Prostaglandin D2 mediates neuronal protection via the DP1 receptor

Cyclo-oxygenases (COXs) catalyze the first committed step in the synthesis of the prostaglandins PGE(2), PGD(2), PGF(2alpha), PGI(2) and thomboxane A(2). Expression and enzymatic activity of COX-2, the inducible isoform of COX, are observed in several neurological diseases and result in significant neuronal injury. The neurotoxic effect of COX-2 is believed to occur through downstream effects of its prostaglandin products. In this study, we examined the function of PGD(2) and its two receptors DP1 and chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) (DP2) in neuronal survival. PGD(2) is the most abundant prostaglandin in brain and regulates sleep, temperature and nociception. It signals through two distinct G protein-coupled receptors, DP1 and DP2, that have opposing effects on cyclic AMP (cAMP) production. Physiological concentrations of PGD(2) potently and unexpectedly rescued neurons in paradigms of glutamate toxicity in cultured hippocampal neurons and organotypic slices. This effect was mimicked by the DP1-selective agonist BW245C but not by the PGD(2) metabolite 15d-PGJ(2), suggesting that neuroprotection was mediated by the DP1 receptor. Conversely, activation of the DP2 receptor promoted neuronal loss. The protein kinase A inhibitors H89 and KT5720 reversed the protective effect of PGD(2), indicating that PGD(2)-mediated neuroprotection was dependent on cAMP signaling. These studies indicate that activation of the PGD(2) DP1 receptor protects against excitotoxic injury in a cAMP-dependent manner, consistent with recent studies of PGE(2) receptors that also suggest a neuroprotective effect of prostaglandin receptors. Taken together, these data support an emerging and paradoxical neuroprotective role of prostaglandins in the CNS.     

Non-redundant functions of cyclooxygenases: oxygenation of endocannabinoids

The two cyclooxygenase (COX) enzymes catalyze the oxygenation of arachidonic acid to prostaglandin endoperoxides, which are the common intermediates in the biosynthesis of the bioactive lipids prostaglandins and thromboxane. COX-1 and COX-2 are approximately 60% identical in amino acid sequence, exhibit highly homologous three-dimensional structures, and appear functionally similar at the biochemical level. Recent work has uncovered a subtle functional difference between the two enzymes, namely the ability of COX-2 to efficiently utilize neutral derivatives (esters and amides) of arachidonic acid as substrates. Foremost among these neutral substrates are the endocannabinoids 2-arachidonoylglycerol and arachidonoylethanolamide. This raises the possibility that COX-2 oxygenation plays a role in a novel signaling pathway dependent on agonist-induced release of endocannabinoids and their selective oxygenation by COX-2. Among the products of COX-2 oxygenation of endocannabinoids are glyceryl prostaglandins, some of which (e.g. glyceryl prostaglandin E(2) and glyceryl prostaglandin I(2)) exhibit interesting biological activities in inflammatory, neurological, and vascular systems. These compounds are produced in intact cells stimulated with physiological agonists and have been isolated from in vivo sources. Important concepts relevant to the hypothesis of a COX-2-selective signaling pathway are presented.     

Oxygenation of the endocannabinoid, 2-arachidonylglycerol, to glyceryl prostaglandins by cyclooxygenase-2

Cyclooxygenases (COX) play an important role in lipid signaling by oxygenating arachidonic acid to endoperoxide precursors of prostaglandins and thromboxane. Two cyclooxygenases exist which differ in tissue distribution and regulation but otherwise carry out identical chemical functions. The neutral arachidonate derivative, 2-arachidonylglycerol (2-AG), is one of two described endocannabinoids and appears to be a ligand for both the central (CB1) and peripheral (CB2) cannabinoid receptors. Here we report that 2-AG is a substrate for COX-2 and that it is metabolized as effectively as arachidonic acid. COX-2-mediated 2-AG oxygenation provides the novel lipid, prostaglandin H(2) glycerol ester (PGH(2)-G), in vitro and in cultured macrophages. PGH(2)-G produced by macrophages is a substrate for cellular PGD synthase, affording PGD(2)-G. Pharmacological studies reveal that macrophage production of PGD(2)-G from endogenous sources of 2-AG is calcium-dependent and mediated by diacylglycerol lipase and COX-2. These results identify a distinct function for COX-2 in endocannabinoid metabolism and in the generation of a new family of prostaglandins derived from diacylglycerol and 2-AG.     

Structural and functional differences between cyclooxygenases: fatty acid oxygenases with a critical role in cell signaling

Cyclooxygenase (COX) catalyzes the first two steps in the conversion of arachidonic acid (AA) to prostaglandins (PGs). The reaction mechanism is well-defined and supported by extensive structural data. There are two isoforms of COX, which are nearly indistinguishable in structure and mechanism, however, COX-2 oxygenates neutral derivatives of AA that are poor substrates for COX-1. The best neutral substrate is 2-arachidonylglycerol, oxygenation of which produces an array of prostaglandin glyceryl esters (PG-Gs) that is nearly as diverse as the PGs. The mobilization of Ca2+ by subnanomolar concentrations of PGE2-G in RAW264.7 cells suggests the existence of a distinct receptor, and the formation of PG-Gs by zymosan-stimulated macrophages indicates that these species may be formed in vivo. These findings suggest that PG-Gs comprise a new class of lipid mediators, and that oxygenation of neutral derivatives of AA is a distinct function for the COX-2 isoform.     

Effect of prostaglandin E2 and prostaglandin I2 on PDGF-induced proliferation of LI90, a human hepatic stellate cell line

Hepatic stellate cells (HSC) are central to liver fibrosis. The eicosanoid pathway and cyclooxygenase-2 (COX-2) may be an important signaling mechanism in HSC. We investigated the role of COX-2, prostaglandin E(2) (PGE(2)) and prostaglandin I(2) (PGI(2)) in proliferation of LI90, an immortalized cell line of HSC. Our results showed that COX-2 was upregulated by platelet-derived growth factor (PDGF), a mitogen in HSC. COX-2 was responsible for the production of PGE(2) and PGI(2) in PDGF-stimulated LI90 cells. Furthermore, we demonstrated that COX-2 and PGE(2) mediated the proliferative response of LI90 to PDGF while synthetic analogue of PGI(2) exhibited anti-proliferative effect. Our findings suggest complex interactions of prostaglandins in liver fibrogenesis. In vivo studies using animal models are needed to elucidate the effect of COX-2 inhibition by non-steroidal anti-inflammatory drugs or COX-2 inhibitor in hepatic fibrosis.     

Metabolism of prostaglandin glycerol esters and prostaglandin ethanolamides in vitro and in vivo.

Prostaglandin glycerol esters (PG-Gs) and prostaglandin ethanolamides (PG-EAs) are generated by the action of cyclooxygenase-2 on the endocannabinoids 2-arachidonylglycerol (2-AG) and arachidonylethanolamide, respectively. These novel eicosanoids may have unique pharmacological properties and/or serve as latent sources of prostaglandins at sites remote from their tissue of origin. Therefore, we investigated the metabolism of PG-Gs and PG-EAs in vitro and in vivo. PGE(2)-G was rapidly hydrolyzed in rat plasma to generate PGE(2) (t(1/2) = 14 s) but was only slowly metabolized in human plasma (t(1/2) > 10 min). An intermediate extent of metabolism of PGE(2)-G was observed in human whole blood (t(1/2) approximately 7 min). The parent arachidonylglycerol, 2-AG, and the more stable regioisomer, 1-AG, also were much more rapidly metabolized in rat plasma compared with human plasma. PGE(2)-EA was not significantly hydrolyzed in plasma, undergoing slow dehydration/isomerization to PGB(2)-EA. Both PGE(2)-G and PGE(2)-EA were stable in canine, bovine, and human cerebrospinal fluid. Human 15-hydroxyprostaglandin dehydrogenase, the enzyme responsible for the initial step in PG inactivation in vivo, oxidized both PGE(2)-G and PGE(2)-EA less efficiently than the free acid. The sterically hindered glyceryl prostaglandin was the poorest substrate examined in the E series. Minimal 15-hydroxyprostaglandin dehydrogenase oxidation of PGF(2 alpha)-G was observed. PGE(2)-G and PGE(2)-EA pharmacokinetics were assessed in rats. PGE(2)-G was not detected in plasma 5 min following an intravenous dose of 2 mg/kg. However, PGE(2)-EA was detectable up to 2 h following an identical dose, displaying a large apparent volume of distribution and a half-life of over 6 min. The results suggest that endocannabinoid-derived PG-like compounds may be sufficiently stable in humans to exert actions systemically. Furthermore, these results suggest that the rat is not an adequate model for investigating the biological activities of 2-arachidonylglycerol or glyceryl prostaglandins in humans.     

The effect of ascorbic acid on Helicobacter pylori induced cyclooxygenase 2 expression and prostaglandin E2 production by gastric epithelial cells in vitro

BACKGROUND: Cyclooxygenase 2 (COX-2) is induced by the presence of Helicobacter pylori (H. pylori) on the gastric mucosa as part of the inflammatory response; this results in the synthesis of prostaglandins that amplify the local inflammatory response. The presence of H. pylori inhibits the secretion of ascorbate into the gastric lumen. Interestingly, ascorbate inhibits the growth of H. pylori and low dietary levels are associated with an increased risk of gastric adenocarcinoma. We therefore investigated the effect of ascorbate on H. pylori mediated COX-2 induction and prostaglandin production in vitro. METHODS: H. pylori was cocultured with gastric epithelial cells in the presence of ascorbate at physiological concentrations. The expression of COX-2 was assessed by Western blotting and prostaglandin E(2) (PGE(2)) was assessed by ELISA. RESULTS: Ascorbate inhibited gastric cell PGE(2) synthesis but not in COX-2 expression in response to H. pylori. In the absence of the organism, ascorbate also reduced PGE(2) expression in cells that constitutively express COX-2, again with no reduction of COX-2 protein expression. CONCLUSIONS: Physiological concentrations of ascorbate inhibit PGE(2) but not COX-2 expression in response to H. pylori in gastric epithelial cells.     

Enhanced expression of cyclooxygenase-2 and prostaglandin E2 in response to endotoxin after trauma is dependent on MAPK and NF-kappaB mechanisms

Macrophage prostaglandin E2 (PGE2) production is important in cellular immune suppression and in affecting the potential development of sepsis after trauma. We hypothesized that macrophage PGE2 production after trauma is regulated by mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-kappaB). Mice were subjected to trauma and splenic macrophages isolated 7 days later. Macrophages from traumatized mice showed increased cyclooxygenase-2 (COX-2) mRNA, protein expression, and PGE2 production compared with controls. Increased phosphorylation of extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38 kinase was observed in macrophages from traumatized mice. Pharmacologic inhibition of MAPK blocked trauma-induced COX-2 expression, and PGE2 production. Trauma macrophages showed increased IkappaBalpha phosphorylation and NF-kappaB binding to DNA. Inhibiting IkappaBalpha blocked trauma-induced NF-kappaB activity, COX-2 expression and PGE2 production. This suggests that trauma-induced PGE2 production is mediated through MAPK and NF-kappaB activation and offers potential for modifying the macrophages' responses following injury.     

Neuronal Cyclooxygenase-2 Activity and Prostaglandins PGE2, PGD2, and PGF2α Exacerbate Hypoxic Neuronal Injury in Neuron-enriched Primary Culture

Cyclooxygenase-2 (COX-2) activity has been implicated in the pathogenesis of cerebral ischemia. To determine whether COX-2 activity within the neuron itself exacerbates hypoxic neuronal injury, neuron-enriched cultures were subjected to anoxia. Treatment with COX-2 selective antagonists decreased cell death. Neurons cultured from homozygous COX-2 gene disrupted mice were resistant to hypoxia compared to those of heterozygotes. Infection of primary neurons with AAV expressing COX-2 exacerbated cell death compared to neurons infected with enhanced green fluorescent protein (EGFP) control vector. Addition of PGE2, PGD2 or PGF2α to the medium exacerbated injury, suggesting that the deleterious effects of COX-2 overexpression in hypoxia could be mediated by direct receptor mediated effects of prostaglandins. Overexpression of COX-2 did not increase expression of cyclin D1 or phosphoretinoblastoma protein (pRb), or cleavage of caspase 3 suggesting that this cell cycle mechanism does not mediate COX-2 toxicity in this model.     

COX-2调节的前列腺素在突触信号传递中的作用

环氧合酶-2 (cyclooxygenase-2,COX-2)是催化花生四烯酸转化为前列腺素的限速酶,广泛参与脑创伤、缺血诱导的神经元损伤、炎症反应及神经变性性疾病等.COX-2在神经病理学中的作用与神经元的突触变化有关.增强或抑制COX-2表达可增强或抑制兴奋性谷氨酸能神经元的神经传递和长时程增强 (LTP),这些效应由COX-2的主要产物前列腺素E2(PGE2)及其受体亚型EP2所介导.因此,阐明COX-2在突触信号中的作用机制将有助于设计新的药物来预防、治疗及减轻神经源性炎症相关的神经紊乱性疾病.     

Inhibited skeletal muscle healing in cyclooxygenase-2 gene-deficient mice: the role of PGE2 and PGF2alpha.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used to treat skeletal muscle injury. However, studies have shown that NSAIDs may be detrimental to the healing process. Mediated by prostaglandin F(2alpha) (PGF(2alpha)) and prostaglandin E(2) (PGE(2)), the cycloxygenase-2 (COX-2) pathway plays an important role in muscle healing. We hypothesize that the COX-2 pathway is important for the fusion of muscle cells and the regeneration of injured muscle. For the in vitro experiments, we isolated myogenic precursor cells from wild-type (Wt) and COX-2 gene-deficient (COX-2(-/-)) mice and examined the effect of PGE(2) and PGF(2alpha) on cell fusion. For the in vivo experiments, we created laceration injury on the tibialis anterior (TA) muscles of Wt and COX-2(-/-) mice. Five and 14 days after injury, we examined the TA muscles histologically and functionally. We found that the secondary fusion between nascent myotubes and myogenic precursor cells isolated from COX-2(-/-) mice was severely compromised compared with that of Wt controls but was restored by the addition of PGF(2alpha) or, to a lesser extent, PGE(2) to the culture. Histological and functional assessments of the TA muscles in COX-2(-/-) mice revealed decreased regeneration relative to that observed in the Wt mice. The findings reported here demonstrate that the COX-2 pathway plays an important role in muscle healing and that prostaglandins are key mediators of the COX-2 pathway. It suggests that the decision to use NSAIDs to treat muscle injuries warrants critical evaluation because NSAIDs might impair muscle healing by inhibiting the fusion of myogenic precursor cells.     

13-Methylarachidonic Acid Is a Positive Allosteric Modulator of Endocannabinoid Oxygenation by Cyclooxygenase

Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid (AA) and the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonylethanolamide to prostaglandins, prostaglandin glyceryl esters, and prostaglandin ethanolamides, respectively. A structural homodimer, COX-2 acts as a conformational heterodimer with a catalytic and an allosteric monomer. Prior studies have demonstrated substrate-selective negative allosteric regulation of 2-AG oxygenation. Here we describe AM-8138 (13(S)-methylarachidonic acid), a substrate-selective allosteric potentiator that augments 2-AG oxygenation by up to 3.5-fold with no effect on AA oxygenation. In the crystal structure of an AM-8138·COX-2 complex, AM-8138 adopts a conformation similar to the unproductive conformation of AA in the substrate binding site. Kinetic analysis suggests that binding of AM-8138 to the allosteric monomer of COX-2 increases 2-AG oxygenation by increasing k_cat and preventing inhibitory binding of 2-AG. AM-8138 restored the activity of COX-2 mutants that exhibited very poor 2-AG oxygenating activity and increased the activity of COX-1 toward 2-AG. Competition of AM-8138 for the allosteric site prevented the inhibition of COX-2-dependent 2-AG oxygenation by substrate-selective inhibitors and blocked the inhibition of AA or 2-AG oxygenation by nonselective time-dependent inhibitors. AM-8138 selectively enhanced 2-AG oxygenation in intact RAW264.7 macrophage-like cells. Thus, AM-8138 is an important new tool compound for the exploration of allosteric modulation of COX enzymes and their role in endocannabinoid metabolism.     

In vivo activation of N-methyl-D-aspartate receptors in the rat hippocampus increases prostaglandin E(2) extracellular levels and triggers lipid peroxidation through cyclooxygenase-mediated mechanisms

Cyclooxygenases (COX) are a family of enzymes involved in the biosynthesis of prostaglandin (PG) and thromboxanes. The inducible enzyme cyclooxygenase-2 (COX-2) is the major isoform found in normal brain, where it is constitutively expressed in neurons and is further up-regulated during several pathological events, including seizures and ischaemia. Emerging evidence suggests that COX-2 is implicated in excitotoxic neurodegenerative phenomena. It remains unclear whether PGs or other products associated to COX activity take part in these processes. Indeed, it has been suggested that reactive oxygen species, produced by COX, could mediate neuronal damage. In order to obtain direct evidence of free radical production during COX activity, we undertook an in vivo microdialysis study to monitor the levels of PGE(2) and 8-epi-PGF(2alpha) following infusion of N-methyl-D-aspartate (NMDA). A 20-min application of 1 mm NMDA caused an immediate, MK-801-sensitive increase of both PGE(2) and 8-epi-PGF(2alpha) basal levels. These effects were largely prevented by the specific cytosolic phospholipase A(2) (cPLA(2) ) inhibitor arachidonyl trifluoromethyl ketone (ATK), by non- selective COX inhibitors indomethacin and flurbiprofen or by the COX-2 selective inhibitor NS-398, suggesting that the NMDA-evoked prostaglandin synthesis and free radical-mediated lipid peroxidation are largely dependent on COX-2 activity. As several lines of evidence suggest that prostaglandins may be potentially neuroprotective, our findings support the hypothesis that free radicals, rather than prostaglandins, mediate the toxicity associated to COX-2 activity.     

Simultaneous analysis of prostaglandin glyceryl esters and prostaglandins by electrospray tandem mass spectrometry

A high-performance liquid chromatography (HPLC) positive-ion electrospray ionization tandem mass spectrometry method for the quantification of prostaglandin glyceryl esters (PG-Gs), a newly discovered class of eicosanoids, is described. All four PG-Gs (PGE(2)-G, PGD(2)-G, PGF(2alpha)-G, and 6-keto-PGF(1alpha)-G) and the prostaglandins (PGs) that are formed by their hydrolysis are simultaneously quantified. Analytes were purified via reverse-phase solid-phase extraction, separated by reverse-phase HPLC, and quantified on a triple-quadrupole mass spectrometer using selected reaction monitoring. Quantification was achieved by stable isotope dilution employing penta-deuterated (PG-Gs) or tetra-deuterated (PGs) analogs. Analyte recovery from cell culture medium was >43% for all analytes at four different concentration levels. The limit of quantification is in the range of 25fmol on-column for each analyte and the analytes exhibit a linear response over approximately a 500-fold range. This method allows simultaneous profiling of several PG-Gs and PGs without multistep sample purification or derivatization.     

Assessment of the relative contribution of COX-1 and COX-2 isoforms to ischemia-induced oxidative damage and neurodegeneration following transient global cerebral ischemia

We investigated the relative contribution of COX-1 and/or COX-2 to oxidative damage, prostaglandin E2 (PGE2) production and hippocampal CA1 neuronal loss in a model of 5 min transient global cerebral ischemia in gerbils. Our results revealed a biphasic and significant increase in PGE2 levels after 2 and 24-48 h of reperfusion. The late increase in PGE2 levels (24 h) was more potently reduced by the highly selective COX-2 inhibitor rofecoxib (20 mg/kg) relative to the COX-1 inhibitor valeryl salicylate (20 mg/kg). The delayed rise in COX catalytic activity preceded the onset of histopathological changes in the CA1 subfield of the hippocampus. Post-ischemia treatment with rofecoxib (starting 6 h after restoration of blood flow) significantly reduced measures of oxidative damage (glutathione depletion and lipid peroxidation) seen at 48 h after the initial ischemic episode, indicating that the late increase in COX-2 activity is involved in the delayed occurrence of oxidative damage in the hippocampus after global ischemia. Interestingly, either selective inhibition of COX-2 with rofecoxib or inhibition of COX-1 with valeryl salicylate significantly increased the number of healthy neurons in the hippocampal CA1 sector even when the treatment began 6 h after ischemia. These results provide the first evidence that both COX isoforms are involved in the progression of neuronal damage following global cerebral ischemia, and have important implications for the potential therapeutic use of COX inhibitors in cerebral ischemia.     

Metabolism of the endocannabinoids, 2-arachidonylglycerol and anandamide,into prostaglandin,thromboxane, and prostacyclin glycerol esters and ethanolamides

Cyclooxygenase-2 (COX-2) action on the endocannabinoids, 2-arachidonylglycerol (2-AG) and anandamide (AEA), generates prostaglandin glycerol esters (PG-G) and ethanolamides (PG-EA), respectively. The diversity of PG-Gs and PG-EAs that can be formed enzymatically following COX-2 oxygenation of endocannabinoids was examined in cellular and subcellular systems. In cellular systems, glycerol esters and ethanolamides of PGE(2), PGD(2), and PGF(2alpha) were major products of the endocannabinoid-derived COX-2 products, PGH(2)-G and PGH(2)-EA. The sequential action of purified COX-2 and thromboxane synthase on AEA and 2-AG provided thromboxane A(2) ethanolamide and glycerol ester, respectively. Similarly, bovine prostacyclin synthase catalyzed the isomerization of the intermediate endoperoxides, PGH(2)-G and PGH(2)-EA, to the corresponding prostacyclin derivatives. Quantification of the efficiency of prostaglandin and thromboxane synthase-directed endoperoxide isomerization demonstrated that PGE, PGD, and PGI synthases catalyze the isomerization of PGH(2)-G at rates approaching those observed with PGH(2). In contrast, thromboxane synthase was far more efficient at catalyzing PGH(2) isomerization than at catalyzing the isomerization of PGH(2)-G. These results define the in vitro diversity of endocannabinoid-derived prostanoids and will permit focused investigations into their production and potential biological actions in vivo.     

Effect of H. pylori infection on the expression of cyclooxygenase-2 in human gastric mucosa

Cyclooxygenase-1 is the primary isoform responsible for the production of cytoprotective prostaglandins (PGE(2) and PGI(2)) in the stomach. In contrast COX-2 is induced at the sites of inflammation. Using Helicobacter pylori infection as a model of inflammation, this study was designed to evaluate the effects of H. pylori infection on prostanoid synthesis and expression of COX-2 in human gastric mucosa.Prostaglandin (PGE(2)) and prostacyclin (PGI(2)) synthesis in gastric biopsies obtained from 21 patients undergoing diagnostic endoscopy, were determined. H. pylori was detected by CLO test, histology and culture. Biopsy samples were incubated either with NS-398, selective COX-2 inhibitor or aspirin. Samples were also treated with endotoxin (LPS) in order to induce COX-2 expression. Tissue was also analysed for COX-2 expression in vivo by immunohistochemistry.In 15 out of 21 patients, H. pylori was detected by at least two of the three methods. Higher levels of PGE(2) and PGI(2) were seen in patients infected with H. pylori (191+/-30 and 245+/-88ng/mg protein, respectively) compared with non-infected patients (77+/-17 and 120+/-36ng/mg protein, respectively). There was significant inhibition of PGE(2) and PGI(2) with aspirin in both H. pylori infected (28+/-6.6 and 53+/-43ng/mg, respectively) and in non-infected patients (16+/-7 and 12.5+/-3.5ng/mg protein, respectively). However, NS-398 and LPS did not alter prostaglandin function significantly. Immunohistochemistry in all patients irrespective of Hp status demonstrated expression of COX-2.Lower concentration of constitutive expression of COX-2 was detected in human gastric mucosa by immunohistochemistry, however, H. pylori infection failed to induce COX-2 protein. In addition, increased prostaglandin synthesis in Hp-infected patients appears to be COX-1 mediated rather than COX-2. Furthermore, failure of endotoxaemia-treated sample to produce more PGE(2) in the face of enhanced COX-2 expression in gastric mucosa further suggests that increased prostanoids in human gastric stomach are COX-1 mediated.