Arachidonic Acid-derived Prostaglandins Research Articles

Set up and validation of a sensitive method to quantify prostaglandins, prostaglandin-glycerol esters and prostaglandin-ethanolamides, as well as their respective precursors

Arachidonic acid-derived prostaglandins are widely studied for their role in inflammation. However, besides arachidonic acid, other arachidonic moiety-containing lipids can be metabolized by COX-2. Indeed, the endocannabinoids 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (anandamide, AEA) can follow the same biochemical pathways than arachidonic acid leading to the formation of prostaglandin-glycerol esters (PG-G) and prostaglandin-ethanolamides (or prostamides, PG-EA), respectively.The data reported so far support the interest of these bioactive lipids in inflammatory conditions. However, there is only a handful of methods described for their quantification in biological matrices. Moreover, given the shared biochemical pathways for arachidonic acid, 2-AG and AEA, a method allowing for the quantification of these precursors and the corresponding prostaglandin derivatives appears as largely needed. Thus, we report here the development and validation of a single run UPLC-MS/MS quantification method allowing the quantification of these endocannabinoids-derived mediators together with the classical prostaglandin. Moreover, we applied the method to the quantification of these lipids in vitro (using lipopolysaccharides-activated J774 macrophage cells) and in vivo in several tissues from DSS-induced colitis mice. This femtomole-range method should improve the understanding of the interaction between these lipid mediators and inflammation.

Identification of Salicylates in Willow Bark (Salix Cortex) for Targeting Peripheral Inflammation.

Salix cortex-containing medicine is used against pain conditions, fever, headaches, and inflammation, which are partly mediated via arachidonic acid-derived prostaglandins (PGs). We used an activity-guided fractionation strategy, followed by structure elucidation experiments using LC-MS/MS, CD-spectroscopy, and 1D/2D NMR techniques, to identify the compounds relevant for the inhibition of PGE2 release from activated human peripheral blood mononuclear cells. Subsequent compound purification by means of preparative and semipreparative HPLC revealed 2′-O-acetylsalicortin (1), 3′-O-acetylsalicortin (2), 2′-O-acetylsalicin (3), 2′,6′-O-diacetylsalicortin (4), lasiandrin (5), tremulacin (6), and cinnamrutinose A (7). In contrast to 3 and 7, compounds 1, 2, 4, 5, and 6 showed inhibitory activity against PGE2 release with different potencies. Polyphenols were not relevant for the bioactivity of the Salix extract but salicylates, which degrade to, e.g., catechol, salicylic acid, salicin, and/or 1-hydroxy-6-oxo-2-cycohexenecarboxylate. Inflammation presents an important therapeutic target for pharmacological interventions; thus, the identification of relevant key drugs in Salix could provide new prospects for the improvement and standardization of existing clinical medicine.

ω-3 Tear Film Lipids Correlate With Clinical Measures of Dry Eye.

Purposeω-3 and ω-6 polyunsaturated fatty acids modulate inflammatory processes throughout the body through distinct classes of lipid mediators that possess both proinflammatory and proresolving properties. The purpose of this cross-sectional study was to explore the relationship between lipid profiles in human tears and dry eye (DE) symptoms and signs.MethodsForty-one patients with normal eyelid and corneal anatomy were prospectively recruited from a Veterans Administration Hospital over 18 months. Symptoms and signs of DE were assessed, and tear samples was analyzed by mass spectrometry–based lipidomics. Statistical analyses comparing the relationship between tear film lipids and DE included Pearson/Spearman correlations and t-tests.ResultsArachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) were present in more than 90% of tear film samples. The ratio of ω-6 (AA) to ω-3 (DHA+EPA) fatty acids was correlated with multiple measures of tear film dysfunction (tear breakup time, Schirmer 2 scores, and corneal staining; all P < 0.05). Arachidonic acid–derived prostaglandin E2 was detected in the majority of samples and correlated with low tear osmolarity, meibomian gland plugging, and corneal staining.ConclusionsBoth ω-3 and ω-6 lipid circuits are activated in the human tear film. The ratio of ω-6:ω-3 tear lipids is elevated in DE patients in proportion to the degree of tear film dysfunction and corneal staining. Metabolic deficiency of ω-3 tear film lipids may be a driver of chronic ocular surface inflammation in DE.

Hypertonic Stress Induces VEGF Production in Human Colon Cancer Cell Line Caco-2: Inhibitory Role of Autocrine PGE2

Vascular Endothelial Growth Factor (VEGF) is a major regulator of angiogenesis. VEGF expression is up regulated in response to micro-environmental cues related to poor blood supply such as hypoxia. However, regulation of VEGF expression in cancer cells is not limited to the stress response due to increased volume of the tumor mass. Lipid mediators in particular arachidonic acid-derived prostaglandin (PG)E2 are regulators of VEGF expression and angiogenesis in colon cancer. In addition, increased osmolarity that is generated during colonic water absorption and feces consolidation seems to activate colon cancer cells and promote PGE2 generation. Such physiological stimulation may provide signaling for cancer promotion. Here we investigated the effect of exposure to a hypertonic medium, to emulate colonic environment, on VEGF production by colon cancer cells. The role of concomitant PGE2 generation and MAPK activation was addressed by specific pharmacological inhibition. Human colon cancer cell line Caco-2 exposed to a hypertonic environment responded with marked VEGF and PGE2 production. VEGF production was inhibited by selective inhibitors of ERK 1/2 and p38 MAPK pathways. To address the regulatory role of PGE2 on VEGF production, Caco-2 cells were treated with cPLA2 (ATK) and COX-2 (NS-398) inhibitors, that completely block PGE2 generation. The Caco-2 cells were also treated with a non selective PGE2 receptor antagonist. Each treatment significantly increased the hypertonic stress-induced VEGF production. Moreover, addition of PGE2 or selective EP2 receptor agonist to activated Caco-2 cells inhibited VEGF production. The autocrine inhibitory role for PGE2 appears to be selective to hypertonic environment since VEGF production induced by exposure to CoCl2 was decreased by inhibition of concomitant PGE2 generation. Our results indicated that hypertonicity stimulates VEGF production in colon cancer cell lines. Also PGE2 plays an inhibitory role on VEGF production by Caco-2 cells exposed to hyperosmotic stress through EP2 activation.

SO2-Induced Neurotoxicity Is Mediated by Cyclooxygenases-2-Derived Prostaglandin E2 and its Downstream Signaling Pathway in Rat Hippocampal Neurons

Sulfur dioxide (SO(2)) pollution in atmospheric environment is involved in neurotoxicity and increased risk for hospitalization and mortality of many brain disorders; however, our understanding of the mechanisms by which SO(2) caused harmful insults on neurons remains elusive. Here, we show that SO(2) exposure produced a neuronal insult, and the neurotoxic effect was likely via stimulating cyclooxygenase-2 (COX-2) elevation by activation of nuclear factor-κB (NF-κB) activity and its acting on the promoter-distal NF-κB-binding site of COX-2 promoter. The action of SO(2) on elevating COX-2 ultimately appeared to be dependent on the increased production of arachidonic acid-derived prostaglandins, mainly prostaglandin E(2) (PGE(2)), and functioning of its EP2/4 receptors. Also, the molecular modulating process might be triggered by free radical attack from SO(2) metabolism in vivo and followed by activating cyclic adenosine monophosphate/protein kinase A pathway and enhancing probability of the release of glutamate, upregulating N-methyl-D-aspartic acid receptor expression and causing neuronal apoptosis. Our results reveal a mechanistic basis for exploring an association between SO(2) inhalation and increased risk for neurological disorders and opening up therapeutic approaches of treating, ameliorating, or preventing brain injuries resulting from SO(2) exposure in atmospheric polluting environment.

Biosynthesis of hemiketal eicosanoids by cross-over of the 5-lipoxygenase and cyclooxygenase-2 pathways

The prostaglandin and leukotriene families of lipid mediators are formed via two distinct biosynthetic pathways that are initiated by the oxygenation of arachidonic acid by either cyclooxygenase-2 (COX-2) or 5-lipoxygenase (5-LOX), respectively. The 5-LOX product 5S-hydroxyeicosatetraenoic acid, however, can also serve as an efficient substrate for COX-2, forming a bicyclic diendoperoxide with structural similarities to the arachidonic acid-derived prostaglandin endoperoxide PGH(2) [Schneider C, et al. (2006) J Am Chem Soc 128:720-721]. Here we identify two cyclic hemiketal (HK) eicosanoids, HKD(2) and HKE(2), as the major nonenzymatic rearrangement products of the diendoperoxide using liquid chromatography-mass spectrometry analyses as well as UV and NMR spectroscopy. HKD(2) and HKE(2) are furoketals formed by spontaneous cyclization of their respective 8,9-dioxo-5S,11R,12S,15S-tetrahydroxy- or 11,12-dioxo-5S,8S,9S,15S-tetrahydroxy-eicosadi-6E,13E-enoic acid precursors, resulting from opening of the 9S,11R- and 8S,12S-peroxide rings of the diendoperoxide. Furthermore, the diendoperoxide is an efficient substrate for the hematopoietic type of prostaglandin D synthase resulting in formation of HKD(2), equivalent to the enzymatic transformation of PGH(2) to PGD(2). HKD(2) and HKE(2) were formed in human blood leukocytes activated with bacterial lipopolysaccharide and calcium ionophore A23187, and biosynthesis was blocked by inhibitors of 5-LOX or COX-2. HKD(2) and HKE(2) stimulated migration and tubulogenesis of microvascular endothelial cells, implicating a proangiogenic role of the hemiketals in inflammatory sites that involve expression of 5-LOX and COX-2. Identification of the highly oxygenated hemiketal eicosanoids provides evidence for a previously unrecognized biosynthetic cross-over of the 5-LOX and COX-2 pathways.

Resolution of inflammation: the beginning programs the end

Acute inflammation normally resolves by mechanisms that have remained somewhat elusive. Emerging evidence now suggests that an active, coordinated program of resolution initiates in the first few hours after an inflammatory response begins. After entering tissues, granulocytes promote the switch of arachidonic acid-derived prostaglandins and leukotrienes to lipoxins, which initiate the termination sequence. Neutrophil recruitment thus ceases and programmed death by apoptosis is engaged. These events coincide with the biosynthesis, from omega-3 polyunsaturated fatty acids, of resolvins and protectins, which critically shorten the period of neutrophil infiltration by initiating apoptosis. Consequently, apoptotic neutrophils undergo phagocytosis by macrophages, leading to neutrophil clearance and release of anti-inflammatory and reparative cytokines such as transforming growth factor-beta1. The anti-inflammatory program ends with the departure of macrophages through the lymphatics. Understanding these and further details of the mechanism required for inflammation resolution may underpin the development of drugs that can resolve inflammatory processes in directed and controlled ways.

The formation of sulfur trioxide radical anion during the prostaglandin hydroperoxidase-catalyzed oxidation of bisulfite (hydrated sulfur dioxide).

The mechanism of prostaglandin synthase-dependent (bi)sulfite (hydrated sulfur dioxide) oxidation was investigated using an enzyme preparation derived from ram seminal vesicles. The horseradish peroxidase-catalyzed oxidation of (bi)sulfite was used as a model system. Incubation of (bi)sulfite with prostaglandin synthase and arachidonic acid, 15-hydroperoxyarachidonic acid, or H2O2 results in the formation of the reactive sulfur trioxide anion radical (SO3(-)). The horseradish peroxidase/H2O2 system also oxidizes (bi)sulfite to SO3(-). This free radical reacts with oxygen resulting in oxygen consumption by these incubations. The free radical was detected with the indirect electron spin resonance technique of spin trapping. The SO3(-) radical adduct formed by the reaction of SO3(-) with the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide, gives a nitroxide free radical with a nearly unique electron spin resonance spectrum (aH = 16.0 G and aN = 14.7 G). Using the spin-trapping technique, the SO3(-) could be detected even in incubations of guinea pig lung microsomes. When arachidonic acid-derived prostaglandin G2 was the source of hydroperoxide, formation of SO3(-) could be inhibited by indomethacin. When 15-hydroperoxyarachidonic acid or hydrogen peroxide was used to drive the enzymatic oxidation of (bi)sulfite, indomethacin had no effect. This hydroperoxidase activity was not nearly as heat-labile as the cyclo-oxygenase reaction which forms prostaglandin G2. Finally, the peroxidatic oxidation of (bi)sulfite may occur in vivo in competition with the mitochondrial sulfite oxidase, which oxidizes (bi)sulfite to sulfate without the formation of free radicals.