-regulated [3H]AA release and JNK1/SAPK activity, suggesting that cytosolic AA in part mediates IL-1-induced JNK1/SAPK activation. Addition of AA stimulated JNK1/SAPK activity in a time- and concentration-dependent manner. This effect was AA specific, as only AA and its precursor linoleic acid stimulated JNK1/SAPK activity. Other fatty acids failed to activate JNK1/SAPK. Pretreatment of MCs with specific inhibitors of AA oxidation by cyclooxygenase, lipoxygenase, and cytochrome P-450 epoxygenase had no effect on either IL-1- or AA-induced JNK1/SAPK activation. Furthermore, stimulation of MCs with the exogenous cyclooxygenase-, lipoxygenase-, phosphodiesterase-, and epoxygenase-derived arachidonate metabolites, in contrast to AA itself, did not activate JNK1/SAPK. CONCLUSION: We conclude that IL-1-stimulated AA release, in part, mediates stimulation of JNK1/SAPK activity and that AA activates JNK1/SAPK by a mechanism that does not require enzymatic oxygenation. JNK1 signaling pathway components may provide molecular switches that mediate structural rearrangements and biochemical processes characteristic of MC activation and could provide a novel target(s) for therapeutic intervention. (+info)
Relationship of arachidonic acid metabolizing enzyme expression in epithelial cancer cell lines to the growth effect of selective biochemical inhibitors.
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Arachidonic acid (AA) metabolizing enzymes are emerging as significant mediators of growth stimulation for epithelial cells. The relative contribution of the various family members of AA metabolizing enzymes to epithelial cancer cell growth is not known. To study this question, we first analyzed a series of epithelial cancer cells to establish the relative frequency of expression for the various enzymes. We analyzed the expression of five AA metabolizing enzymes as well as 5-lipoxygenase activating protein (FLAP) in a panel of human epithelial cancer cell lines (n = 20) using reverse transcription-PCR. From this analysis, we found that cyclooxygenase-1 (COX-1), 5-lipoxygenase (5-LOX), and FLAP were universally expressed in all cancer cell lines tested. For the remaining enzymes, the expression of COX-2, 12-LOX, and 15-LOX varied among cell lines, 60, 35, and 90%, respectively. Although the pattern of expression varied among the different cell types, all of the enzymes were expressed in all major cancer histologies. Using a panel of selective biochemical AA metabolizing enzyme inhibitors, we then evaluated the effect of these agents on cell lines with known expression status for the AA metabolizing enzymes. For the enzymes that were not universally expressed, growth inhibition by selective biochemical inhibitors did not closely correlate with the expression status of specific enzymes (P > 0.05). For the universally expressed enzymes, the LOX inhibitors were more potent growth inhibitors than the COX inhibitors. The frequent expression of the AA metabolizing enzymes suggests that AA metabolism pathway may be modulated in response to xenobiotic exposure during carcinogenesis. Although establishing a priori AA metabolizing enzyme status was not consistently informative about what AA metabolizing enzyme inhibition would be most growth inhibitory, the frequent inhibition of many epithelial cancers by these biochemical inhibitors opens a new avenue for cancer therapy and intervention in carcinogenesis. (+info)
Arachidonic acid, but not its metabolites, is essential for FcgammaR-stimulated intracellular killing of Staphylococcus aureus by human monocytes.
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Since arachidonic acid (AA) production by phospholipase A2 (PLA2) is essential for the Fcgamma receptor (FcgammaR)-mediated respiratory burst and phagocytosis of opsonized erythrocytes by monocytes and macrophages, we focused in this study on the role of AA and its metabolites in the FcgammaR-stimulated intracellular killing of Staphylococcus aureus by human monocytes. The results revealed that the PLA2 inhibitors, but not inhibitors of cyclo-oxygenase and lipoxygenase, markedly suppressed the FcgammaR-mediated killing process. The production of O-2 by monocytes upon FcgammaR cross-linking was inhibited by 4-bromophenacyl bromide in a dose-dependent fashion, indicating that inhibition of PLA2 activity impairs the oxygen-dependent bactericidal mechanisms of monocytes, which could be partially restored by addition of exogenous AA and docosahexaenoic acid, but not myristic acid. These polyunsaturated fatty acids, but not myristic acid, stimulated the intracellular killing of S. aureus by monocytes, although not as effectively as FcgammaR cross-linking. Furthermore, FcgammaR cross-linking stimulated the release of AA from monocytes. Studies with selective inhibitors revealed that the FcgammaR-mediated activation of PLA2 is dependent on Ca2+ and tyrosine kinase activity. Together these results indicate a key role for PLA2/AA, but not its major metabolites, in mediating the FcgammaR-stimulated intracellular killing of S. aureus by monocytes. (+info)