The opposing effects of n-3 and n-6 fatty acids

Polyunsaturated fatty acids (PUFAs) can be classified in n-3 fatty acids and n-6 fatty acids, and in westernized diet the predominant dietary PUFAs are n-6 fatty acids. Both types of fatty acids are precursors of signaling molecules with opposing effects, that modulate membrane microdomain composition, receptor signaling and gene expression. The predominant n-6 fatty acid is arachidonic acid, which is converted to prostaglandins, leukotrienes and other lipoxygenase or cyclooxygenase products. These products are important regulators of cellular functions with inflammatory, atherogenic and prothrombotic effects. Typical n-3 fatty acids are docosahexaenoic acid and eicosapentaenoic acid, which are competitive substrates for the enzymes and products of arachidonic acid metabolism. Docosahexaenoic acid- and eicosapentaenoic acid-derived eicosanoids antagonize the pro-inflammatory effects of n-6 fatty acids. n-3 and n-6 fatty acids are ligands/modulators for the nuclear receptors NFkappaB, PPAR and SREBP-1c, which control various genes of inflammatory signaling and lipid metabolism. n-3 Fatty acids down-regulate inflammatory genes and lipid synthesis, and stimulate fatty acid degradation. In addition, the n-3/n-6 PUFA content of cell and organelle membranes, as well as membrane microdomains strongly influences membrane function and numerous cellular processes such as cell death and survival.     

Synthesis and relevance of cardiac eicosanoids with particular emphasis on ischemia and reperfusion

Eicosanoids represent a family of compounds derived primarily from arachidonic acid. It is now known that arachidonic acid can undergo metabolism via at least three distinct pathways, although the most readily understood are those resulting in prostaglandin or leukotriene formation via cyclooxygenase and 5-lipoxygenase, respectively. These products can be synthesized by the heart or can be released from accumulating neutrophils under various pathological conditions. Eicosanoids possess a wide array of pharmacological actions that could be of importance either in the initiation or modulation of various cardiac diseases. Here, we review the potential importance of eicosanoids to ischemic heart disease. Data are cited that examine the potential importance of these compounds to experimentally induced cardiac injury as well as clinically observed ischemic heart disease. Particular emphasis is placed on recent studies that document the relevance of endogenously synthesized arachidonic acid metabolites as well as the consequence of modulating eicosanoid synthesis through pharmacological or dietary means on cardiac injury under experimental or clinical situations.     

The enzymes in cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism in human corpora lutea: dependence on luteal phase, cellular and subcellular distribution

Eicosanoids synthesized within corpus luteum are presumed to regulate luteal function in women. However, the potential cellular source(s) of the eicosanoids, whether small and large luteal cells differ in eicosanoid synthesis and whether eicosanoids other than prostaglandin (PG)E2, PGF2 alpha and 6-keto-PGI1 alpha can be synthesized, have not been investigated. The present immunocytochemical studies were undertaken to answer these questions using mono and polyclonal antibodies to several enzymes in arachidonic acid metabolism by cyclooxygenase and lipoxygenase pathways. Human corpora lutea from early (n = 5), mid (n = 6) and late (n = 3) luteal phases were specifically immunostained for all the enzymes. All the enzymes were present in small and large luteal cells as well as in non luteal cells. However, small luteal cells contained more immunoreactive 5-lipoxygenase, PGD2 and PGF2 alpha synthases; large luteal cells contained more TXA2 synthase and 12-lipoxygenase; small and large luteal cells contained similar amounts of cyclooxygenase and PGI2 synthase. In all the cells, immunoreactive PGD2, PGI2 and TXA2 synthases increased from early to mid luteal phase and then declined in late luteal phase. Cyclooxygenase, 5- and 12-lipoxygenases and PGF2 alpha synthase, on the other hand, increased from early to mid and mid to late luteal phases. Immunoreactive cyclooxygenase and 5- and 12-lipoxygenases were present primarily in rough endoplasmic reticulum (ER) and/or smooth ER and cytoplasm. Quite unexpectedly, all three enzymes were also found in nuclear membranes, condensed chromatin and especially at the perimeter of condensed chromatin. Dispersed chromatin contained very little or no immunoreactive enzyme. These results indicate that regulation of human luteal function by eicosanoids synthesized within the corpus luteum is complex involving perhaps a) small and large luteal as well as non luteal cells, b) eicosanoids which have not been previously considered to play a role in luteal function and c) coordinate regulation of more than one enzyme in the pathways of arachidonic acid metabolism.     

Eicosanoids mediate nodulation reactions to bacterial infections in adults of the cricket,Gryllus assimilis

Nodulation is the temporally and quantitatively most important cellular defense reaction to bacterial infections in insects. Inhibition of eicosanoid biosynthesis in adults of the cricket, Gryllus assimilis, immediately prior to intrahemocoelic injections of the bacterium, Serratia marcescens, sharply reduced the nodulation response. Separate treatments with specific inhibitors of phospholipase A(2), cyclooxygenase, and lipoxygenase reduced nodulation, supporting our view that nodule formation is a complex process involving lipoxygenase and cyclooxygenase products. The inhibitory influence of dexamethasone was apparent within 2h of injection, and nodulation was significantly reduced, relative to control crickets, over 22h. The dexamethasone effects were reversed by treating bacteria-injected insects with the eicosanoid-precursor polyunsaturated fatty acid, arachidonic acid. Low levels of arachidonic acid were detected in fat body phospholipids, and fat body preparations were shown to be competent to biosynthesize eicosanoids from exogenous radioactive arachidonic acid. These findings in a hemimetabolous insect broaden our hypothesis that eicosanoids mediate cellular immune reactions to bacterial infections in most, if not all, insects.     

Essential fatty acids and immune response

The implication that essential fatty acids (EFA) can affect immune response was based on the observation that EFA deficiency can accentuate or improve symptoms of certain autoimmune diseases in animals, and that supplementation of linoleic acid to animals reversed such effects. Furthermore, treatment of animals with cyclooxygenase inhibitors abrogated the effect of linoleic acid. Administration of cyclooxygenase inhibitors to animals enhanced both cell-mediated and humoral immune responses. In vitro studies have shown that prostaglandin E (PGE) group inhibits both T and B lymphocyte functions; it is suggested that effects of EFA on immune response are, in part, mediated through eicosanoids. Growing evidence now suggests that the PGE group of prostaglandins can serve as a negative feedback modulator of immune response. However, in vitro effects of other cyclooxygenase-derived products, such as PGI2 and thromboxane A2 (TXA2) have not been well established, perhaps because of their instability in aqueous media. Unlike the PGE group, some of lipoxygenase-derived products of arachidonic acid have shown immunostimulatory effects, as assessed by lymphokine production in vitro. Whether such effects can be seen in vivo remains to be determined. Some lipoxygenase-derived products with strong chemotactic action may indirectly influence immune response by modulating the population of antigen-presenting macrophages in tissues. Thus, the net effect of eicosanoids synthesized in macrophages on modulating immune response may depend on relative amounts of cyclooxygenase-derived products as compared with lipoxygenase-derived products. Macrophages are the major source of eicosanoids among immunocompetent cells. The profile of eicosanoids, produced in vitro by macrophages, varies with type of stimuli and anatomical sites. It can also be affected by the fatty acid composition of tissue lipids, which in turn can be modified by the composition of dietary EFA. Whether manipulating dietary EFA can modulate immune response in normal humans and animals needs to be determined.     

Eicosanoids and the esophagus

Eicosanoids are products of arachidonic acid metabolism. Among the products produced are the prostaglandins and leukotrienes, products which are known to play important roles in health and disease of many gastrointestinal tissues. Here, we review current knowledge about eicosanoids in the esophagus, including production in healthy and diseased tissues and potential physiologic and pathophysiologic effects in two important esophageal mucosal disorders, reflux esophagitis and esophageal cancer.     

Nitric oxide stimulated vascular smooth muscle cells undergo apoptosis induced in part by arachidonic acid derived eicosanoids

The role of eicosanoids in atherogenesis has not been thoroughly explained. This is partly due to the numerous eicosanoids and the variable effects that each has on different systems. Apoptosis of vascular smooth muscle cells has been shown to play a role in the atherosclerotic disease leading to lesion formation and further destabilization of the formed lesion. In this study, we have investigated the role of arachidonic acid derived eicosanoids in nitric oxide (NO)-stimulated vascular smooth muscle cells. We have shown previously that the nitric oxide (NO)-induced apoptosis of vascular smooth muscle cells was accompanied by arachidonic acid release via cytoplasmic phospholipase A(2) (cPLA(2)) activation. Also, arachidonic acid, but not oleic acid, induced apoptosis of these cells at low concentrations (5-10 microM). Our results revealed that the cPLA(2) specific inhibitor, arachidonyl trifluoromethyl ketone (AACOCF(3)), blocked NO-induced eicosanoid production, while the presence of arachidonic acid enhanced the ability of the cells to make prostaglandin E(2) (PGE(2)). Also, inhibitors of the cyclo-oxygenase (Cox) enzymes, such as N-[2-cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS-398), a specific Cox-2 inhibitor, or indomethacin, a non-specific Cox inhibitor, blocked NO-induced PGE(2) production and apoptosis of vascular smooth muscle cells to the same extent, indicating that apoptosis might be induced by a Cox-2 metabolic product. In addition to these observations, the eicosanoids investigated, namely, PGE(2), PGI(2) LTB(4), and PGJ(2), showed different effects on vascular smooth muscle cells. Both PGJ(2) and LTB(4) decreased the percentage of viable cells and induced apoptosis of vascular smooth muscle cells, while PGE(2) and PGI(2) had no effect on cell viability and failed to induce apoptosis. These data suggest that eicosanoids, such as PGJ(2), but not PGE(2) or PGI(2), are involved in NO-induced apoptosis of vascular smooth muscle cells and that the eicosanoid synthesis pathways might be utilized for vascular therapeutic strategies.     

Cyclooxygenase enzymes and prostaglandins in reproductive tract physiology and pathology

Prostaglandins, thromboxanes (TX) and leukotrienes, collectively referred to as eicosanoids, are cyclooxygenase (COX) metabolites of arachidonic acid (AA). Prostaglandins, have been recognised for many years as key molecules in regulating reproductive tract physiology and pathology. Numerous recent studies in in vitro model systems and knockout mouse models have demonstrated specific functional roles for the respective cyclooxygenase enzymes, prostaglandins and prostanoid receptors. Here we review the findings obtained in several of these studies with emphasis on the roles played by cyclooxygenase enzymes and prostaglandins, specifically prostaglandin E2 (PGE2) and F2alpha in reproductive tract physiology and pathology.     

The complex role of eicosanoids in the brain: Implications for brain tumor development and therapeutic opportunities

Eicosanoids are a family of bioactive lipids that play diverse roles in the normal physiology of the brain, including neuronal signaling, synaptic plasticity, and regulation of cerebral blood flow. In the brain, eicosanoids are primarily derived from arachidonic acid, which is released from membrane phospholipids in response to various stimuli. Prostaglandins (PGs) and leukotrienes (LTs) are the major classes of eicosanoids produced in the brain, and they act through specific receptors to modulate various physiological and pathological processes. Dysregulation of eicosanoids has been implicated in the development and progression of brain tumors, including glioblastoma (GBM), meningioma, and medulloblastoma. Eicosanoids have been shown to promote tumor cell proliferation, migration, invasion, angiogenesis, and resistance to therapy. Particularly, PGE2 promotes GBM cell survival and resistance to chemotherapy. Understanding the role of eicosanoids in brain tumors can inform the development of diagnostic and prognostic biomarkers, as well as therapeutic strategies that target eicosanoid pathways. Cyclooxygenase (COX)-2 and 5-lipoxygenase (LOX) inhibitors have been shown to reduce the growth and invasiveness of GBM cells. Moreover, eicosanoids have immunomodulatory effects that can impact the immune response to brain tumors. Understanding the role of eicosanoids in the immune response to brain tumors can inform the development of immunotherapy approaches for these tumors. Overall, the complex role of eicosanoids in the brain underscores the importance of further research to elucidate their functions in normal physiology and disease, and highlights the potential for developing novel therapeutic approaches that target eicosanoid pathways in brain tumors.     

Nitrogen dioxide induces cis-trans-isomerization of arachidonic acid within cellular phospholipids. Detection of trans-arachidonic acids in vivo.

Oxygen free radicals oxidize arachidonic acid to a complex mixture of metabolites termed isoeicosanoids that share structural similarity to enzymatically derived eicosanoids. However, little is known about oxidations of arachidonic acid mediated by reactive radical nitrogen oxides. We have studied the reaction of arachidonic acid with NO2, a free radical generated by nitric oxide and nitrite oxidations. A major group of products appeared to be a mixture of arachidonic acid isomers having one trans-bond and three cis-double bonds. We have termed these new products trans-arachidonic acids. These isomers were chromatographically distinct from arachidonic acid and produced mass spectra that were nearly identical with mass spectra of arachidonic acid. The lack of ultraviolet absorbance above 205 nm and the similarity of mass spectra of dimethyloxazoline derivatives suggested that the trans-bond was not conjugated with any of the cis-bonds, and the C=C bonds were located at carbons 5, 8, 11, and 14. Further identification was based on comparison of chromatographic properties with synthetic standards and revealed that NO2 generated 14-trans-eicosatetraenoic acid and a mixture containing 11-trans-, 8-trans-, and 5-trans-eicosatetraenoic acids. Exposure of human platelets to submicromolar levels of NO2 resulted in a dose-dependent formation of 14-trans-eicosatetraenoic acid and other isomers within platelet glycerophospholipids. Using a sensitive isotopic dilution assay we detected trans-arachidonic acids in human plasma (50.3 +/- 10 ng/ml) and urine (122 +/- 50 pg/ml). We proposed a mechanism of arachidonic acid isomerization that involves a reversible attachment of NO2 to a double bond with formation of a nitroarachidonyl radical. Thus, free radical processes mediated by NO2 lead to generation of trans-arachidonic acid isomers, including biologically active 14-trans-eicosatetraenoic acid, within membrane phospholipids from which they can be released and excreted into urine.     

New frontiers of inositide-specific phospholipase C in nuclear signalling

Strong evidence has been obtained during the last 16 years suggesting that phosphoinositides, which are involved in the regulation of a large variety of cellular processes in the cytoplasm and in the plasma membrane, are present within the nucleus. A number of advances has resulted in the discovery that nuclear phosphoinositides and their metabolizing enzymes are deeply involved in cell growth and differentiation. Remarkably, the nuclear inositide metabolism is regulated independently from that present elsewhere in the cell. Even though nuclear inositol lipids generate second messengers such as diacyglycerol and inositol 1,4,5-trisphosphate, it is becoming increasingly clear that in the nucleus polyphosphoinositides may act by themselves to influence functions such as pre-mRNA splicing and chromatin structure. This review aims at highlighting the most significant and up-dated findings about inositol lipid metabolism in the nucleus.     

Regulation of inflammation in cancer by eicosanoids

Inflammation in the tumor microenvironment is now recognized as one of the hallmarks of cancer. Endogenously produced lipid autacoids, locally acting small molecule lipid mediators, play a central role in inflammation and tissue homeostasis, and have recently been implicated in cancer. A well-studied group of autacoid mediators that are the products of arachidonic acid metabolism include: the prostaglandins, leukotrienes, lipoxins and cytochrome P450 (CYP) derived bioactive products. These lipid mediators are collectively referred to as eicosanoids and are generated by distinct enzymatic systems initiated by cyclooxygenases (COX 1 and 2), lipoxygenases (5-LOX, 12-LOX, 15-LOXa, 15-LOXb), and cytochrome P450s, respectively. These pathways are the target of approved drugs for the treatment of inflammation, pain, asthma, allergies, and cardiovascular disorders. Beyond their potent anti-inflammatory and anti-cancer effects, non-steroidal anti-inflammatory drugs (NSAIDs) and COX-2 specific inhibitors have been evaluated in both preclinical tumor models and clinical trials. Eicosanoid biosynthesis and actions can also be directly influenced by nutrients in the diet, as evidenced by the emerging role of omega-3 fatty acids in cancer prevention and treatment. Most research dedicated to using eicosanoids to inhibit tumor-associated inflammation has focused on the COX and LOX pathways. Novel experimental approaches that demonstrate the anti-tumor effects of inhibiting cancer-associated inflammation currently include: eicosanoid receptor antagonism, overexpression of eicosanoid metabolizing enzymes, and the use of endogenous anti-inflammatory lipid mediators. Here we review the actions of eicosanoids on inflammation in the context of tumorigenesis. Eicosanoids may represent a missing link between inflammation and cancer and thus could serve as therapeutic target(s) for inhibiting tumor growth.     

Cytochrome P450 and arachidonic acid bioactivation. Molecular and functional properties of the arachidonate monooxygenase

The demonstration of in vivo arachidonic acid epoxidation and omega-hydroxylation established the cytochrome P450 epoxygenase and omega/omega-1 hydroxylase as formal metabolic pathways and as members of the arachidonate metabolic cascade. The characterization of the potent biological activities associated with several of the cytochrome P450-derived eicosanoids suggested new and important functional roles for these enzymes in cellular, organ, and body physiology, including the control of vascular reactivity and systemic blood pressures. Past and current advances in cytochrome P450 biochemistry and molecular biology facilitate the characterization of cytochrome P450 isoforms responsible for tissue/organ specific arachidonic acid epoxidation and omega/omega-1 hydroxylation, and thus, the analysis of cDNA and/or gene specific functional phenotypes. The combined application of physiological, biochemical, molecular, and genetic approaches is beginning to provide new insights into the physiological and/or pathophysiological significance of these enzymes, their endogenous substrates, and products.     

Precolumn extraction and reversed-phase high-pressure liquid chromatography of prostaglandins and leukotrienes

Prostaglandins, leukotrienes, and other metabolites of arachidonic acid can be conveniently and efficiently extracted from biological media using a precolumn containing octadecylsilyl silica connected to a 6-port switching valve that is in line with an analytical HPLC column. This procedure makes it possible to extract complex mixtures of eicosanoids and to analyze them by reversed-phase HPLC in a single step. The requirement to evaporate solvents from extracts prior to HPLC is therefore eliminated, saving time and reducing the possibilities for loss and contamination. The effects on recoveries of various media for loading the sample onto the precolumn were investigated, and it was concluded that 15% methanol at neutral pH gives the best overall results. It is therefore not necessary to acidity the sample prior to extraction, which simplifies the procedure and improves the recoveries of acid-labile eicosanoids. Following extraction, eicosanoids can be introduced onto the HPLC column by changing the position of the 6-port switching valve. We have investigated several approaches to the analysis of complex mixtures of these products by reversed-phase HPLC. The best results were obtained using a ternary gradient with a non-end-capped column of octadecylsilyl silica. Metabolites of arachidonic acid other than peptido-leukotrienes were first eluted by increasing the concentrations of acetonitrile and methanol in the mobile phase, which contained a constant concentration of trifluoroacetic acid (0.001%). Peptido-leukotrienes were then eluted with a second gradient, in which the concentrations of acetonitrile and methanol were kept constant, but the concentration of trifluoroacetic acid was increased to 0.0091%. Leukotrienes C4, D4, and E4 appear as sharp peaks at the end of the chromatogram and are completely separated from other types of arachidonic acid metabolites.     

Biosynthesis, catabolism, and biological properties of HPETEs, hydroperoxide derivatives of arachidonic acid

The oxygenation of arachidonic acid by lipoxygenases results in the formation of HPETEs (hydroperoxyeicosatetraenoic acids), the first products of the LOX pathway. These compounds are short lived and are catabolised into various families of more stable compounds of which the HETEs, hepoxilins, lipoxins and leukotrienes have been identified so far. The development of new techniques have helped to identify and understand the structures of various HPETEs and only recently the biological effects of HPETEs and their various catabolites are being unraveled. Although lipoxygenases are ubiquitous, not all tissues possess the same spectrum of lipoxygenase enzymes. Hence different HPETEs can be formed in different tissues. Recent studies have revealed that HPETEs or products derived from them possess a diversity of important biological properties including the regulation of electrolyte flux and eicosanoid and corticosterone syntheses, release of histamine, regulation of oocyte maturation and release of various reproductive hormones. HPETEs appear to be involved in some pathological conditions viz, skin psoriasis, Clarkson's disease, nerve injury and spinal cord ischemia. These novel eicosanoids are associated with the release of insulin as well as renin. Recently HPETEs have been suggested to act as second messengers in the Aplysia sensory neurons and its catabolite, hepoxilin, has been demonstrated to have effects on mammalian hippocampal neurons. The purpose of this review is to provide a brief summary of the formation of the HPETEs and the various families of compounds derived from them as well as the various types of biological activities for these products described so far.     

Light and electron microscope immunocytochemical localization of 5- and 12-lipoxygenases and cyclooxygenase enzymes in human granulosa cells from preovulatory follicles

Cellular and subcellular distribution of 5- and 12-lipoxygenases and cyclooxygenase enzymes were investigated in human granulosa cells from preovulatory follicles using light and electron microscope immunocytochemistry. The results demonstrated that all three enzymes are present in granulosa cells but not in minor contaminating red blood cells. While the distribution of cyclooxygenase and 12-lipoxygenase was relatively uniform among the granulosa cells, 5-lipoxygenase was not uniformly distributed among these cells. All three enzymes are present in microvillus plasma membranes, rough endoplasmic reticulum, cytoplasm, nuclear membranes and chromatin. In summary, 5- and 12-lipoxygenases and cyclooxygenase enzymes, which catalyze the transformation of arachidonic acid into different eicosanoids, are present in several subcellular organelles including nuclei of granulosa cells from preovulatory follicles.     

Ionophore-induced metabolism of phospholipids and eicosanoid production in porcine aortic endothelial cells: selective release of arachidonic acid from diacyl and ether phospholipids

Confluent cultures of porcine aortic endothelial cells were prelabeled with 1 microM [14C]arachidonic acid complexed to 1 microM bovine serum albumin. After washing, the cells were stimulated with 1 microM A23187 for time intervals between 30 s and 30 min. Cellular lipids were extracted and separated into major lipid classes and phospholipid subclasses. The external medium was analyzed for released radioactive eicosanoids. The time-course of total release of 14C radioactivity demonstrated a biphasic nature of A23187-induced changes in endothelial cell lipids. Early, from 30 s to 5 min, substantial losses of [14C]arachidonic acid from diacylphosphatidylethanolamine and phosphatidylinositol, as well as an abrupt increase in diacylphosphatidylcholine-associated radioactivity were observed. These initial changes coincided with the release of 14C-labeled cyclooxygenase products. Later changes (5-30 min) included a sustained progressive loss of 14C radioactivity from alkenyl (alk-1-enyl) acylphosphatidylethanolamine and diacylphosphatidylcholine. These later changes coincided with the elaboration of 14C-labeled lipoxygenase products. Although unequivocal assignments cannot be made, the data suggest that specific pools of arachidonic acid provide precursors for individual classes of eicosanoids.