The mechanism of enhancement by fatty acid hydroperoxides of anaphylactic mediator release.

Indomethacin augmented the release of histamine and SRS-A but abolished synthesis of TxB2. Compound CLI that inhibited both cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism did not augment release of anaphylactic mediators. 13-HPLA enhanced mediator release from lungs in which arachidonic acid metabolism was blocked by compount CLI. Thus, it is concluded that 13-HPLA enhances mediator release not by altering the balance of arachidonic acid metabolites, e.g. by inhibiting synthesis of prostacyclin, but by a direct effect on lung mast cells. A corollary to this conclusion is that the fatty acid hydroperoxide (HPETE) formed by lipoxygenase from arachidonic acid may also augment the release of anaphylactic mediators. Thus, the enhancement of mediator release by indomethacin may be attributed to increased synthesis of HPETE following inhibition of cyclo-oxygenase.     

The role of marine omega-3 (n-3) fatty acids in inflammatory processes, atherosclerosis and plaque stability

Atherosclerosis has an important inflammatory component and acute cardiovascular events can be initiated by inflammatory processes occurring in advanced plaques. Fatty acids influence inflammation through a variety of mechanisms; many of these are mediated by, or associated with, the fatty acid composition of cell membranes. Human inflammatory cells are typically rich in the n-6 fatty acid arachidonic acid, but the contents of arachidonic acid and of the marine n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) can be altered through oral administration of EPA and DHA. Eicosanoids produced from arachidonic acid have roles in inflammation. EPA also gives rise to eicosanoids and these are usually biologically weak. EPA and DHA give rise to resolvins which are anti-inflammatory and inflammation resolving. EPA and DHA also affect production of peptide mediators of inflammation (adhesion molecules, cytokines, etc.). Thus, the fatty acid composition of human inflammatory cells influences their function; the contents of arachidonic acid, EPA and DHA appear to be especially important. The anti-inflammatory effects of marine n-3 polyunsaturated fatty acids (PUFAs) may contribute to their protective actions towards atherosclerosis and plaque rupture.     

Regulation of arachidonic acid availability for eicosanoid production.

Mammalian cells have developed specific pathways for the incorporation, remodeling, and release of arachidonic acid. Acyltransferase and transacylase pathways function to regulate the levels of esterified arachidonic acid in specific phospholipid pools. There are several distinct, differentially regulated phospholipases A2 in cells that mediate agonist-induced release of arachidonic acid. These pathways are important in controlling cellular levels of free arachidonic acid. Both arachidonic acid and its oxygenated metabolites are potent bioactive mediators that regulate a myriad of physiological and pathophysiological processes.     

Decrease in uptake of arachidonic acid by indomethacin in LS174T human colon cancer cells; a novel cyclooxygenase-2-inhibition-independent effect

Nonsteroidal anti-inflammatory drugs (NSAIDs) have chemopreventive activity and may be suitable for treatment of colorectal cancer. A popular and potent NSAID, indomethacin, is known to cause serious side-effects, for this reason its therapeutic usefulness is limited. However, these side-effects are likely to be attributed to the additional effects of indomethacin besides its cyclooxygenase inhibition. In this study, we examined the effect of indomethacin on arachidonic acid uptake using LS174T human colon cancer cells. We here show that treatment of LS174T cells with indomethacin reduced arachidonic acid uptake as well as reduced expressions of fatty acid translocase/CD36 and peroxisome proliferators-activated receptor γ. Since arachidonic acid is a major substrate of inflammatory mediators such as prostaglandins and leukotrienes, we believe this novel effect of indomethacin may apply to new treatment strategies that aim to suppress these mediators by decreasing the uptake of their substrates, which would eventually inhibit colorectal cancer malignancy.     

Prostaglandin-generating factor of anaphylaxis induces mucous glycoprotein release and the formation of lipoxygenase products of arachidonate from human airways

The effects of prostaglandin-generating factor of anaphylaxis (PGF-A) upon the lipoxygenaton of arachidonic acid and the promotion of mucous glycoprotein secretion by human airways were analyzed concurrently in order to determine the role that lipoxygenase products play in the secretion of mucus which accompanies immediate hypersensitivity reactions of airways. PGF-A enhanced both mucous glycoprotein relesae and the 5- and 15-lipoxygenation of arachidonic acid as well as the formation of leukotrien B₄ (LTB₄) with similar dose-response relationships. The capacity of PGF-A to stimulate mucous glycoprotein release was inhibited by ETYA but not by indomethacin, suggesting that PGF-A stimulated lipoxygenase products may be involved. Lipoxygenase products of arachidonic acid thus may serve as mediators of the enhancement of mucus secretion from human airways in response to PGF-A.     

Leukocyte recognition and metabolism of leukotrienes

The lipoxygenation of arachidonic acid in many different types of cells generates diverse mediators of hypersensitivity and inflammation. The leukotrienes represent one such family of mediators, which exert potent effects on smooth muscle, the microcirculation, and leukocytes. Leukocytes express distinct subsets of receptors for different leukotrienes. Transpeptidatic, peptidolytic, oxidative, and peroxidative pathways of leukocytes contribute substantially to the interconversion and biodegradation of leukotrienes. The 5-lipoxygenation of endogenous arachidonic acid appears to be a critical prerequisite for the activation of the function of leukocytes and some other cells. Natural and pharmacological inhibitors of 5-lipoxygenation in T lymphocytes noncytotoxically suppress the migration and transformation of the lymphocytes in response to antigens and mitogens. Lipoxygenase products of arachidonic acid thus fulfill important roles both as extracellular mediators and as functional intracellular constituents.     

Receptor-mediated action of hepoxilin A3 releases diacylglycerol and arachidonic acid from human neutrophils

We have previously shown that hepoxilin A3 increases the intracellular concentration of Ca+2 in human neutrophils. Herein we address the initial events of hepoxilin action on the neutrophil which precede the rise in intracellular calcium. We show that hepoxilin A3 at 10-1000 nM concentrations releases from [1-14C]-arachidonic acid labeled neutrophils diacylglycerol and unesterified arachidonic acid in a time and concentration dependent fashion. The release of arachidonic acid and diacyglycerol are receptor-mediated events which are blocked by pertussis toxin. This data shows that hepoxilin A3 stimulates phospholipases C and A2 in the cell which may be involved in the rise in cytosolic calcium. Thus, hepoxilins may represent a hitherto unrecognised class of cellular mediators.     

A calcium-dependent mechanism for associating a soluble arachidonoyl-hydrolyzing phospholipase A2 with membrane in the macrophage cell line RAW 264.7

Arachidonoyl-hydrolyzing phospholipase A2 plays a central role in providing substrate for the synthesis of the potent lipid mediators of inflammation, the eicosanoids, and platelet-activating factor. Although Ca2+ is required for arachidonic acid release in vivo and most phospholipase A2 enzymes require Ca2+ for activity in vitro, the role of Ca2+ in phospholipase A2 activation is not understood. We have found that an arachidonoyl-hydrolyzing phospholipase A2 from the macrophage-like cell line, RAW 264.7, exhibits Ca2(+)-dependent association with membrane. The intracellular distribution of the enzyme was studied as a function of the Ca2+ concentration present in homogenization buffer. The enzyme was found almost completely in the 100,000 x g soluble fraction when cells were homogenized in the presence of Ca2+ chelators and there was a slight decrease in soluble fraction activity when cells were homogenized at the level of Ca2+ in an unstimulated cell (80 nM). When cells were homogenized at Ca2+ concentrations expected in stimulated cells (230-450 nM), 60-70% of the phospholipase A2 activity was lost from the soluble fraction and became associated with the particulate fraction in a manner that was partly reversible with EGTA. Membrane-associated phospholipase A2 activity was demonstrated by [3H]arachidonic acid release both from exogenous liposomes and from radiolabeled membranes. With radiolabeled particulate fraction as substrate, this enzyme hydrolyzed arachidonic acid but not oleic acid from membrane phospholipid, and [3H]arachidonic acid was derived from phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol/phosphatidylserine. We suggest a mechanism in which the activity of phospholipase A2 is regulated by Ca2+: in an unstimulated cell phospholipase A2 is found in the cytosol; upon receptor ligation the cytosolic Ca2+ concentration increases, and the enzyme becomes membrane-associated which facilitates arachidonic acid hydrolysis.     

Arachidonic acid stimulates TNFα production in Kupffer cells via a reactive oxygen species-pERK1/2-Egr1-dependent mechanism

Kupffer cells are a key source of mediators of alcohol-induced liver damage such as reactive oxygen species, chemokines, growth factors, and eicosanoids. Since diets rich in polyunsaturated fatty acids are a requirement for the development of alcoholic liver disease, we hypothesized that polyunsaturated fatty acids could synergize with ethanol to promote Kupffer cell activation and TNFα production, hence, contributing to liver injury. Primary Kupffer cells from control and from ethanol-fed rats incubated with arachidonic acid showed similar proliferation rates than nontreated cells; however, arachidonic acid induced phenotypic changes, lipid peroxidation, hydroperoxides, and superoxide radical generation. Similar effects occurred in human Kupffer cells. These events were greater in Kupffer cells from ethanol-fed rats, and antioxidants and inhibitors of arachidonic acid metabolism prevented them. Arachidonic acid treatment increased NADPH oxidase activity. Inhibitors of NADPH oxidase and of arachidonic acid metabolism partially prevented the increase in oxidant stress. Upon arachidonic acid stimulation, there was a rapid and sustained increase in TNFα, which was greater in Kupffer cells from ethanol-fed rats than in Kupffer cells from control rats. Arachidonic acid induced ERK1/2 phosphorylation and nuclear translocation of early growth response-1 (Egr1), and ethanol synergized with arachidonic acid to promote this effect. PD98059, a mitogen extracellular kinase 1/2 inhibitor, and curcumin, an Egr1 inhibitor, blocked the arachidonic acid-mediated upregulation of TNFα in Kupffer cells. This study unveils the mechanism whereby arachidonic acid and ethanol increase TNFα production in Kupffer cells, thus contributing to alcoholic liver disease.     

Reprint of "oxidative alterations of cyclooxygenase during atherogenesis" [Prostag. Oth. Lipid. M. 80 (2006) 1-14

Nitric oxide (*NO) and eicosanoids are critical mediators of physiological and pathophysiological processes. They include inflammation and atherosclerosis. *NO production and eicosanoid synthesis become disrupted during atherosclerosis and thus, it is important to understand the mechanisms that may contribute to this outcome. We, and others, have shown that nitrogen oxide (NOx) species modulate cyclooxygenase (COX; also known as prostaglandin H2 synthase) activity and alter eicosanoid production. We have determined that peroxynitrite (ONOO-) has multiple effects on COX activity. ONOO- can provide the peroxide tone necessary for COX activation, such that simultaneous exposure of COX to its arachidonic acid substrate and ONOO- results in increased eicosanoid production. Alternatively, in the absence of arachidonic acid, ONOO- can modify COX through nitration of an essential tyrosine residue (Tyr385) such that it is incapable of catalysis. In this regard, we have shown that COX nitration occurs in human atherosclerotic tissue and in aortic lesions from ApoE-/- mice kept on a high fat diet. Additionally, we have demonstrated that Tyr nitration in ApoE-/- mice is dependent on the inducible form of NO synthase (iNOS). Under conditions where ONOO- persists and arachidonic acid is not immediately available, the cell may try to correct the situation by responding to ONOO- and releasing arachidonic acid via a signaling pathway to favor COX activation. Other post-translational modifications of COX by NOx species include S-nitrosation of cysteine (Cys) residues (which may have an activating effect) and Cys oxidation. The central focus of this review will include a discussion of how NOx species alter COX activity at the molecular level and how these modifications may contribute to altered eicosanoid output during atherosclerosis and lesion development.     

Stimulation of arachidonic acid metabolism: differences in potencies of recombinant human interleukin-1 alpha and interleukin-1 beta on two cell types

Recombinant human interleukin-l (rIL-1) alpha and beta, which have 26% homology in their amino acid sequence, stimulated arachidonic acid metabolism by squirrel monkey smooth muscle cells and rat liver cells; their relative effectiveness, however, varied with the two cells. Recombinant IL-1 alpha was 3 times more effective than rIL-1 beta at stimulating arachidonic acid metabolism by the primate smooth muscle cells. Recombinant IL-1 alpha was 3 times less effective than rIL-1 beta when measured by their capacity to synergistically stimulate arachidonic acid metabolism of rat liver cells in the presence of palytoxin and anti-diuretic hormone (ADH). The rIL-1 alpha and rIL-1 beta also stimulated the release of radiolabelled arachidonic acid from the smooth muscle cells prelabelled with [3H]arachidonic acid. The two recombinant IL-1s have different heat stabilities, again when measured by their capacity to stimulate arachidonic acid metabolism; IL-1 alpha was more heat stable than IL-1 beta.     

Uptake, release and novel species-dependent oxygenation of arachidonic acid in human and animal airway epithelial cells

To determine identities of mediators and mechanisms for their release from pulmonary airway epithelial cells, we examined the capacities of epithelial cells from human, dog and sheep airways to incorporate, release and oxygenate arachidonic acid. Purified cell suspensions were incubated with radiolabeled arachidonic acid and/or ionophore A23187; fatty acid esterification and hydrolysis were traced chromatographically, and oxygenated metabolites were identified using high-pressure liquid chromatography and mass-spectrometry. In each species, cellular uptake of 10 nM arachidonic acid was concentrated in the phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine fractions, and subsequent incubation with 5 microM A23187 caused release of 10-12% of the radiolabeled pool selectively from phosphatidylcholine and phosphatidylinositol. By contrast, the products of arachidonic acid oxygenation were species-dependent and in the case of human cells were also novel: A23187-stimulated human epithelial cells converted arachidonic acid predominantly to 15-hydroxyeicosatetraenoic acid (15-HETE) and two distinct 8,15-diols in addition to prostaglandin (PG) E2 and PGF2 alpha. Cell incubation with exogenous arachidonic acid (2.0-300 microM) led to progressively larger amounts of 15-HETE and the dihydroxy, epoxyhydroxy and keto acids characteristic of arachidonate 15-lipoxygenase. Both dog and sheep cells converted exogenous or endogenous arachidonic acid to low levels of 5-lipoxygenase products, including leukotriene B4 without significant 15-lipoxygenase activity. In the cyclooxygenase series, sheep cells selectively released PGE2, while dog cells generated predominantly PGD2. The findings demonstrate that stereotyped esterification and phospholipase activities are expressed at uniform levels among airway epithelial cells from these species, but pathways for oxygenating arachidonic acid allow mediator diversity depending greatly on species and little on arachidonic acid presentation.     

Identification of receptors for platelet-activating factor in rat Kupffer cells

Ligand binding studies demonstrated that isolated rat Kupffer cells possess high affinity binding sites for platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, AGEPC). AGEPC binding reached saturation within 10 min at 25 degrees C and was reversible. A Scatchard analysis revealed a single class of AGEPC receptors numbering about 10,600 sites/cell and possessing a dissociation constant of 0.45 nM. Similar values for the dissociation constant for AGEPC (0.12 and 0.34 nM) were obtained independently by kinetic analysis of specific AGEPC binding. AGEPC binding was stereospecific and was inhibited by Zn2+ and AGEPC receptor antagonists including BN52021 and U66985. The AGEPC receptor was functionally active since it was shown to mediate arachidonic acid release and eicosanoid production in Kupffer cells, and these events were inhibited by AGEPC receptor antagonist BN52021. The receptor-mediated arachidonic acid release was extracellular calcium-dependent and was abolished by calcium channel blocker prenylamine and by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, indicating that calcium influx through a receptor-regulated calcium channel in the plasma membrane is involved in the AGEPC-induced arachidonic acid release. It is suggested that rat Kupffer cells have specific and functionally active AGEPC receptors which are involved in signaling mechanisms which govern the production of several other autacoid-type mediators in the liver.     

Oxidative alterations of cyclooxygenase during atherogenesis

Nitric oxide (*NO) and eicosanoids are critical mediators of physiological and pathophysiological processes. They include inflammation and atherosclerosis. *NO production and eicosanoid synthesis become disrupted during atherosclerosis and thus, it is important to understand the mechanisms that may contribute to this outcome. We, and others, have shown that nitrogen oxide (NO(x)) species modulate cyclooxygenase (COX; also known as prostaglandin H(2) synthase) activity and alter eicosanoid production. We have determined that peroxynitrite (ONOO(-)) has multiple effects on COX activity. ONOO(-) can provide the peroxide tone necessary for COX activation, such that simultaneous exposure of COX to its arachidonic acid substrate and ONOO(-) results in increased eicosanoid production. Alternatively, in the absence of arachidonic acid, ONOO(-) can modify COX through nitration of an essential tyrosine residue (Tyr385) such that it is incapable of catalysis. In this regard, we have shown that COX nitration occurs in human atherosclerotic tissue and in aortic lesions from ApoE(-/-) mice kept on a high fat diet. Additionally, we have demonstrated that Tyr nitration in ApoE(-/-) mice is dependent on the inducible form of NO synthase (iNOS). Under conditions where ONOO(-) persists and arachidonic acid is not immediately available, the cell may try to correct the situation by responding to ONOO(-) and releasing arachidonic acid via a signaling pathway to favor COX activation. Other post-translational modifications of COX by NO(x) species include S-nitrosation of cysteine (Cys) residues (which may have an activating effect) and Cys oxidation. The central focus of this review will include a discussion of how NO(x) species alter COX activity at the molecular level and how these modifications may contribute to altered eicosanoid output during atherosclerosis and lesion development.     

Inhibition of phospholipase A(2) as a therapeutic target

The hydrolysis of cell membrane phospholipids by phospholipase A(2) (PLA(2)) leads to the production of numerous lipid mediators of diverse pathological conditions, mainly inflammatory diseases. These include lysophospholipids and their derivatives, and arachidonic acid and its derivatives (the eicosanoids). Both these groups of mediators are produced predominantly by the secretory PLA(2)s (sPLA(2)s) which hydrolyze the phospholipids of the cell surface membrane. Protection of cell membrane from these 'inflammatory enzymes' can therefore be used for the treatment of inflammatory processes. A prototype of cell-impermeable PLA(2) inhibitors, which protect the cell membrane from different sPLA(2)s without affecting vital phospholipid metabolism, is presented and discussed in the present review.     

Arachidonic acid turnover in response to lipopolysaccharide and opsonized zymosan in human monocyte-derived macrophages.

Macrophages are an important source of the lipid mediators, arachidonic acid metabolites and platelet-activating factor (PAF), produced during inflammation. Studies were undertaken to identify the phospholipid substrates that can serve as a source of arachidonic acid in human monocyte-derived macrophages exposed to the inflammatory stimuli bacterial lipopolysaccharide (LPS) and opsonized zymosan (OpZ). Since PAF is derived from 1-alkyl-2-acyl-glycerophosphocholine, it was of interest to determine if this phospholipid precursor could also serve as a source of arachidonic acid. The day-5 macrophages incorporated 38% of the available [3H]arachidonic acid into lipid by 4 h, 54% of which was in phospholipid [phosphatidylcholine (PC) greater than phosphatidylethanolamine (PE) greater than phosphatidylinositol (PI)]. The proportion of label incorporated into ether-linked PC and PE increased with time. After prelabelling with [3H]arachidonic acid, the effect of stimuli on the redistribution of label within phospholipids was followed. Without stimulus there was a loss of label from PC, PI and phosphatidic acid by 3 h, but an increase of label in PE. The [3H]arachidonic acid that was lost from PC in the absence of stimulus was derived solely from the 1-acyl-linked species of PC, whereas an increase in label occurred in the 1-alkyl-linked species of PC. By contrast, LPS stimulation resulted in a preferential, dose-dependent loss of label from PC and PI, which was maximal between 1 and 3 h after adding the LPS. In addition, LPS induced a 35% decrease in the molar quantity of PI in the macrophages but had no effect on the quantity of PC, PE or phosphatidylserine. Stimulation with OpZ also resulted in a loss of label, mainly from PC and PI. Of the total label lost from PC in response to LPS or OpZ, approx. 50% was derived from the 1-alkyl-linked species. The results suggest that phospholipase C- and phospholipase A2-mediated mechanisms for arachidonic acid release are activated in human macrophages exposed to the inflammatory stimuli LPS and OpZ. In addition, 1-alkyl-linked PC can serve as a source of arachidonic acid and as a precursor for PAF production in the stimulated macrophages.     

Diacylglycerol provides arachidonic acid for lipoxygenase products that mediate angiotensin II-induced aldosterone synthesis

The lipoxygenase products of arachidonic acid metabolism have been shown to be important mediators of stimulus secretion coupling in various endocrine tissues. We have recently shown that the 12-lipoxygenase product, 12-hydroxyeicosatetraenoic acid plays a key role as a new specific mediator of angiotensin II-induced aldosterone secretion in the adrenal. In view of the several pathways by which cellular arachidonate can be generated and the important role of diacylglycerol in angiotensin II-responses, we studied the role of diacylglycerol as the source of arachidonic acid for 12-hydroxyeicosatetraenoic formation. Treatment of normal human adrenal glomerulosa cells with the selective diacylglycerol-lipase inhibitor, RHC 80267, resulted in a dose-dependent inhibition of angiotensin II-induced aldosterone as well as 12-hydroxyeicosatetraenoic formation. These results suggest that AA derived from diacylglycerol is the precursor of 12-hydroxyeicosatetraenoic involved in angiotensin II-induced aldosterone secretion. These results reveal a new second messenger role for diacylglycerol in addition to activation of protein kinase C.     

Selective expression of 15-lipoxygenase activity by cultured human keratinocytes

Human keratinocytes isolated from neonatal skin express 15-lipoxygenase activity at a level far greater than that of any of the other pathways for lipoxygenation of arachidonic acid. The 10,000 x g supernatant of sonicates of 10(6) keratinocytes generates 15-hydroxy-eicosatetraenoic acid from 5 micrograms/ml of arachidonic acid at a mean maximum rate of 38 ng/30 min at 37 degrees C, that is similar to the activity of the 15-lipoxygenase of human airway epithelial cells and greater than that of endothelial cells and leukocytes. The unique mediators derived from the 15-lipoxygenation of arachidonic acid, that stimulate secretion and exert hyperalgesic effects, may achieve a concentration in skin sufficient to regulate local cellular and neural functions.     

Cyclooxygenase metabolites are compartmentalized in the human lower respiratory tract

Cyclooxygenase metabolites of arachidonic acid are thought to play an important role in the regulation of diverse physiological functions in the lung. Although the concentration of these metabolites required to have effects is several orders of magnitude greater than the concentration of these mediators in the blood, it has been postulated that local concentrations within tissues are much higher. In a direct test of this hypothesis, the concentrations of the cyclooxygenase products of arachidonic acid including prostaglandin (PG) E, PGF2 alpha, 6-keto-PGF1 alpha, and thromboxane B2, were measured in a specialized tissue compartment, the epithelial surface of the lower respiratory tract. The concentration of these mediators within this compartment was 50- to 80-fold greater than concurrent blood levels and are sufficient to likely have physiological effects. Thus the epithelial surface of the lower respiratory tract represents a specialized compartment with high local levels of cyclooxygenase products of arachidonic acid.