Lipid body function in eicosanoid synthesis: an update

Eicosanoids (prostaglandins, leukotrienes and lipoxins) are signaling lipids derived from arachidonic acid metabolism that have important roles in physiological and pathological processes. Lately, intracellular compartmentalization of eicosanoid-synthetic machinery has emerged as a key component in the regulation of eicosanoid synthesis and functions. Over the past years substantial progresses have been made demonstrating that precursors and enzymes involved in eicosanoid synthesis localize at lipid bodies (also known as lipid droplets) and lipid bodies are distinct sites for eicosanoid generation. Here we will review the current knowledge on the functions of lipid bodies as specialized intracellular sites of compartmentalization of signaling with major roles in eicosanoid formation within cells engaged in inflammatory, infectious and neoplastic process.     

Phospholipid synthesis in isolated alveolar type II cells exposed in vitro to paraquat and hyperoxia.

Isolated alveolar epithelial type II cells were exposed to paraquat and to hyperoxia by gas diffusion through the thin Teflon bottom of culture dishes. After exposure, type II cells were further incubated in the presence of labelled substrates to assess their capacity to synthesize lipids. Hyperoxia alone (90% O2; 5 h) had minor effects on lipid metabolism in the type II cells. At low paraquat concentrations (5 and 10 microM), hyperoxia enhanced the paraquat-induced decrease of [Me-14C]choline incorporation into phosphatidylcholines. The incorporation rates of [Me-14C]choline, [1-14C]palmitate, [1-14C]glucose and [1,3-3H]glycerol into various phospholipid classes and neutral lipids were decreased by paraquat, depending on the concentration and duration of the exposure. The incorporation of [1-14C]acetate into phosphatidylcholines, phosphatidylglycerols and neutral lipids appeared to be very sensitive to inactivation by paraquat. At 5 microM-paraquat the rate of [1-14C]acetate incorporation was decreased to 50% of the control values. The rate of [1-14C]palmitate incorporation into lipids was much less sensitive; it even increased at low paraquat concentrations. At 10 microM-paraquat both NADPH and ATP were significantly decreased. It is concluded that lipid synthesis in isolated alveolar type II cells is extremely sensitive to paraquat. At low concentrations of this herbicide, lipid synthesis, and particularly fatty acid synthesis, is decreased. The effects on lipid metabolism may be partly related to altered NADPH and ATP concentrations.     

Effects of heavy metal ions on phospholipid metabolism in human neutrophils: relationship to ionophore-mediated cytotoxicity

This study examined the effects of 0.1mM heavy metal ions (Au3+, Zn2+, Cr3+, Mn2+, and Cu2+) on ionophore-treated human neutrophils. Treatment of human neutrophils with 5-10 microM ionophore A23187 resulted in phospholipid deacylation and eicosanoid release within 5 min. After approximately 20 min, viability decreased significantly with near total cell death by 50 min. Heavy metal ions altered phospholipid metabolism, eicosanoid synthesis, and cytotoxicity in parallel fashion. Radioimmunoassays for 5-HETE and LTB4 demonstrated that Au3+ and Zn2+ stimulated, Cr3+ had little effect on, and Mn2+ and Cu2+ inhibited eicosanoid release from ionophore-treated neutrophils. Cells prelabelled with [3H]arachidonic acid exhibited similar metal-mediated effects on lipid metabolism. Strong negative correlations between metal effects on viability and the metabolism of arachidonic acid suggest that eicosanoids participate in ionophore-induced cytotoxicity.     

Calcium ionophore potentiates chemotactic peptide and platelet activating factor in stimulating thromboxane B2 and leukotriene B4 biosynthesis in human neutrophils

Formyl-Met-Leu-Phe (FMLP) and platelet activating factor (PAF) stimulated the synthesis of thromboxane B2 (TXB2) and leukotriene B4 (LTB4) to a small degree in human neutrophils. Calcium ionophore A-23187 enhanced synergistically both FMLP and PAF induced eicosanoid synthesis, whereas phorbol ester PMA attenuated PAF but not FMLP stimulated arachidonate metabolism. These results suggest that calcium mobilization may be a rate limiting step in FMLP and PAF induced synthesis of TXB2 and LTB4 and that protein kinase C activation may play a negative regulatory role in PAF stimulated eicosanoid synthesis.     

Cyclic-AMP mediated drugs: differential or global reduction of eicosanoid synthesis in the isolated rat lung?

In this study the question was addressed whether cAMP mediated drugs induce a differential reduction of branches of the arachidonic acid metabolism rather than a global reduction of eicosanoid synthesis. The isolated lungs of actively sensitized rats were employed to study prostaglandin and leukotriene release in the presence and absence of the cAMP mediated drugs theophylline, milrinone, sulmazole, isobutyl-methylxanthine and salbutamol. The release of eicosanoids as measured by RIA was predominantly basal and continuous, with a mild antigen induced stimulation only for TXB(2) and the leukotrienes. All drugs reduced eicosanoid release globally. It is concluded that cAMP mediated drugs interfere with arachidonic acid metabolism at a site proximal to the branching into lipoxygenase and cyclo-oxygenase pathways.     

Tumour necrosis factor (cachectin) induces phospholipase A2 activity and synthesis of a phospholipase A2-activating protein in endothelial cells.

Tumour necrosis factor (TNF) is an important mediator of endotoxin-induced vascular collapse and other inflammatory reactions. Eicosanoids have been implicated in the pathogeensis of these responses. In order to explore further the potential interactions between TNF and eicosanoid metabolism in eliciting vascular responses, we studied the effects of TNF on the bovine endothelial cell line CPAE. TNF induced cellular retraction observed by light microscope. This morphological change was monitored by the passage of iodinated protein A between adjacent cells and by release of [3H]arachidonic acid metabolites from cells. Both the morphological and functional responses were abrogated by inhibition of eicosanoid synthesis with BW755c. The release of [3H]arachidonic acid metabolites appeared to be mediated by a transient increase in phospholipase A2 activity. Phospholipase C activity was not affected by TNF. The maximal increase in phospholipase A2 activity occurred at 5 min following the addition of TNF. Phospholipase A2 activation, [3H]arachidonic acid-metabolite synthesis and passage of iodinated protein A, required both RNA and protein synthesis and were associated with an increase in the synthesis of a recently described phospholipase A2-activating protein. The Bordetella pertussis toxin, islet-activating protein, also inhibited the increase in phospholipase A2 activity, the release of [3H]arachidonic acid metabolites and the passage of iodinated protein A, suggesting that the TNF receptor-ligand interaction resulting in cellular retraction, phospholipase A2 activation and eicosanoid synthesis, is coupled through the Ni guanine nucleotide regulatory protein in these cells.     

Arachidonic acid metabolism in fibroblasts from patients with peroxisomal diseases: response to interleukin 1

Prostaglandin E2 synthesis and eicosanoid biosynthetic enzyme activities (arachidonyl CoA synthetase, cyclooxygenase and phospholipase A2) were measured in dermal fibroblasts from patients with metabolic disorders of peroxisomal origin and compared to those from normal subjects and patients with other metabolic disorders of lipid metabolism. Basal- as well as interleukin 1-stimulated prostaglandin E2 syntheses were higher in fibroblasts from patients with X-linked adrenoleukodystrophy, the Zellweger cerebrohepatorenal syndrome and rhizomelic chondrodysplasia punctata than in normals. Basal cyclooxygenase and phospholipase A2 activities were elevated in most of the peroxisomal disease cells. Cells from patients with adrenomyeloneuropathy, however, had significantly lower cytokine-stimulated cyclooxygenase and phospholipase A2 activities than normals, as well as lower prostaglandin E2 synthesis in response to interleukin 1. The peroxisomal disease lines exhibited dose-response curves to interleukin 1 similar to controls. Receptor-binding analysis indicated that cells from patients with rhizomelic chondrodysplasia punctata expressed 5-times fewer interleukin 1 receptors than normals and the other disease lines. Exaggerated arachidonic acid metabolism in response to interleukin 1 suggests that cells from patients with peroxisomal enzyme defects may be useful in elucidating pathways for arachidonate release and eicosanoid synthesis.     

Differential regulation of prostaglandin E2 and thromboxane A2 production in human monocytes: implications for the use of cyclooxygenase inhibitors

There is an autocrine relationship between eicosanoid and cytokine synthesis, with the ratio of prostaglandin E2 (PGE2)/thromboxane A2 (TXA2) being one of the determinants of the level of cytokine synthesis. In monocytes, cyclooxygenase type 1 (COX-1) activity appears to favor TXA2 production and COX-2 activity appears to favor PGE2 production. This has led to speculation regarding possible linkage of COX isozymes with PGE and TXA synthase. We have studied the kinetics of PGE2 and TXA2 synthesis under conditions that rely on COX-1 or -2 activity. With small amounts of endogenously generated prostaglandin H2 (PGH2), TXA2 synthesis was greater than PGE2. With greater amounts of endogenously generated PGH2, PGE2 synthesis was greater than TXA2. Also, TXA synthase was saturated at lower substrate concentrations than PGE synthase. This pattern was observed irrespective of whether PGH2 was produced by COX-1 or COX-2 or whether it was added directly. Furthermore, the inhibition of eicosanoid production by the action of nonsteroidal anti-inflammatory drugs or by the prevention of COX-2 induction with the p38 mitogen-activated protein kinase inhibitor SKF86002 was greater for PGE2 than for TXA2. It is proposed that different kinetics of PGE synthase and TXA synthase account for the patterns of production of these eicosanoids in monocytes under a variety of experimental conditions. These properties provide an alternative explanation to notional linkage or compartmentalization of COX-1 or -2 with the respective terminal synthases and that therapeutically induced changes in eicosanoid ratios toward predominance of TXA2 may have unwanted effects in long-term anti-inflammatory and anti-arthritic therapy.     

Paraquat detoxicative system in the mouse liver postmitochondrial fraction

We examined the paraquat detoxicative system in mouse livers. The survival rate of mice receiving 50 mg/kg paraquat was 41% at 7 days and significantly rose to 88, 64, 69% with pretreatment with phenytoin, phenobarbital, and rifampicin, respectively. Phenytoin induced activity in NADPH-cytochrome P450 reductase, CYP3A, CYP2B, and CYP2C that was 3 to 4 times higher than that of the controls. Phenobarbital induced CYP2B and rifampicin induced CYP3A, respectively, in addition to NADPH-cytochrome P450 reductase. 3-Methylcholanthrene did not induce these enzymes and did not alter the survival rate. All the mice pretreated with CoCl(2) (a CYP synthesis inhibitor) or SKF 525-A (a CYP inhibitor) were dead after 5 days, and troleandomycin (a CYP3A-specific inhibitor) also reduced the survival rate. When cell homogenates were incubated with paraquat and NADPH, paraquat decreased and its metabolic intermediate paraquat-monopyridone was formed. Troleandomycin inhibited the decrease in paraquat and increased the monopyridone. After making a subfraction of the homogenate, monopyridone was produced in the postmicrosomal 105,000g supernatant, but not in the microsomes. The pretreatment of mice with phenytoin decreased the monopyridone in the postmitochondrial fraction, but did not affect the supernatant. These results indicated that paraquat was first metabolized in the postmicrosomal supernatant into monopyridone, and that may have been subsequently hydroxylated by the microsomes. Repeated intravenous injections of alpha-tocopherol to paraquat-loaded mice significantly reduced the paraquat mortality and when these mice were pretreated with rifampicin, 100% of them survived. These studies demonstrate that postmitochondrial fractions play an important role in paraquat detoxication metabolism, and that the combination of CYP induction and alpha-tocopherol administration is highly useful for the survival of paraquat-exposed mice.     

Regulation of superoxide dismutase synthesis in Escherichia coli: glucose effect.

Growth of Escherichia coli, based upon the fermentation of glucose, is associated with a low intracellular level of superoxide dismutase. Exhaustion of glucose, or depression of the pH due to accumulation of organic acids, causes these organisms to then obtain energy from the oxidative degradation of other substances present in a rich medium. This shift in metabolism is associated with a marked increase in the rate of synthesis of superoxide dismutase. Depression of the synthesis of superoxide dismutase by glucose is not due to catabolite repression since it is not eliminated by cyclic adenosine 3',5'-monophosphate and since alpha-methyl glucoside does not mimic the effect of glucose. Moreover, glucose itself no longer depresses superoxide dismutase synthesis when the pH has fallen low enough to cause a shift to a non-fermentative metabolism. It appears likely that superoxide dismutase is controlled directly or indirectly by the intracellular level of O2- and that glucose depressed the level of this enzyme because glucose metabolism is not associated with as rapid a production of O2- as is the metabolsim of many other substances. In accord with this view is the observation that paraquat, which can increase the rate of production of O2- by redox cycling, caused a rapid and marked increase in superoxide dismutase.     

Regulation of arachidonic acid metabolism in macrophages by immune and nonimmune interferons

Mouse resident peritoneal M phi release AAA and metabolize it into cyclooxygenase- and lipoxygenase-derived eicosanoids, when triggered in vitro with different stimuli. Pretreatment of M phi with nonimmune IFN-alpha and IFN-beta dramatically decreased AA liberation from M phi phospholipids and eicosanoid formation after stimulation of M phi with Zy, A23187, or PMA. M phi exposed to immune IFN-gamma also showed a substantial impairment of both AA liberation and eicosanoid production upon exposure to Zy. However, AA and eicosanoid release was increased by IFN-gamma, rather than depressed, in PMA-triggered M phi. In addition, IFN-gamma showed differential effects on M phi stimulated with A23187. In fact, it inhibited AA release as well as formation of lipoxygenase-derived LTC4, but it highly increased the release of the cyclooxygenase products PGE2 and 6-keto PGF1 alpha. The ability of IFN-gamma to differentially modulate AA metabolism of M phi, depending on the nature of the triggering agent, sets forth the high specificity of the regulatory capacity of this molecule. This is at variance with the down regulation of AA metabolism that is generally observed with nonimmune IFN.     

Transforming growth factor-beta modulates eicosanoid metabolism in osteogenic osteosarcoma cells

The metabolites of cycloxyegenase and lipoxygenase pathways are known to play an important role in the bone metabolism involving osteoclast and osteoblast interaction, bone resorption and morphogenesis. The recently discovered growth factor TGF-beta is abundant in bone and some of its intracellular and extracellular effects depend on de novo synthesis of eicosanoids. However, the effect of TGF-beta on the synthesis and the release of eicosanoid by bone cells is essentially unknown. In the present study we have identified the main eicosanoid metabolites produced by osteogenic osteosarcoma cell-line SAOS1 and investigated how production and release of these is affected by TGF-beta. We found that the leukotriene C4 is the main metabolite produced by these cells and that TGF-beta induces concentration-dependent, quantitative and qualitative alterations in eicosanoid production and release by human osteogenic osteosarcoma cells SAOS1.     

Ethanol induces release of arachidonic acid but not synthesis of eicosanoids in mouse peritoneal macrophages

Exposure of mouse peritoneal macrophages to ethanol induces a rapid release of arachidonic acid to the extracellular medium. All major classes of phospholipids, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol contribute to this release. Ethanol-induced mobilization of arachidonic acid occurs by deacylation, but it is not accompanied by eicosanoid synthesis. These data suggest that at least two signals are necessary for the release and metabolism of arachidonic acid. Ethanol also activates a phospholipase C which hydrolyzes only phosphatidylinositol, and not its phosphorylated derivatives.     

Dietary unsaturated fatty acids: interactions and possible needs in relation to eicosanoid synthesis

In addition to providing energy and essential fatty acids, dietary fatty acids can affect numerous biochemical and physiologic reactions related to secretory, cardiovascular, and immune functions. The major dietary unsaturated fatty acid, linoleic acid, affects tissue arachidonic acid and can influence eicosanoid-mediated reactions. Chronic, excess, or imbalanced eicosanoid synthesis may be conductive to excessive inflammation, thrombotic tendencies, atherosclerosis, and immune suppression. Dietary n-3 polyunsaturated fatty acids (PUFAs) may ameliorate eicosanoid-related phenomena by reducing tissue arachidonic acid and by inhibiting eicosanoid synthesis. This review summarizes information concerning the metabolism of unsaturated fatty acids, with emphasis on tissue arachidonic acid levels and eicosanoids, and discusses the need for data concerning the appropriate intake of dietary n-6 and n-3 PUFAs to modulate arachidonic acid and eicosanoid synthesis and to minimize possible adverse reactions.     

The stimulation of arachidonic acid metabolism in human platelets by hydrodynamic stresses

Even though shear-induced platelet activation and aggregation have been studied for about 20 years, there remains some controversy concerning the arachidonic acid metabolites formed during stress activation and the role of thromboxane A2 in shear-induced platelet aggregation. In this study, platelets were labelled with [1-14C]arachidonic acid to follow the metabolism of arachidonic acid in stimulated platelets using HPLC and scintillation counting. Platelets activated by thrombin formed principally thromboxane A2, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE). In contrast, for platelets activated by shear--though arachidonic acid metabolism was stimulated--only 12-HETE was formed and essentially no cyclooxygenase metabolites were detected. This indicates that physical forces may initiate a different pathway for eicosanoid metabolism than most commonly used chemical stimuli and perhaps also implies that regulation of the cyclooxygenase activity may be a secondary level of regulation in eicosanoid metabolism.     

Leukocyte lipid bodies — Biogenesis and functions in inflammation

Lipid body accumulation within leukocytes is a common feature in both clinical and experimental infectious, neoplasic and other inflammatory conditions. Here, we will review the contemporary evidence related to the biogenesis and structure of leukocyte lipid bodies (also known as lipid droplets) as inflammatory organelles. Studies of leukocyte lipid bodies are providing functional, ultrastructural and protein compositional evidences that lipid bodies are not solely storage depots of neutral lipid. Over the past years substantial progresses have been made to demonstrate that lipid body biogenesis is a highly regulated process, that culminate in the compartmentalization of a specific set of proteins and lipids, that place leukocyte lipid bodies as inducible cytoplasmic organelles with roles in cell signaling and activation, regulation of lipid metabolism, membrane trafficking and control of the synthesis and secretion of inflammatory mediators. Pertinent to the roles of lipid bodies in inflammation and cell signaling, enzymes involved in eicosanoid synthesis are localized at lipid bodies and lipid bodies are sites for eicosanoid generation. Collectively, lipid bodies in leukocytes are emerging as critical regulators of different inflammatory diseases, key markers of leukocyte activation and attractive targets for novel anti-inflammatory therapies.     

Microsomal interactions between iron, paraquat, and menadione: effect on hydroxyl radical production and alcohol oxidation

The addition of menadione or paraquat to rat liver microsomes resulted in about a threefold increase in the production of hydroxyl radical (.OH) as reflected by the increased oxidation of 2-keto-4-thiomethylbutyric acid (KMBA) to ethylene. This increase was not sensitive to superoxide dismutase but was blocked by catalase. The increase occurred in the absence of added iron and was not affected by the potent iron chelating agent, desferrioxamine, which suggests the possibility that .OH was produced from an interaction between H2O2 and the paraquat or menadione radical. Menadione and paraquat were especially effective in stimulating the oxidation of KMBA in the presence of certain iron chelates such as ferric-ADP, -ATP, or -EDTA, but not ferric-desferrioxamine, -citrate, or -histidine, or unchelated iron. In fact, ferric-ADP or -ATP only stimulated .OH production in the presence of menadione or paraquat. In the presence of ferric-EDTA, the greater than additive increase of .OH production was sensitive to catalase, but not to superoxide dismutase, suggesting the possibility of reduction of ferric-EDTA by paraquat or menadione radical. The interactions with ferric adenine nucleotides may increase the catalytic effectiveness of menadione or paraquat in producing potent oxidants such as the hydroxyl radical, and thus play a role in the toxicity associated with these agents. Paraquat and menadione had little effect on the overall oxidation of ethanol by microsomes. Microsomal drug metabolism was decreased by menadione or paraquat. As a consequence, the effect of these agents on the microsomal oxidation of ethanol was complex since it appeared that paraquat and menadione stimulated the oxidation of ethanol by a .OH-dependent mechanism, but inhibited the oxidation of ethanol by a cytochrome P-450-dependent oxidation pathway. Experiments with carbon monoxide, ferric-EDTA, and 2-butanol plus catalase tended to verify that microsomal oxidation of alcohols was increased by a .OH-dependent pathway when menadione or paraquat were added to microsomes.     

An Integrated Model of Eicosanoid Metabolism and Signaling Based on Lipidomics Flux Analysis

There is increasing evidence for a major and critical involvement of lipids in signal transduction and cellular trafficking, and this has motivated large-scale studies on lipid pathways. The Lipid Metabolites and Pathways Strategy consortium is actively investigating lipid metabolism in mammalian cells and has made available time-course data on various lipids in response to treatment with KDO₂-lipid A (a lipopolysaccharide analog) of macrophage RAW 264.7 cells. The lipids known as eicosanoids play an important role in inflammation. We have reconstructed an integrated network of eicosanoid metabolism and signaling based on the KEGG pathway database and the literature and have developed a kinetic model. A matrix-based approach was used to estimate the rate constants from experimental data and these were further refined using generalized constrained nonlinear optimization. The resulting model fits the experimental data well for all species, and simulated enzyme activities were similar to their literature values. The quantitative model for eicosanoid metabolism that we have developed can be used to design experimental studies utilizing genetic and pharmacological perturbations to probe fluxes in lipid pathways.     

Eicosanoid Release Is Increased by Membrane Destabilization and CFTR Inhibition in Calu-3 Cells

The antiinflammatory protein annexin-1 (ANXA1) and the adaptor S100A10 (p11), inhibit cytosolic phospholipase A2 (cPLA2α) by direct interaction. Since the latter is responsible for the cleavage of arachidonic acid at membrane phospholipids, all three proteins modulate eicosanoid production. We have previously shown the association of ANXA1 expression with that of CFTR, the multifactorial protein mutated in cystic fibrosis. This could in part account for the abnormal inflammatory status characteristic of this disease. We postulated that CFTR participates in the regulation of eicosanoid release by direct interaction with a complex containing ANXA1, p11 and cPLA2α. We first analyzed by plasmon surface resonance the in vitro binding of CFTR to the three proteins. A significant interaction between p11 and the NBD1 domain of CFTR was found. We observed in Calu-3 cells a rapid and partial redistribution of all four proteins in detergent resistant membranes (DRM) induced by TNF-α. This was concomitant with increased IL-8 synthesis and cPLA2α activation, ultimately resulting in eicosanoid (PGE2 and LTB4) overproduction. DRM destabilizing agent methyl-β-cyclodextrin induced further cPLA2α activation and eicosanoid release, but inhibited IL-8 synthesis. We tested in parallel the effect of short exposure of cells to CFTR inhibitors Inh172 and Gly-101. Both inhibitors induced a rapid increase in eicosanoid production. Longer exposure to Inh172 did not increase further eicosanoid release, but inhibited TNF-α-induced relocalization to DRM. These results show that (i) CFTR may form a complex with cPLA2α and ANXA1 via interaction with p11, (ii) CFTR inhibition and DRM disruption induce eicosanoid synthesis, and (iii) suggest that the putative cPLA2/ANXA1/p11/CFTR complex may participate in the modulation of the TNF-α-induced production of eicosanoids, pointing to the importance of membrane composition and CFTR function in the regulation of inflammation mediator synthesis.