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Menopause is a normal stage in the human female aging process characterized by the cessation of menstruation and the ovarian production of estrogen and progesterone hormones. Menopause is associated with an increased risk of several different diseases. Cardiovascular diseases are generally less common in females than in age-matched males. However, this female advantage is lost after menopause. Cardiac hypertrophy is a disease characterized by increased cardiac size that develops as a response to chronic overload or stress. Similar to other cardiovascular diseases, the risk of cardiac hypertrophy significantly increases after menopause. However, the exact underlying mechanisms are not yet fully elucidated. Several studies have shown that surgical or chemical induction of menopause in experimental animals is associated with cardiac hypertrophy, or aggravates cardiac hypertrophy induced by other stressors. Arachidonic acid (AA) released from the myocardial phospholipids is metabolized by cardiac cytochrome P450 (CYP), cyclooxygenase (COX), and lipoxygenase (LOX) enzymes to produce several eicosanoids. AA-metabolizing enzymes and their respective metabolites play an important role in the pathogenesis of cardiac hypertrophy. Menopause is associated with changes in the cardiovascular levels of CYP, COX, and LOX enzymes and the levels of their metabolites. It is possible that these changes might play a role in the increased risk of cardiac hypertrophy after menopause., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The effect of elicitation with arachidonic and jasmonic acids on the production of phenolic compounds as well as the antioxidant and anti-inflammatory properties of phenolic extracts of wheatgrass was evaluated. The qualitative and quantitative analysis of phenolic compounds carried out with the UPLC-MS technique indicated that luteolin and apigenin derivatives were the dominant flavonoids, while ferulic acid derivatives and syringic acid were the main components in the phenolic acid fraction in the wheatgrass. No qualitative changes in the examined phenolic compounds were observed in the case of the control and elicited plants, while there was an increase in the content of some compounds. The antioxidant activity increased in the elicited samples (with the exception of reducing power) and this elevation was partially correlated with the increase in the polyphenol content in the studied plants. Elicitation with 0.01 µM arachidonic acid also caused improvement of potential anti-inflammatory properties of the wheatgrass., (Copyright © 2019. Published by Elsevier Ltd.)

Asthenozoospermia is a common cause of male infertility, the aetiology of which remains unclear in 50-60% of cases. The current study aimed to characterize metabolic alterations in asthenozoospermic seminal plasma and to explore the signalling pathways involved in sperm motility regulation. At first, high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry was used to detect the targeted metabolic network of arachidonic acid (AA). Metabolomic multivariate data analysis showed significant distinction of AA metabolites between asthenozoospermic and healthy seminal plasma. AA as well as its lipoxygenase (LOX) and cytochrome P450 metabolites were found to be abnormally increased, while cyclooxygenase (COX) metabolites were complicatedly disturbed in asthenozoospermic volunteers compared with those in healthy ones. In vitro experiments and western blot analysis of sperm cells revealed a decrease in sperm motility and upregulation of sperm phosphor-P38 induced by AA. P38 inhibitor could increase AA-reduced sperm motility. Also, all the inhibitors of the three metabolic pathways of AA could block AA-induced P38 mitogen-activated protein kinase (MAPK) activation and further improve sperm motility. We report here for the first time that an abnormal AA metabolic network could reduce sperm motility via P38 MAPK activation through the LOX, cytochrome P450 and COX metabolic pathways, which might be an underlying pathomechanism of asthenozoospermia.

Ethnopharmacological Relevance: Huo Luo Xiao Ling Dan (HLXLD), a traditional Chinese medicine (TCM), is commonly used for the treatment of rheumatoid arthritis (RA)., Aim of the Study: To explore the potential therapeutic mechanism of HLXLD on anti-inflammatory activity., Materials and Methods: A metabolomic approach based on UFLC-MS/MS to profile arachidonic acid (AA) metabolic changes was used. The cyclooxygenase (COX) and lipoxygenase (LOX) catalyzed metabolites in plasma were quantified on 7, 14, 21, and 28 days after the rats injected with Complete Freund's adjuvant and orally administrated with HLXLD, methotrexate and dexamethasone in parallel as the positive control drugs., Results: Nineteen metabolites involved in COX and LOX pathways in RA model group were significant increased compared with normal group (P < 0.05), including 12-hydroxyeicosatetraenoic acid (12-HETE), 15-HETE, 8-HETE, leukotriene B 4 (LTB 4 ), prostaglandin E 2 (PGE 2 ), PGI 2 , PGD 2 , PGF 2α , thromboxane B 2 (TXB 2 ), etc. From day 7 to day 28, the trajectory direction of HLXLD group and positive control groups gradually moved towards the initial space, and the concentrations of AA and its metabolites after HLXLD treatment were significantly reduced in dual pathways compared to control groups., Conclusion: HLXLD induced a substantial change in the AA metabolic profiles through refrain the expression of COX and LOX. The present investigation also highlights that distinct ingredients of this formula tend to inhibit different target to achieve a therapeutic effect., (Copyright © 2018. Published by Elsevier B.V.)

Arachidonic acid (AA, 20:4) is an omega-6 polyunsaturated fatty acid (PUFA) and the main precursor to the class of lipid mediators known as eicosanoids. The enzymes that catalyze the oxygenation of AA begin by abstracting hydrogen from one of three bis-allylic carbons within 1,4-cis,cis-diene units. Substitution of deuterium for hydrogen has been shown to lead to massive kinetic isotope effects (KIE) for soybean lipoxygenase (sLOX) oxygenation of linoleic acid (LA, 18:2). Yet, experimental determination of the KIE during oxygenation of AA and LA by mammalian enzymes including cyclooxygenase (COX) and lipoxygenase (LOX) has revealed far lower values. All prior studies investigating the KIE of PUFA oxygenation have relied on in vitro systems using purified enzymes and were limited by availability of deuterated substrates. Here we demonstrate the use of macrophages as an ex vivo model system to study the physiological KIE (PKIE) during enzymatic AA oxygenation by living cells using a newly synthesized library of deuterated AA isotopologues. By extending lipidomic UPLC-MS/MS approaches to simultaneously quantify native and deuterated lipid products, we were able to demonstrate that the magnitude of the PKIE measured in macrophages for COX and LOX oxygenation of AA is similar to KIEs determined in previous reports using the AA isotopologue deuterated at carbon 13 (C13). However, for the first time we show that increasing the number of deuterated bis-allylic carbons to include both C10 and C13 leads to a massive increase in the PKIE for COX oxygenation of AA. We provide evidence that hydrogen(s) present at C10 of AA play a critical role in the catalysis of prostaglandin and thromboxane synthesis. Furthermore, we discovered that deuteration of C10 promotes the formation of the resolving lipid mediator lipoxin B4, likely by interfering with AA cyclization and shunting AA to the LOX pathway under physiological conditions.

The gene encoding a novel acidic lipoxygenase from Myxococcus xanthus DK1622 (accession: WP&#95;011551853.1) was cloned into vector pET-28a and expressed in Escherichia coli BL21(DE3). The recombinant enzyme (rMxLOX), with a molecular weight of approximately 80 kDa, was purified to homogeneity using one-step nickel-affinity chromatography and showed an activity of 5.6 × 10 4 U/mg. The optimum pH and temperature for rMxLOX activity were found to be 3.0 and 30 °C, respectively. Purified rMxLOX exhibited activity towards linoleic acid and arachidonic acid as substrates, with linoleic acid being the better substrate (K m and k cat values of 0.048 mM and 13.3/s, respectively). The synthetic dye aniline blue was decolorized 69.7 ± 3.5%, following incubation with rMxLOX for 35 min. These results reveal the potential for the use of rMxLOX in the pulp, textile, and wastewater treatment industries., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The differentiation of resident tissue macrophages from embryonic precursors and that of inflammatory macrophages from bone marrow cells leads to macrophage heterogeneity. Further plasticity is displayed through their ability to be polarized as subtypes M1 and M2 in a cell culture microenvironment. However, the detailed regulation of eicosanoid production and its involvement in macrophage biology remains unclear. Using a lipidomics approach, we demonstrated that eicosanoid production profiles between bone marrow-derived (BMDM) and peritoneal macrophages differed drastically. In polarized BMDMs, M1 and M2 phenotypes were distinguished by thromboxane B 2 , prostaglandin (PG) E 2 , and PGD 2 production, in addition to lysophospholipid acyltransferase activity. Although Alox5 expression and the presence of 5-lipoxygenase (5-LO) protein in BMDMs was observed, the absence of leukotrienes production reflected an impairment in 5-LO activity, which could be triggered by addition of exogenous arachidonic acid (AA). The BMDM 5-LO regulatory mechanism was not responsive to PGE 2 /cAMP pathway modulation; however, treatment to reduce glutathione peroxidase activity increased 5-LO metabolite production after AA stimulation. Understanding the relationship between the eicosanoids pathway and macrophage biology may offer novel strategies for macrophage-associated disease therapy.

Most terrestrial plants form green leaf volatiles (GLVs), which are mainly composed of six-carbon (C6) compounds. In our effort to study the distribution of the ability of lipoxygenase (LOX) to form GLVs, we found that a liverwort, Marchantia polymorpha, formed n-hexanal and (Z)-3-hexenal. Some LOXs execute a secondary reaction to form short chain volatiles. One of the LOXs from M. polymorpha (MpLOX7) oxygenized arachidonic and α-linolenic acids at almost equivalent efficiency and formed C6-aldehydes during its catalysis; these are likely formed from hydroperoxides of arachidonic and α-linolenic acids, with a cleavage of the bond between carbon at the base of the hydroperoxy group and carbon of double bond, which is energetically unfavorable. These lines of evidence suggest that one of the LOXs in liverwort employs an unprecedented reaction to form C6 aldehydes as by-products of its reaction with fatty acid substrates.

Background: Oxylipids are oxygenated polyunsaturated fatty acid (PUFA) metabolites that are responsible for the onset and resolution of the inflammatory response. Enzymatic oxygenation through the lipoxygenase (LOX) or cytochrome P450 (CYP) pathways can form oxylipids that have either proinflammatory or proresolving functions depending on the type of PUFA substrate and degree of metabolism. The objective of this study was to determine how PUFA substrates and their corresponding oxylipids are associated with obesity., Methods: Plasma non-esterified FA and oxylipids were isolated from 123 Caucasian males using solid phase extraction and quantified using high performance liquid chromatography-tandem mass spectrometry. Statistical analyses included linear regressions and polytomous logistic regressions, and the responses were body mass index (BMI) and waist circumference (WC), and serum leptin, total adiponectin, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and C-peptide. Models were adjusted for age and smoking, and p-values were corrected for false discovery per Benjamini-Hochberg and Bonferroni., Results: We report that BMI, WC, and several serum cytokines were highly associated arachidonic acid (ARA)-derived hydroxyeicosatetraenoic acids (HETEs), and vicinal diols (i.e., alcohols on adjacent carbon atoms) derived from several PUFAs. There was a significant linear relationship between BMI, WC, and serum leptin, and ARA-derived 5-, 11-, and 15-HETE. Specifically, BMI and WC were positively associated with proinflammatory 5- and 11-hydroxyeicosatetraenoic acid (HETE), even after normalization to ARA concentrations and false discovery p-value correction. Individuals with 5-HETE concentrations >5.01nmol/L or 11-HETE concentrations and >0.89nmol/L were over 5 times more likely to be obese compared to those with ≤1.86nmol/L and ≤0.39nmol/L, respectively. Vicinal diols from linoleic, eicosapentaenoic, and docosahexaenoic acid were inversely associated with obesity. Across all statistical tests, vicinal diols were inversely associated with obesity whether normalized to parent PUFA concentrations or normalized to precursor epoxides. Interestingly, the proinflammatory cytokines IL-6 and TNF-α were not associated with any oxylipids. Since 5-HETE is a 5LOX product, 11-HETE is marker of lipid peroxidation, and vicinal diols are formed through soluble epoxide hydrolase (sEH) metabolism of CYP epoxygenated PUFAs, therefore, these results indicate that obesity is likely associated with altered metabolism with distinct oxygenating pathways. Taken together, our results indicate that obesity is associated with specific oxylipids indicative of altered PUFA metabolism through several pathways (i.e., LOX, reactive oxygen species, and sEH and CYP epoxygenase), rather than attributed solely to altered dietary PUFA intake., (Copyright © 2017 Elsevier Inc. All rights reserved.)

We describe a method for the targeted analysis of bioactive arachidonic acid metabolites through cyclooxygenase (COX) and lipoxygenase (LOX) pathway in knee joint, liver, kidney, spleen and heart using an ultra-fast liquid chromatography-tandem mass (UFLC-MS/MS) method. Method validation was investigated, including linearity, precision, accuracy, matrix effect, extraction recovery and stability for the simultaneous analysis of prostaglandins (PGs), thromboxanes (TXs), leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs). The method enables us to chromatographically separate branched-chain species from their straight-chain isomers as well as separate biologically important eicosanoids. The concentrations of the following major eicosanoids were significantly increased in rheumatoid arthritis model rats than in normal ones: 5-HETE, 8-HETE, 12-HETE, 15-HETE, PGF 2α , TXB 2 , 5-HpETE, LTE 4 , PGE 2 , PGD 2 , LTB 4 . Further multivariate data analysis (partial least square-discriminant analysis) showed COX products (PGs, TXs) were readily distributed towards liver and kidney, LOX products (LTs, HETEs) towards knee joint and spleen, and heart had no characteristic metabolites. The method described here offers a useful tool for the evaluation of complex regulatory eicosanoids responses in RA disease states and provides support for use of dual inhibitors of COX and LOX enzymes on RA treatment., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In this study, we explored the function of arachidonic acid (ARA) in adipogenesis in the grass carp (Ctenopharyngodon idellus) using in vivo and in vitro models. An 8-week feeding trial was performed using three isonitrogenous and isoenergetic purified diets: ARA-free, ARA, and ARA + acetylsalicylic acid [ASA, a cyclooxygenase (COX) inhibitor]. Fish were sampled after 4 and 8 weeks of feeding. Results showed that ARA-fed fish had a significantly lower intraperitoneal fat index (IPFI) and smaller adipocytes; these decreases were reversed by ASA after 8 weeks of feeding. Nevertheless, at week 4, the IPFI and adipocyte size were higher in the ARA group, and they were comparable to those of fish fed ARA + ASA. To further investigate the influence of ARA on adipocyte differentiation, confluent pre-adipocytes of grass carp were incubated with ARA for 3 days. This in vitro experiment demonstrated that ARA promoted adipogenesis in a dose-dependent manner. Pre-treatment with the lipoxygenase (LOX) inhibitor nordihydroguaiaretic acid attenuated the pro-adipogenic function of ARA. However, after treatment with ARA for 8 days, adipocytes had a lower lipid content than cells treated with oleic acid, and ASA could suppress this effect. We thus revealed the dual function of ARA in adipogenesis in grass carp. The LOX pathway may play a key role in pro-adipogenesis after short-term treatment with ARA, whereas the COX pathway is possibly responsible for the inhibition of adipogenesis after long-term treatment.

Zishen pill (ZSP) is a traditional Chinese medicine (TCM) used to treat benign prostatic hyperplasia (BPH). The study used a metabolomic approach based on UHPLC-MS/MS to profile arachidonic acid (AA) metabolic changes and to investigate the interventional mechanisms of ZSP in testosterone- induced BPH rats. In order to explore the potential therapeutic effect of ZSP, rat models were constructed and orally administrated with ZSP. Plasma and urine samples were collected after four weeks and then eleven potential biomarkers (15-HETE, 12-HETE, TXA2, 5-HETE, AA, PGI2, PGF2α, 8-HETE, PGD2, PGE2 and LTB4) were identified and quantified by UHPLC-MS/MS. The chromatographic separation was carried out with gradient elution using a mobile phase comprised of 0.05% formic acid aqueous solution (pH=3.3) (A) and acetonitrile: methanol (80:20, V/V) (B), and each AA metabolites was measured using electrospray ionization source with negative mode and multiple reaction monitoring. The eleven biomarkers in BPH group rat plasma and urine were significant higher than those in sham group rats. Using the potential biomarkers as a screening index, the results suggest that ZSP can potentially reverse the process of BPH by partially regulating AA metabolism through refrain the expression of cyclooxygenase (COX) and lipoxygenase (LOX). This study demonstrates that a metabolomic strategy is useful for identifying potential BPH biomarkers and investigating the underlying mechanisms of a TCM in BPH treatment., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Studying phospholipases A2 (PLA2s) is a challenging task since they act on membrane-like aggregated substrates and not on monomeric phospholipids. Multidisciplinary approaches that include hydrogen/deuterium exchange mass spectrometry (DXMS) and computational techniques have been employed with great success in order to address important questions about the mode of interactions of PLA2 enzymes with membranes, phospholipid substrates and inhibitors. Understanding the interactions of PLA2s is crucial since these enzymes are the upstream regulators of the eicosanoid pathway liberating free arachidonic acid (AA) and other polyunsaturated fatty acids (PUFA). The liberation of AA by PLA2 enzymes sets off a cascade of molecular events that involves downstream regulators such as cyclooxygenase (COX) and lipoxygenase (LOX) metabolites leading to inflammation. Aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) work by inhibiting COX, while Zileuton inhibits LOX and both rely on PLA2 enzymes to provide them with AA. That means PLA2 enzymes can potentially also be targeted to diminish inflammation at an earlier point in the process. In this review we describe extensive efforts reported in the past to define the interactions of PLA2 enzymes with membranes, substrate phospholipids and inhibitors using DXMS, molecular docking, and molecular dynamics (MD) simulations., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The regulation of opioid receptor system function in peripheral sensory neurons is not well understood. Opioid agonist efficacy to inhibit nociceptor function and to promote antinociception is generally weak under basal conditions and frequently no response occurs. However, in response to a cyclooxygenase-dependent metabolite of arachidonic acid (AA) after exposure to inflammatory mediators, such as bradykinin (BK) or exogenous AA, peripheral opioid receptor systems become much more responsive to opioid agonists. In this study, we examined the time course for the induction and maintenance of functional competence of the δ-opioid receptor (DOR) system in adult rat nociceptors in culture and in vivo. We found that the responsive state of DOR after pretreatment with BK or exogenous AA is transient (30-60 minutes) and persists for 15-30 minutes after a 15-minute exposure of nociceptors to BK or AA. Interestingly, whereas functional competence of the DOR system could be reinduced with a second application of BK 60 minutes after the first, responsiveness of the DOR system could not be reinduced after an initial exposure to AA. This nonresponsive state of DOR after exogenous AA was mediated by a lipoxygenase (LOX)-dependent metabolite of AA. Intraplantar carrageenan also produced transient DOR functional competence and responsiveness was also reinduced by inhibition of LOX. Thus, the DOR system expressed by peripheral sensory neurons is under dual regulation by cyclooxygenase- and LOX-dependent metabolites of AA., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Eicosanoids play an important role in inducing complex and crucial physiological processes in animals. Eicosanoid biosynthesis in animals is widely reported; however, eicosanoid production in invertebrate tissue is remarkably different to vertebrates and in certain respects remains elusive. We, for the first time, compared the orthologs involved in arachidonic acid (AA) metabolism in 14 species of invertebrates and 3 species of vertebrates. Based on parsimony, a complex AA-metabolic system may have existed in the common ancestor of the Metazoa, and then expanded and diversified through invertebrate lineages. A primary vertebrate-like AA-metabolic system via cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) pathways was further identified in the basal chordate, amphioxus. The expression profiling of AA-metabolic enzymes and lipidomic analysis of eicosanoid production in the tissues of amphioxus supported our supposition. Thus, we proposed that the ancestral complexity of AA-metabolic network diversified with the different lineages of invertebrates, adapting with the diversity of body plans and ecological opportunity, and arriving at the vertebrate-like pattern in the basal chordate, amphioxus., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Arachidonic acid (ARA) metabolites maintain pregnancy and control parturition. We generated a network of 77 proteins involved in placental ARA metabolism to identify novel proteins in this pathway. We identified a long pathway within this network which showed that secretory and cytosolic phospholipase A2 proteins act in concert. The functions of all network proteins expressed in the placental decidua were determined by database searches. Thus ARA metabolism was linked to carbohydrate metabolism. One protein, transmembrane protein 62 (TMEM62), expressed in decidua was previously uncharacterized, and was identified as a putative lipoxygenase. TMEM62 may play a role in pregnancy and/or parturition., (Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.)

15-Hydroxyeicosatetraenoic acid (HETE), as a mammalian biologically active metabolite, has anticarcinogenic effect. The conditions of producing 15-HETE from arachidonic acid by using soybean lipoxygenase were optimal at pH 8.5 and 20°C with 9 g/l arachidonic acid, 54.4 U/ml soybean lipoxygenase, and 4% methanol. Under these optimized conditions, the enzyme produced 9.5 g/l 15-HETE after 25 min, with a molar conversion yield of 99% and a productivity of 22.8 g l(-1) h(-1). To the best of our knowledge, this is the first biotechnological production of 15-HETE.

Late phase airflow obstruction and reduction in forced vital capacity are characteristic features of human asthma. Airway microvascular leakage and lung edema are also present in the inflammatory phase of asthma, but the impact of this vascular response on lung functions has not been precisely defined. This study was designed to evaluate the role of increased lung microvascular leakage and edema on the late phase changes in forced vital capacity (FVC) and peak expiratory flow (PEF) in allergen-challenged Brown Norway rats using pharmacological inhibitors of the allergic inflammatory response. Rats were sensitized and challenged with ovalbumin aerosol and forced expiratory lung functions (FVC, PEF) and wet and dry lung weights were measured 48 h after antigen challenge. Ovalbumin challenge reduced FVC (63% reduction) and PEF (33% reduction) and increased wet (65% increase) and dry (51% increase) lung weights. The antigen-induced reduction in FVC and PEF was completely inhibited by oral treatment with betamethasone and partially attenuated by inhibitors of arachidonic acid metabolism including indomethacin (cyclooxygenase inhibitor), 7-TM and MK-7246 (CRTH2 antagonists) and montelukast (CysLT1 receptor antagonist). Antagonists of histamine H1 receptors (mepyramine) and 5-HT receptors (methysergide) had no significant effects indicating that these pre-formed mast cell mediators were not involved. There was a highly significant (P < 0.005) correlation for the inhibition of FVC reduction and increase in wet and dry lung weights by these pharmacological agents. These results strongly support the hypothesis that lung microvascular leakage and the associated lung edema contribute to the reduction in forced expiratory lung functions in antigen-challenged Brown Norway rats and identify an important role for the cyclooxygenase and lipoxygenase products of arachidonic acid metabolism in these responses., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Mammalian lipoxygenases (LOXs) play an important role in physiological and pathological processes through the biosynthesis of lipid mediators-leukotrienes, lipoxins and other arachidonic acid derivatives.There are four major families of LOXs that can be analyzed through the production of hydroxyeicosatetraenoic acids (HETEs). No analytical method to detect 5-, 8-, 12- and 15-HETE in one run has been published to date. The HPLC method combines reversed-phase separative column Nucleosil 120-5 C18 and NP column Zorbax Rx.SIL for identification. This conjunction enables separation of 12-HETE and 15-HETE to the baseline which is essential in 12/15-LOX research and elution of all four HETEs in one run. The method was successfully tested on partially purified LOXs from rat lung cytosol., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Increasingly evidence indicates that enzymes, receptors and metabolites of the arachidonic acid (AA) cascade play a role in intestinal epithelial cell proliferation and colorectal tumorigenesis. However, the information available does not provide a complete picture and contains a number of discrepancies. For this reason it might be appropriate a thorough study into the impacts of the AA cascade on intestinal epithelial cell growth. Our data show that non-differentiated Caco-2 cells cultured with 10% fetal bovine serum (FBS) synthesize appreciable amounts of prostaglandin E2 (PGE2), leukotriene B4 (LTB4) and 5-, 12 and 15-hydroxyeicosatetraenoic acid (HETE) but not LTD4, 20-HETE and epoxyeicosatrienoic acids. We also found that inhibitors of PGE2, LTB4 and 5-, 12-, 15-HETE synthesis as well as receptor antagonists of PGE2 and LTB4 blocked Caco-2 cell growth and DNA synthesis induced by 10% FBS without cytotoxic or apoptotic activity. Interestingly, PGE2, LTB4 and 5-, 12- and 15-HETE at concentrations reached in 10% FBS Caco-2 cultures (1-10nM) were able to induce Caco-2 cell growth and DNA synthesis. This was due to the interaction of PGE2 with EP1 and EP4 receptors and LTB4 and HETEs with BLT1 and BLT2 receptors. Moreover, we provide evidence that PGE2 stimulates several cell signaling pathways such as ERK, P38α, CREB and GSKβ/β-catenin involved in the regulation of Caco-2 growth. Finally, we provide evidence that the mitogenic effects of LTB4 and HETEs can be dependent, at least in part, on PGE2 synthesis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The mammalian target of rapamycin (mTOR) signaling pathway is upregulated in the pathogenesis of many cancers. Arachidonic acid (AA) and its metabolites play critical role in the development of breast cancer, but the mechanisms through which AA promotes mammary tumorigenesis and progression are poorly understood. We found that the levels of AA and cytosolic phospholipase A2 (cPLA2) strongly correlated with the signaling activity of mTORC1 and mTORC2 as well as the expression levels of vascular epithelial growth factor (VEGF) in human breast tumor tissues. In cultured breast cancer cells, AA effectively activated both mTOR complex 1 (mTORC1) and mTORC2. Interestingly, AA-stimulated mTORC1 activation was independent of amino acids, phosphatidylinositol 3-kinase (PI3-K) and tuberous sclerosis complex 2 (TSC2), which suggests a novel mechanism for mTORC1 activation. Further studies revealed that AA stimulated mTORC1 activity through destabilization of mTOR-raptor association in ras homolog enriched in brain (Rheb)-dependent mechanism. Moreover, we showed that AA-stimulated cell proliferation and angiogenesis required mTOR activity and that the effect of AA was mediated by lipoxygenase (LOX) but not cyclooxygenase-2 (COX-2). In animal models, AA-enhanced incidences of rat mammary tumorigenesis, tumor weights and angiogenesis were inhibited by rapamycin. Our findings suggest that AA is an effective intracellular stimulus of mTOR and that AA-activated mTOR plays critical roles in angiogenesis and tumorigenesis of breast cancer.

The purpose of the study was to examine whether arachidonic acid inhibits vascular Na(+)-K(+)-ATPase in pulmonary vasculature and if so, what are the mechanisms involved. Functional Na(+)-K(+)-ATPase activity was studied in terms of K(+)-induced relaxation in sheep pulmonary arterial rings contracted with K(+)-free solution and 5-HT. Arachidonic acid (10-100 μM) caused concentration-dependent inhibition of KCl-induced relaxations and also increased basal arterial tone. Cytochrome P-450 inhibitor, 17-octadecynoic acid (17-ODYA) completely reversed the arachidonic acid (30 μM)-induced inhibition of KCl relaxation. Further, in the presence of HET0016, a selective blocker of 20-hydroxyeicosatetraenoic acid (20-HETE), arachidonic acid-induced inhibition of KCl relaxation was not evident. Accordingly, 20-HETE, a cytochrome P-450 metabolite of arachidonic acid, also significantly attenuated KCl-induced relaxations. Norhydihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, however, partially restored the relaxation to K(+), impaired in the presence of arachidonic acid (30 μM). On the other hand, cyclooxygenase inhibitor indomethacin failed to reverse the inhibitory effect of arachidonic acid on KCl-induced relaxation. Staurosporin, a protein kinase C inhibitor, completely reversed the inhibitory effect of arachidonic acid and 20-HETE on K(+)-induced relaxation. In conclusion, the results suggest that 20-HETE, a cytochrome P-450 metabolite of arachidonic acid has a predominant role in the inhibition of functional Na(+)-K(+)-ATPase activity in the sheep pulmonary artery, while the lipooxygenase pathway has a secondary role. It is also evident that protein kinase C is involved in the inhibition of Na(+)-K(+)-ATPase by arachidonic acid/20-HETE in sheep pulmonary artery., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The present study was designed to clarify whether the arachidonic acid cascade contributes to the decreased threshold for pentylenetetrazole-induced seizure under benzodiazepine withdrawal in mice. The seizure threshold for pentylenetetrazole was significantly decreased by the discontinuation of chronic treatment with diazepam. The decrease in the seizure threshold for pentylenetetrazole during diazepam withdrawal was significantly suppressed by intracerebroventricular (i.c.v.) pretreatment with the phospholipase A(2) inhibitor quinacrine (30, 100 nmol) and the lipoxygenase inhibitor nordihydroguaiaretic acid (10, 30 nmol). In contrast, the decreased seizure threshold in the diazepam-withdrawal group was intensified by pretreatment with the cyclooxygenase inhibitor diclofenac (56 nmol). These compounds did not alter the threshold for seizure in a control group. These findings suggest that enhancement of the arachidonic acid cascade may contribute to the hypersusceptibility to pentylenetetrazole-induced seizure during diazepam withdrawal.

Animal lipoxygenases (LOXs) are classified according to their specificity of arachidonic acid oxygenation, and previous sequence alignments suggested that S-LOXs contain a conserved Ala at a critical position at the active site but R-LOXs carry a Gly instead. Here we cloned, expressed, and characterized a novel LOX isoform from the model vertebrate Danio rerio (zebrafish) that carries a Gly at this critical position, classifying this enzyme as putative arachidonic acid R-LOX. Surprisingly, the almost exclusive arachidonic acid oxygenation product was 12S-H(p)ETE (hydro(pero)xyeicosatetraenoic acid), and extensive mutation around Gly-410 failed to induce R-lipoxygenation. This finding prompted us to explore the importance of the corresponding amino acids in other vertebrate S-LOXs. We found that Ala-to-Gly exchange in human 15-LOX2 and human platelet 12-LOX induced major alterations in the reaction specificity with an increase of specific R-oxygenation products. For mouse 5-LOX and 12/15-LOX from rabbits, men, rhesus monkeys, orangutans, and mice, only minor alterations in the reaction specificity were observed. For these enzymes, S-HETE (hydroxyeicosatetraenoic acid) isomers remained the major oxygenation products, whereas chiral R-HETEs contributed only 10-30% to the total product mixture. Taken together these data indicate that the Ala-versus-Gly concept may not always predict the reaction specificity of vertebrate LOX isoforms.

Although the importance of the macrophage complement receptor immunoglobulin (CRIg) in the phagocytosis of complement opsonized bacteria and in inflammation has been established, the regulation of CRIg expression remains undefined. Because cellular activation during inflammation leads to the release of arachidonate, a stimulator of leukocyte function, we sought to determine whether arachidonate regulates CRIg expression. Adding arachidonate to maturing human macrophages and to prematured CRIg(+) macrophages caused a significant decrease in the expression of cell-surface CRIg and CRIg mRNA. This effect was independent of the metabolism of arachidonate via the cyclooxygenase and lipoxygenase pathways, because it was not inhibited by the nonsteroidal anti-inflammatory drugs indomethacin and nordihydroguaiaretic acid. Studies with specific pharmacological inhibitors of arachidonate-mediated signaling pathways showed that protein kinase C was involved. Administration of dexamethasone to macrophages caused an increase in CRIg expression. Studies with proinflammatory and immunosuppressive cytokines showed that IL-10 increased, but interferon-γ, IL-4, and transforming growth factor-β1 decreased CRIg expression on macrophages. This down- and up-regulation of CRIg expression was reflected in a decrease and increase, respectively, in the phagocytosis of complement opsonized Candida albicans. These data suggest that a unique inflammatory mediator network regulates CRIg expression and point to a mechanism by which arachidonate and dexamethasone have reciprocal effects on inflammation., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Head and neck cancers are known to synthesize arachidonic acid metabolites. Interfering with arachidonic acid metabolism may inhibit growth and invasiveness of cancer cells. In this study we investigate effects of sulindac (the non-selective COX inhibitor), aspirin (the irreversible, preferential COX-1 inhibitor), NS-398 (the selective COX-2 inhibitor), NDGA (nordihydroguaiaretic acid, the selective LOX inhibitor) and ETYA (5,8,11,14-eicosatetraynoic acid, the COX and LOX inhibitor) on cell viability, MMP-2 and MMP-9 activities, and in vitro invasion of cancer cells derived from primary and metastatic head and neck, and colon cancers. The inhibitors of COX and/or LOX could inhibit cell proliferation, MMP activity and invasion in head and neck and colon cancer cells. However, the inhibitory effect was obviously observed in colon cancer cells. Inhibition of arachidonic acid metabolism caused a decrease in cancer cell motility, which partially explained by the inhibition of MMPs. Therefore, COX and LOX pathways play important roles in head and neck cancer cell growth., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Cytosolic phospholipase A(2)-inhibited astrocytes respond to the cocktail lipopolysaccharide/interferon-gamma with an immediate formation of peroxynitrite (ONOO(-)) and a delayed lethal response. Low concentrations of arachidonic acid (ARA; i.e., <0.1 microM) cause tyrosine kinase-dependent inhibition of neuronal nitric oxide synthase (nNOS) activity, thereby suppressing formation of ONOO(-) and the ensuing lethal response. ARA promoted its effects only when given to the cultures just prior to, or in parallel with, the proinflammatory mixture. High concentrations of ARA, i.e., >3 microM, promoted cytoprotection when applied to the cultures up to 50 min after the formation of endogenous ONOO(-) had been completed or up to 30 min after addition of exogenous ONOO(-). The mechanism(s) involved in these responses was, however, independent of tyrosine kinase activation and was in fact mediated by ARA metabolites of the lipoxygenase pathway. These results are consistent with a scenario in which astrocytes respond to low or high amounts of ARA with the triggering of different pathways involved in the inflammatory response. Early nNOS inhibition mediated by very low levels of ARA is indeed critical for nuclear factor-kappaB activation, which is otherwise effectively inhibited by constitutive nitric oxide, and for preventing early formation of ONOO(-). Greater ARA concentrations promote survival in astrocytes committed to death by ONOO(-), a species extensively released under inflammatory conditions, via a mechanism dependent on lipoxygenase metabolism and inhibition of downstream events leading to cell demise., ((c) 2010 Wiley-Liss, Inc.)

We have sought evidence that arachidonic acid (AA) induces mitochondrial depolarization in isolated myocytes by a lipoxygenase (LOX)-dependent mechanism and that such depolarization might contribute to arrhythmogenesis following ischemia-reperfusion injury. A method was developed for measuring mitochondrial depolarization in isolated adult rat myocytes in suspension, using tetramethylrhodamine ethyl ester. The addition of AA to myocytes resulted in mitochondrial depolarization that was inhibited by the LOX inhibitor baicalein, by the reactive oxygen species (ROS) scavenger mercaptoproprionylglycine, and by the anion channel inhibitor diisothiocyanatostilbene-disulfonic acid (DIDS). AA induced mitochondrial uncoupling and mitochondrial ATPase activity in myocytes, but both were insensitive to baicalein. We conclude that the metabolic effect of AA in myocytes puts mitochondria into an energetically compromised state where membrane potential is easily changed by the DIDS-sensitive LOX/ROS-mediated opening of an inner membrane anion channel. In an in vivo anesthetized rat model of coronary artery occlusion, baicalein was found to strongly inhibit arrhythmias induced by ischemia-reperfusion injury. Arrhythmias following ischemia-reperfusion injury have been previously associated with DIDS-sensitive ROS-mediated mitochondrial depolarization, and free fatty acids including AA were previously found to accumulate during such injury. We therefore conclude that arrhythmias following ischemia-reperfusion injury might originate from mitochondrial depolarization mediated by LOX and AA.

Background: Gliomas are tumors of the central nervous system derived from glial cells. They show cellular heterogeneity and lack specific diagnostic markers. Although a possible role for the eicosanoid cascade has been suggested in glioma tumorigenesis, the relationship between enzymes and receptors implicated in arachidonic acid metabolism, with histological tumor type has not yet been determined., Design and Methods: Quantitative real-time reverse transcription-polymerase chain reaction was performed to measure and compare transcript levels of enzymes and receptors implicated in both lipoxygenase and cyclooxygenase pathways between oligodendrogliomas, astrocytomas, glioblastomas and mixed oligoastrocytomas., Results: Arachidonic acid metabolism-related enzymes and receptor transcripts (i) were underexpressed in classical oligodendrogliomas compared to astrocytomas and/or glioblastomas, (ii) differed between astrocytomas and glioblastomas and (iii) had an intermediate expression in mixed oligoastrocytomas., Conclusions: mRNA levels of enzymes and receptors implicated both in lipoxygenase and cyclooxygenase pathways differed significantly in gliomas according to the histological type., (Copyright 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

The lipoxygenases (LOX) are a family of non-heme iron-containing dioxygenases which catalyze the stereospecific insertion of molecular oxygen into arachidonic acid, leading to hydroxy derivatives as end products. In this work, we docked arachidonic acid and two of its competitive inhibitors, flavonoids baicalein and quercetin, into the binding pockets of human 12- and 15-lipoxygenase. Steered molecular dynamics (SMD) simulations were employed to study the unbinding processes of the substrate and inhibitors from the two isoforms.

Arachidonic acid (AA) reaction with cyclooxygenase (COX) and lipoxygenases (LOX) yield eicosanoids that can mediate prostate cancer proliferation and enhance both tumour vascularization and metastasis. Increasingly measurement of eicosanoids with liquid chromatography is employed to implicate LOX activity in different biological systems and in particular link LOX activity to the progression of cancer in experimental models. This study demonstrates that simply identifying patterns of eicosanoid regio-isomerism is insufficient to designate LOX activity in prostate cancer cells and the analysis must include complete stereochemical assignment of the various isomers in order to validate the assignment of LOX activity.

We have shown that melatonin immediately and transiently stimulates intracellular free radical production on a set of leukocytes, possibly as a consequence of calmodulin binding. We show here that melatonin-induced ROS are produced by lipoxygenase (LOX), since they are prevented by a set of LOX inhibitors, and are accompanied by increase of the 5-LOX product 5-HETE. LOX activation is accompanied by strong liberation of AA; inhibition of Ca(2+)-independent, but not Ca(2+)-dependent, phospholipase A2 (PLA2), prevents both melatonin-induced arachidonic acid and ROS production, whereas LOX inhibition only prevents ROS, indicating that PLA2 is upstream with respect to LOX, as occurs in many signaling pathways. Chlorpromazine, an inhibitor of melatonin-calmodulin interaction, inhibits both ROS and arachidonic acid production, thus possibly placing calmodulin at the origin of a melatonin-induced pro-radical pathway. Interestingly, it is known that Ca(2+)-independent PLA2 binds to calmodulin: our results are compatible with PLA2 being liberated by melatonin from a steady-state calmodulin sequestration, thus initiating an arachidonate signal transduction. These results delineate a novel molecular pathway through which melatonin may participate to the inflammatory response.

The reaction of soybean lipoxygenase-1 with linoleic acid has been extensively studied and displays very large kinetic isotope effects. In this work, substrate and solvent kinetic isotope effects as well as the viscosity dependence of the oxidation of arachidonic acid were investigated. The hydrogen atom abstraction step was rate-determining at all temperatures, but was partially masked by a viscosity-dependent step at ambient temperatures. The observed KIEs on k(cat) were large ( approximately 100 at 25 degrees C).

Arachidonic acid (AA), a polyunsaturated fatty acid with four double bonds, has multiple actions on living cells. Many of these effects are mediated by an action of AA or its metabolites on ion channels. During the last 10 years, new types of ion channels, transient receptor potential (TRP) channels, store-operated calcium entry (SOCE) channels and non-SOCE channels have been studied. This review summarizes our current knowledge about the effects of AA on TRP and non-SOCE channels as well as classical ion channels. It aims to distinguish between effects of AA itself and effects of AA metabolites. Lipid mediators are of clinical interest because some of them (for example, leukotrienes) play a role in various diseases, others (such as prostaglandins) are targets for pharmacological therapeutic intervention.

Purpose: The arachidonic acid pathway incorporates phospholipase, cyclooxygenase, lipoxygenase and epoxygenase enzymes. This pathway has been shown to have a major role in the development and progression of a number of cancers, including prostate cancer. We discuss the current status of research of this pathway in the area of prostate cancer, ranging from preclinical in vitro studies to human clinical trials., Materials and Methods: We performed an online search of the current and past peer reviewed literature on prostate cancer and arachidonic acid, phospholipase, cyclooxygenase, lipoxygenase, epoxygenase, platelet activating factor, prostaglandin and eicosanoid. We retrieved and evaluated all full-length articles published in English from the 1980s to January 2007., Results: Epidemiological evidence suggested that nonsteroidal anti-inflammatory drugs may decrease the risk of prostate cancer. This effect, presumably through the inhibition of cyclooxygenase-2, has been validated in preclinical studies. Cyclooxygenase-2 inhibition has also decreased the rate of prostate specific antigen increase in men with biochemical recurrence after treatment for prostate cancer. Although lipoxygenase and secretory phospholipase A2 inhibition was also effective for decreasing prostate cancer growth in preclinical studies, to our knowledge these strategies have not yet been used in clinical trials. Cytosolic phospholipase A2, platelet activating factor and epoxygenase need further investigation to determine a role in prostate cancer., Conclusions: Evolving data suggest a significant role for some areas of the arachidonic acid pathway in prostate cancer. Inhibiting 1 or a number of these enzymes in combination may hold promise for future prostate cancer treatment.

Oxygenation of arachidonoyl lysophosphatidylcholine (lysoPC) or arachidonoyl lysophosphatidic acid (lysoPA) by lipoxygenase (LOX) was examined. The oxidized products were identified by HPLC/UV spectrophotometry/mass spectrometry analyses. Straight-phase and chiral-phase HPLC analyses indicated that soybean LOX-1 and rabbit reticulocyte LOX oxygenated arachidonoyl lysophospholipids mainly at C-15 with the S form as major enantiomer, whereas porcine leukocyte LOX oxygenated at C-12 with the S form. Next, the sequential exposure of arachidonoyl-lysoPC to soybean LOX-1 and porcine leukocyte LOX afforded two major isomers of dihydroxy derivatives with conjugated triene structure, suggesting that 15(S)-hydroperoxyeicosatetraenoyl derivatives were converted to 8,15(S)-dihydroxyeicosatetraenoyl derivatives. Separately, arachidonoyl-lysoPA, but not arachidonoyl-lysoPC, was found to be susceptible to double oxygenation by soybean LOX-1 to generate a dihydroperoxyeicosatetraenoyl derivative. Overall, arachidonoyl lysophospholipids were more efficient than arachidonic acid as LOX substrate. Moreover, the catalytic efficiency of arachidonoyl-lysoPC as substrate of three lipoxygenases was much greater than that of arachidonoyl-lysoPA or arachidonic acid. Taken together, it is proposed that arachidonoyl-lysoPC or arachidonoyl-lysoPA is efficiently oxygenated by plant or animal lipoxygenases, C12- or C15-specific, to generate oxidized products with conjugated diene or triene structure.

Vascular remodeling refers to lasting structural alterations in the vessel wall that are initiated in response to external and internal stimuli. These changes are distinct from acute functional responses of blood vessels when challenged by increased blood pressure, altered hemodynamics, or vasoactive mediators. In early atherogenesis, when lesion formation is starting to impact local hemodynamics, the vessel wall responds with outward vascular remodeling to maintain normal blood flow. However, inward remodeling may also occur during the time course of plaque formation, contributing to vascular stenosis. Lipoxygenases form a heterogeneous family of lipid-peroxidizing enzymes, which have been implicated in atherogenesis. Several lines of in vitro and in vivo evidence indicated their involvement in disease development, but the precise function of different lipoxygenase isoforms is still a matter of discussion. Vascular remodeling is an early response during plaque development; therefore, lipoxygenases may be involved in this process. Unfortunately, little is known about the potential role of lipoxygenase isoforms in vascular remodeling. This review will briefly summarize our knowledge of the role of lipoxygenases in vascular biology and will critically review the activities of the 3 most athero-relevant lipoxygenase isoforms in atherogenesis, with particular emphasis on vascular remodeling.

Human peroxiredoxins serve dual roles as anti-oxidants and regulators of H(2)O(2)-mediated cell signaling. The functional versatility of peroxiredoxins depends on progressive oxidation of key cysteine residues. The sulfinic or sulfonic forms of peroxiredoxin lose their peroxidase activity, which allows cells to accumulate H(2)O(2) for signaling or pathogenesis in inflammation, cancer, and other disorders. We report that arachidonic acid lipid hydroperoxide metabolites of 5-, 12-, 15-lipoxygenase-1, and cyclooxygenase-2 oxidize the 2-Cys-peroxiredoxins 1, 2, and 3 to their sulfinic and sulfonic forms. When added exogenously to cells, 5-, 12- and 15-hydroperoxy-eicosatetraenoic acids also over-oxidized peroxiredoxins. Our results suggest that lipoxygenases and cyclooxygenases may affect 2-Cys peroxiredoxin signaling, analogous to NADPH oxidases in the "floodgate" model (Wood, Z. A., Poole, L. B, and Karplus P. A. (2003) Science 300, 600-653). Peroxiredoxin-dependent mechanisms may modulate the receptor-dependent actions of autocoids derived from cellular lipoxygenase and cyclooxygenase catalysis.

Leukotrienes are metabolites of arachidonic acid derived from the action of 5-LO (5-lipoxygenase). The immediate product of 5-LO is LTA4 (leukotriene A4), which is enzymatically converted into either LTB4 (leukotriene B4) by LTA4 hydrolase or LTC4 (leukotriene C4) by LTC4 synthase. The regulation of leukotriene production occurs at various levels, including expression of 5-LO, translocation of 5-LO to the perinuclear region and phosphorylation to either enhance or inhibit the activity of 5-LO. Several other proteins, including cPLA2a (cytosolic phospholipase A2a) and FLAP (5-LO-activating protein) also assemble at the perinuclear region before production of LTA4. LTC4 synthase is an integral membrane protein that is present at the nuclear envelope; however, LTA4 hydrolase remains cytosolic. Biologically active LTB4 is metabolized by w-oxidation carried out by specific cytochrome P450s (CYP4F) followed by b-oxidation from the w-carboxy position and after CoA ester formation. Other specific pathways of leukotriene metabolism include the 12-hydroxydehydrogenase/15-oxo-prostaglandin-13-reductase that forms a series of conjugated diene metabolites that have been observed to be excreted into human urine. Metabolism of LTC4 occurs by sequential peptide cleavage reactions involving a g-glutamyl transpeptidase that forms LTD4 (leukotriene D4) and a membrane-bound dipeptidase that converts LTD4 into LTE4 (leukotriene E4) before w-oxidation. These metabolic transformations of the primary leukotrienes are critical for termination of their biological activity, and defects in expression of participating enzymes may be involved in specific genetic disease.

Arachidonic acid (AA) metabolism in the non-pregnant sheep uterus was studied in vitro using conventional chromatographic and HPLC techniques. High expression of both lipoxygenase (LOX) as well as cyclooxygenase (COX) enzymes and their activities was found in the uterine tissues. On incubation of uterine enymes with AA, the LOX products formed were identified as 5-hydroperoxyeicosatetraenoic acid (5-HPETE), 12- and 15-hydroxyeicosatetraenoic acids (12- and 15-HETEs), based on their separation on TLC and HPLC. By employing differential salt precipitation techniques, the LOXs generating products 5-HPETE (5-LOX), 12-HETE and 15-HETE (12- and 15-dual LOX) were isolated. Based on their analysis on TLC, the COX products formed were identified as prostaglandins - PGF2alpha and prostacyclin derivative 6-keto PGF1alpha. The study forms the first report on the comprehensive analysis on the metabolism of AA in sheep uterus in vitro via the LOX and COX pathways.

A sensitive, specific, and robust liquid chromatography/mass spectrometric (LC/MS) method was developed and validated that allows simultaneous analysis of arachidonic acid (AA) and its cyclooxygenase, cytochrome P450, and lipoxygenase pathway metabolites prostaglandins (PGs), dihydroxyeicosatrienoic acids (DiHETrEs), hydroxyeicosatetraenoic acids (HETEs) and epoxyeicosatrienoic acids (EETs), including PGF(2alpha), PGE(2), PGD(2), PGJ(2), 14,15-DiHETrE, 11,12-DiHETrE, 8,9-DiHETrE, 5,6-DiHETrE, 20-HETE, 15-HETE, 12-HETE, 9-HETE, 8-HETE, 5-HETE, 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET in rat brain tissues. Deuterium labeled PGF(2alpha)-d(4), PGD(2)-d(4), 15(S)-HETE-d(8), 14,15-EET-d(8), 11,12-EET-d(8), 8,9-EET-d(8), and AA-d(8) were used as internal standards. Solid phase extraction was used for sample preparation. A gradient LC/MS method using a C18 column and electrospray ionization source under negative ion mode was optimized for the best sensitivity and separation within 35 min. The method validation, including LC/MS instrument qualification, specificity, calibration model, accuracy, precision (without brain matrix and with brain matrix), and extraction efficiency were performed. The linear ranges of the calibration curves were 2-1000 pg for PGs, DiHETrEs, HETEs, and EETs, 10-2400 pg for PGE(2) and PGD(2), and 20-2000 ng for AA, respectively.

Three enzyme systems, cyclooxygenases that generate prostaglandins, lipoxygenases that form hydroxy derivatives and leukotrienes, and epoxygenases that give rise to epoxyeicosatrienoic products, metabolize arachidonic acid after its release from neural membrane phospholipids by the action of phospholipase A(2). Lysophospholipids, the other products of phospholipase A(2) reactions, are either reacylated or metabolized to platelet-activating factor. Under normal conditions, these metabolites play important roles in synaptic function, cerebral blood flow regulation, apoptosis, angiogenesis, and gene expression. Increased activities of cyclooxygenases, lipoxygenases, and epoxygenases under pathological situations such as ischemia, epilepsy, Alzheimer's disease, Parkinson disease, amyotrophic lateral sclerosis, and Creutzfeldt-Jakob disease produce neuroinflammation involving vasodilation and vasoconstriction, platelet aggregation, leukocyte chemotaxis and release of cytokines, and oxidative stress. These are closely associated with the neural cell injury which occurs in these neurological conditions. The metabolic products of docosahexaenoic acid, through these enzymes, generate a new class of lipid mediators, namely docosatrienes and resolvins. These metabolites antagonize the effect of metabolites derived from arachidonic acid. Recent studies provide insight into how these arachidonic acid metabolites interact with each other and other bioactive mediators such as platelet-activating factor, endocannabinoids, and docosatrienes under normal and pathological conditions. Here, we review present knowledge of the functions of cyclooxygenases, lipoxygenases, and epoxygenases in brain and their association with neurodegenerative diseases.

The metabolism of arachidonic acid may follow multiple, inter-related pathways, leading to the generation or release of a wide variety of biologically active substances (including bradykinins, serotonin, histamine, prostaglandins, and cytokines) that produce pain and/or inflammation. Therapeutic agents that directly affect one particular pathway, such as cyclooxygenase inhibitors (typically, nonsteroidal anti-inflammatory agents), may indirectly affect one or more other pathways, as well as the resolution of inflammation. Rather than taking the traditional approach to treating multiple types of pain by inhibiting a single pathway, future treatments of pain and inflammation may target specific pathways and pain mechanisms and/or address two or more pathways simultaneously. A thorough understanding and appreciation of the mechanisms contributing to various pain and inflammatory states, the metabolic fates of arachidonic acid, the functions of its many metabolites, and the interrelatedness of the various metabolic pathways involved in nociception may lead to more rational and optimal approaches to addressing patient suffering.

In addition to their role in many vital cellular functions, arachidonic acid (AA) and its eicosanoid metabolites are involved in the pathogenesis of several diseases, including atherosclerosis and cancer. To understand the potential mechanisms by which these lipid molecules could influence the disease processes, particularly cardiovascular diseases, we studied AA's effects on vascular smooth muscle cell (VSMC) motility and the role of cAMP-response element binding protein-1 (CREB-1) in this process. AA exerted differential effects on VSMC motility; at lower doses, it stimulated motility, whereas at higher doses, it was inhibitory. AA-induced VSMC motility requires its conversion via the lipoxygenase (LOX) and cyclooxygenase (COX) pathways. AA stimulated the phosphorylation of extracellular signal-regulated kinases (ERKs), Jun N-terminal kinases (JNKs), and p38 mitogen-activated protein kinase (p38MAPK) in a time-dependent manner, and blockade of these serine/threonine kinases significantly attenuated AA-induced VSMC motility. In addition, AA stimulated CREB-1 phosphorylation and activity in a manner that was also dependent on its metabolic conversion via the LOX and COX pathways and the activation of ERKs and p38MAPK but not JNKs. Furthermore, suppression of CREB-1 activation inhibited AA-induced VSMC motility. 15(S)-Hydroxyeicosatetraenoic acid and prostaglandin F2alpha, the 15-LOX and COX metabolites of AA, respectively, that are produced by VSMC at lower doses, were also found to stimulate motility in these cells. Together, these results suggest that AA induces VSMC motility by complex mechanisms involving its metabolism via the LOX and COX pathways as well as the ERK- and p38MAPK-dependent and JNK-independent activation of CREB-1.

Tea has been shown to possess several health beneficial properties primarily due to its polyphenolic content. The major polyphenolic compounds in black tea leaves are theaflavins (TFs) formed by oxidative coupling of catechins in tea leaves during its processing. In this paper, we report the characterization of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse ear inflammatory model and the inhibitory effects of major black tea TFs derivatives on this inflammation. In addition, the effect on inflammatory biomarkers, such as proinflammatory cytokines and arachidonic acid metabolites, are reported as well. A single topical application of TPA to ears of CD-1 mice induced a time- and dose-dependent increase in edema as well as formation of proinflammatory cytokine proteins interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) in mouse ears. A single topical application of equimolar of black tea constituents (TF, theaflavin-3-gallate, theaflavin-3'-gallate, and theaflavin-3,3'-digallate) strongly inhibited TPA-induced edema of mouse ears. Application of TFs mixture to mouse ears 20 min prior to each TPA application once a day for 4 days inhibited TPA-induced persistent inflammation, as well as TPA-induced increase in IL-1beta and IL-6 protein levels. TFs also inhibited arachidonic acid (AA) metabolism via both cyclooxygenase (COX) and lipoxygenase pathways. This observation was substantiated by decreased amounts of AA metabolites prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) levels. Combined application of TF and sulindac, a nonsteroidal anti-inflammatory drug resulted a significant synergetic anti-inflammatory effect. Oral administration of TFs or the hot water extract of black tea leaves also significantly inhibited TPA-induced edema in mouse ears. In conclusion, proinflammatory cytokines, IL-1beta and IL-6, as well as the intermediated metabolites of AA, PGE2, and LTB4 are good biomarkers for inflammation. Black tea constituents, TF and its derivatives, had strongly anti-inflammatory activity in vivo which may be due to their ability to inhibit AA metabolism via lipoxygenase and COX pathways.

Dietary fats, which increase the risk of prostate cancer, stimulate release of intestinal neurotensin (NT), a growth-promoting peptide that enhances the formation of arachidonic acid metabolites in animal blood. This led us to use PC3 cells to examine the involvement of lipoxygenase (LOX) and cyclooxygenase (COX) in the growth effects of NT, including activation of EGF receptor (EGFR) and downstream kinases (ERK, AKT), and stimulation of DNA synthesis. NT and EGF enhanced [3H]-AA release, which was diminished by inhibitors of PLA2 (quinacrine), EGFR (AG1478) and MEK (U0126). NT and EGF phosphorylated EGFR, ERK and AKT, and stimulated DNA synthesis. These effects were diminished by PLA2 inhibitor (quinacrine), general LOX inhibitors (NDGA, ETYA), 5-LOX inhibitors (Rev 5901, AA861), 12-LOX inhibitor (baicalein) and FLAP inhibitor (MK886), while COX inhibitor (indomethacin) was without effect. Cells treated with NT and EGF showed an increase in 5-HETE levels by HPLC. PKC inhibitor (bisindolylmaleimide) blocked the stimulatory effects of NT, EGF and 5-HETE on DNA synthesis. We propose that 5-LOX activity is required for NT to stimulate growth via EGFR and its downstream kinases. The mechanism may involve an effect of 5-HETE on PKC, which is known to facilitate MEK-ERK activation. NT may enhance 5-HETE formation by Ca2+-mediated and ERK-mediated activation of DAG lipase and cPLA2. NT also upregulates cPLA2 and 5-LOX protein expression. Thus, the growth effects of NT and EGF involve a feed-forward system that requires cooperative interactions of the 5-LOX, ERK and AKT pathways.

The epidermis expresses cyclooxygenases, lipoxygenases, and cytochromes P450, which utilize arachidonic acid to generate a diverse array of lipid mediators affecting epidermal cellular differentiation and functions. Recent studies show that mouse epidermis expresses CYP2B19, a keratinocyte-specific epoxygenase that generates 11,12- and 14,15-epoxyeicosatrienoic (EET) acids from arachidonate. We studied CYP2B19-dependent metabolism in mouse epidermal microsomes, reconstituted in the presence of [1-(14)C]arachidonic acid. The majority of the (14)C products formed independently of NADPH, indicative of robust epidermal cyclooxygenase and lipoxygenase activities. We studied two NADPH-dependent products generated in a highly reproducible manner from arachidonate. One of these (product I) coeluted with the CYP2B19 product 14,15-EET on a reversed-phase high-performance liquid chromatography (HPLC) system; there was no evidence for other regioisomeric EET products. Further analyses proved that product I was not an epoxy fatty acid, based on different retention times on a normal-phase HPLC system and failure of product I to undergo hydrolysis in acidic solution. We analyzed purified epidermal (14)C products by liquid chromatography negative electrospray ionization mass spectrometry. Structures of the NADPH-dependent products were confirmed to be 12-oxo-5,8,14-eicosatrienoic acid (I) and 12-hydroxy-5,8,14-eicosatrienoic acid (II). This was the first evidence for a 12-hydroxy-5,8,14-eicosatrienoic acid biosynthetic pathway in mouse epidermis. Epidermal microsomes also generated 12-hydroperoxy, 12-hydroxy, and 12-oxo eicosatetraenoic acids from arachidonate, possible intermediates in the 12-hydroxy-5,8,14-eicosatrienoic acid biosynthetic pathway. These results predict that hydroxyeicosatrienoic acids are synthesized from arachidonate in human epidermis. This would have important implications for human skin diseases given the known pro- and anti-inflammatory activities of stereo- and regioisomeric hydroxyeicosatrienoic acids.

Objective: Preeclampsia is associated with oxidative stress, elevated plasma levels of linoleic acid (LA), and increased vascular smooth muscle expression of the inflammatory chemokine, interleukin-8 (IL-8). We hypothesized that increased levels of LA under conditions of oxidative stress would increased production of IL-8 by vascular smooth muscle cells because LA is the dietary precursor to arachidonic acid (AA) and its metabolites that mediate inflammation. We also hypothesized that oleic acid (OA), which is not metabolized to AA metabolites, would not increase IL-8 under conditions of oxidative stress., Methods: To test this hypothesis, we cultured placental arterial smooth muscle (PASM) cells with an oxidizing solution enriched with LA (OxLA) or OA (OxOA). Media concentrations were analyzed for IL-8 and AA metabolites. Inhibitors were used to block the lipoxygenase and cyclooxygenase pathways., Results: Exposure of cells to OxLA, but not to OxOA, significantly increased production of IL-8. OxLA also significantly increased production of AA metabolites. Nordihydroguaiaretic acid, an inhibitor of the lipoxygenase pathway, blocked IL-8 and leukotriene B4 (LTB4) production induced by OxLA, whereas indomethacin, an inhibitor of the cyclooxygenase pathway, blocked IL-8, prostaglandin E2 (PGE2), and thromboxane B2 (TXB2) production. Reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated gene expression in PASM cells for representative lipoxygenase (LTB4) and cyclooxygenase (thromboxane) metabolite receptors., Conclusion: PASM cells produced IL-8 in response to LA, but not OA, under conditions of oxidative stress. The IL-8 response was mediated by AA metabolites.

We tested the hypotheses that EDHF in rat middle cerebral arteries (MCAs) involves 1) metabolism of arachidonic acid through the epoxygenase pathway, 2) metabolism of arachidonic acid through the lipoxygenase pathway, or 3) reactive oxygen species. EDHF-mediated dilations were elicited in isolated and pressurized rat MCAs by activation of endothelial P2Y(2) receptors with either UTP or ATP. All studies were conducted after the inhibition of nitric oxide synthase and cyclooxygenase with N(omega)-nitro-l-arginine methyl ester (10 microM) and indomethacin (10 microM), respectively. The inhibition of epoxygenase with miconazole (30 microM) did not alter EDHF dilations to UTP, whereas the structurally different epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanoic acid (20 or 40 microM) only modestly inhibited EDHF at the highest concentration of UTP. An antagonist of epoxyeicosatrienoic acids, 14,15-epoxyeicosa-5(Z)-enoic acid, had no effect on EDHF dilations to UTP. Chronic inhibition of epoxygenase in the rat with 1-aminobenzotriazol (50 mg/kg twice daily for 5 days) did not alter EDHF dilations. The inhibition of the lipoxygenase pathway with either 10 microM baicalein or 10 microM nordihydroguaiaretic acid produced no major inhibitory effects on EDHF dilations. The combination of superoxide dismutase (200 U/ml) and catalase (140 U/ml) had no effect on EDHF dilations. Neither tiron (10 mM), a cell-permeable scavenger of reactive oxygen species, nor deferoxamine (1 or 10 mM), an iron chelator that blocks the formation of hydroxyl radicals, altered EDHF dilations in rat MCAs. We conclude that EDHF dilations in the rat MCA do not involve the epoxygenase pathway, lipoxygenase pathway, or reactive oxygen species including H(2)O(2).