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.     

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.     

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.     

COX-2 oxidative metabolite of endocannabinoid 2-AG enhances excitatory glutamatergic synaptic transmission and induces neurotoxicity

Neuroinflammation has been implicated in the pathogenesis of neurodegenerative diseases. Cyclooxygenase-2 (COX-2), an inducible enzyme converting arachidonic acid (AA) to prostaglandins, is the key player in neuroinflammation. It has been long thought that the COX-2-mediated neuronal injury/degeneration is attributed to the increased production of AA-derived prostaglandins. Recent studies show that endogenous cannabinoid 2-arachidonoylglycerol (2-AG) is a natural substrate for COX-2, and it can be oxygenated by COX-2 to form prostaglandin glyceryl esters. In this study, we demonstrate that prostaglandin E(2) glyceryl ester (PGE(2)-G), a major COX-2 oxidative metabolite of 2-arachidonoylglycerol, enhanced hippocampal glutamatergic synaptic transmission indicated by the increased frequency of miniature excitatory post-synaptic currents, and induced neuronal injury/death revealed by the terminal transferase dUTP nick end labeling staining and caspase 3 activation. The actions of PGE(2)-G are not mediated via a cannabinoid receptor 1, but mediated through ERK, p38 mitogen-activated protein kinase, IP(3), and NF-kappaB signal transduction pathways. In addition, the PGE(2)-G-induced neurotoxicity is attenuated by blockade of the NMDA receptors. Our results suggest that the COX-2 oxidative metabolism of endocannabinoids is an important mechanism contributing to the inflammation-induced neurodegeneration.     

Hydrolysis of prostaglandin glycerol esters by the endocannabinoid-hydrolyzing enzymes, monoacylglycerol lipase and fatty acid amide hydrolase

Cyclooxygenase-2 (COX-2) can oxygenate the endocannabinoids, arachidonyl ethanolamide (AEA) and 2-arachidonylglycerol (2-AG), to prostaglandin-H2-ethanolamide (PGH2-EA) and -glycerol ester (PGH2-G), respectively. Further metabolism of PGH2-EA and PGH2-G by prostaglandin synthases produces a variety of prostaglandin-EA's and prostaglandin-G's nearly as diverse as those derived from arachidonic acid. Thus, COX-2 may regulate endocannabinoid levels in neurons during retrograde signaling or produce novel endocannabinoid metabolites for receptor activation. Endocannabinoid-metabolizing enzymes are important regulators of their action, so we tested whether PG-G levels may be regulated by monoacylglycerol lipase (MGL) and fatty acid amide hydrolase (FAAH). We found that PG-Gs are poor substrates for purified MGL and FAAH compared to 2-AG and/or AEA. Determination of substrate specificity demonstrates a 30-100- and 150-200-fold preference of MGL and FAAH for 2-AG over PG-Gs, respectively. The substrate specificity of AEA compared to those of PG-Gs was approximately 200-300 fold higher for FAAH. Thus, PG-Gs are poor substrates for the major endocannabinoid-degrading enzymes, MGL and FAAH.     

Fishing for prostanoids: deciphering the developmental functions of cyclooxygenase-derived prostaglandins

Prostaglandin G/H synthases (PGHS), commonly referred to as cyclooxygenases (COX-1 and COX-2), catalyze a key step in the synthesis of biologically active prostaglandins (PGs), the conversion of arachidonic acid (AA) into prostaglandin H(2) (PGH(2)). PGs have important functions in a variety of physiologic and pathologic settings, including inflammation, cardiovascular homeostasis, reproduction, and carcinogenesis. However, an evaluation of prostaglandin function in early development has been difficult due to the maternal contribution of prostaglandins from the uterus. The emergence of zebrafish as a model system has begun to provide some insights into the roles of this signaling cascade during vertebrate development. In zebrafish, COX-1 derived prostaglandins are required for two distinct stages of development, namely during gastrulation and segmentation. During gastrulation, PGE(2) signaling promotes cell motility, without altering the cell shape or directional migration of gastrulating cells. During segmentation, COX-1 signaling is also required for posterior mesoderm development, including the formation of vascular tube structures, angiogenesis of intersomitic vessels, and pronephros morphogenesis. We propose that deciphering the role for prostaglandin signaling in zebrafish development could yield insight and ultimately address the mechanistic details underlying various disease processes that result from perturbation of this pathway.     

Stereospecificity of hydrogen abstraction in the conversion of arachidonic acid to 15R-HETE by aspirin-treated cyclooxygenase-2. Implications for the alignment of substrate in the active site

The initial and rate-limiting step in prostaglandin biosynthesis is stereoselective removal of the pro-S hydrogen from the 13-carbon of arachidonic acid. This is followed by oxygenation at C-11, formation of the five-membered ring, and a second oxygenation at C-15 to yield the endoperoxide product, prostaglandin G(2). Aspirin treatment of cyclooxygenase-2 is known to acetylate an active site serine, block prostaglandin biosynthesis, and give 15R-hydroxyeicosatetraenoic acid (15R-HETE) as the only product. 15R-HETE and prostaglandins have opposite stereoconfigurations of the 15-hydroxyl. To understand the changes that lead to 15R-HETE synthesis in aspirin-treated COX-2, we employed pro-R- and pro-S-labeled [13-(3)H]arachidonic acids to investigate the selectivity of the initial hydrogen abstraction. Remarkably, aspirin-treated COX-2 formed 15R-HETE with removal of the pro-S hydrogen at C-13 (3-9% retention of pro-S tritium label), the same stereoselectivity as in the formation of prostaglandins by native cyclooxygenase. To account for this result and the change in oxygenase specificity, we suggest that the bulky serine acetyl group forces a realignment of the omega end of the arachidonic acid carbon chain. This can rationalize abstraction of the C-13 pro-S hydrogen, the blocking of prostaglandin synthesis, and the formation of 15R-HETE as the sole enzymatic product.     

(R)-Profens are substrate-selective inhibitors of endocannabinoid oxygenation by COX-2

Cyclooxygenase-2 (COX-2) catalyzes the oxygenation of arachidonic acid and the endocannabinoids 2-arachidonoylglycerol and arachidonoylethanolamide. Evaluation of a series of COX-2 inhibitors revealed that many weak competitive inhibitors of arachidonic acid oxygenation are potent inhibitors of endocannabinoid oxygenation. (R) enantiomers of ibuprofen, naproxen and flurbiprofen, which are considered to be inactive as COX-2 inhibitors, are potent 'substrate-selective inhibitors' of endocannabinoid oxygenation. Crystal structures of the COX-2–(R)-naproxen and COX-2–(R)-flurbiprofen complexes verified this unexpected binding and defined the orientation of the (R) enantiomers relative to (S) enantiomers. (R)-Profens selectively inhibited endocannabinoid oxygenation by lipopolysaccharide-stimulated dorsal root ganglion (DRG) cells. Substrate-selective inhibition provides new tools for investigating the role of COX-2 in endocannabinoid oxygenation and a possible explanation for the ability of (R)-profens to maintain endocannabinoid tone in models of neuropathic pain.     

Amino acid determinants in cyclooxygenase-2 oxygenation of the endocannabinoid anandamide

The endocannabinoid arachidonylethanolamide (AEA, anandamide) is an endogenous ligand for the cannabinoid receptors and has been shown to be oxygenated by cyclooxygenase-2 (COX-2). We examined the structural requirements for COX-mediated, AEA oxygenation using a number of substrate analogues and site-directed mutants of COX-2. Fourteen AEA analogues were synthesized and tested as COX substrates. These studies identified the hydroxyl moiety of AEA as a critical determinant in the ability of COX enzymes to effect robust endocannabinoid oxygenation. In addition, these studies suggest that subtle structural modifications of AEA analogues near the ethanolamide moiety can result in pronounced changes in their ability to serve as COX-2 substrates. Site-directed mutagenesis studies have permitted the development of a model of AEA binding within the COX-2 active site. As with arachidonic acid, the omega-terminus of AEA binds in a hydrophobic alcove near the top of the COX-2 active site. The polar ethanolamide moiety of AEA, like the carboxylate of arachidonate, interacts with Arg-120 at the bottom of the COX-2 active site. Mutation of Tyr-385 prevents AEA oxygenation, suggesting that, as in the case of other COX substrates, AEA metabolism is initiated by Tyr-385-mediated hydrogen abstraction. Thus, AEA binds within the COX-2 active site in a conformation roughly similar to that of arachidonic acid. However, important differences have been identified that account for the isoform selectivity of AEA oxygenation. Importantly, the COX-2 side pocket and Arg-513 in particular are critical determinants of the ability of COX-2 to efficiently generate prostaglandin H(2) ethanolamide. The reduced efficiency of COX-1-mediated, AEA oxygenation can thus be explained by the absence of an arginine residue at position 513 in this isoform. Mutational analysis of Leu-531, an amino acid located directly across from the COX-2 side pocket, suggests that AEA is shifted away from this hydrophobic residue and toward Arg-513 relative to arachidonic acid. Coupled with earlier observations with the endocannabinoid 2-arachidonylglycerol, these results indicate that one possible function of the highly conserved COX-2 active site side pocket is to promote endocannabinoid oxygenation.     

Many actions of cyclooxygenase-2 in cellular dynamics and in cancer

Cyclooxygenase-2 (COX-2) is the inducible isoform of cyclooxygenase, the enzyme that catalyzes the rate-limiting step in prostaglandin synthesis from arachidonic acid. Various prostaglandins are produced in a cell type-specific manner, and they elicit cellular functions via signaling through G-protein coupled membrane receptors, and in some cases, through the nuclear receptor PPAR. COX-2 utilization of arachidonic acid also perturbs the level of intracellular free arachidonic acid and subsequently affects cellular functions. In a number of cell and animal models, induction of COX-2 has been shown to promote cell growth, inhibit apoptosis and enhance cell motility and adhesion. The mechanisms behind these multiple actions of COX-2 are largely unknown. Compelling evidence from genetic and clinical studies indicates that COX-2 upregulation is a key step in carcinogenesis. Overexpression of COX-2 is sufficient to cause tumorigenesis in animal models and inhibition of the COX-2 pathway results in reduction in tumor incidence and progression. Therefore, the potential for application of non-steroidal anti-inflammatory drugs as well as the recently developed COX-2 specific inhibitors in cancer clinical practice has drawn tremendous attention in the past few years. Inhibition of COX-2 promises to be an effective approach in the prevention and treatment of cancer, especially colorectal cancer.     

Inhibition of Endocannabinoid Metabolism by the Metabolites of Ibuprofen and Flurbiprofen

Background

In addition to their effects upon prostaglandin synthesis, the non-steroidal anti-inflammatory drugs ibuprofen and flurbiprofen inhibit the metabolism of the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (AEA) by cyclooxygenase-2 (COX-2) and fatty acid amide hydrolase (FAAH), respectively. Here, we investigated whether these effects upon endocannabinoid metabolism are shared by the main metabolites of ibuprofen and flurbiprofen.

Methodology/Principal Findings

COX activities were measured via changes in oxygen consumption due to oxygenation of arachidonic acid (for COX-1) and arachidonic acid and 2-AG (for COX-2). FAAH activity was quantified by measuring hydrolysis of tritium labelled AEA in rat brain homogenates. The ability of ibuprofen and flurbiprofen to inhibit COX-2-catalysed oxygenation of 2-AG at lower concentrations than the oxygenation of arachidonic acid was seen with 4′-hydroxyflurbiprofen and possibly also 3′-hydroxyibuprofen, albeit at lower potencies than the parent compounds. All ibuprofen and flurbiprofen metabolites retained the ability to inhibit FAAH in a pH-dependent manner, although the potency was lower than seen with the parent compounds.

Conclusions/Significance

It is concluded that the primary metabolites of ibuprofen and flurbiprofen retain some of the properties of the parent compound with respect to inhibition of endocannabinoid metabolism. However, these effects are unlikely to contribute to the actions of the parent compounds in vivo.     

Control of prostaglandin stereochemistry at the 15-carbon by cyclooxygenases-1 and -2. A critical role for serine 530 and valine 349.

Prostaglandin synthesis by cyclooxygenases-1 and -2 (COX-1 and COX-2) involves an initial oxygenation of arachidonic acid at C-11, followed by endoperoxide and cyclopentane ring formation, and then a second reaction with molecular oxygen in the S configuration at C-15. The resulting 15S-hydroxyl group of prostaglandins is crucial for their bioactivity. Using human COX-1 and human and murine COX-2, we have identified two amino acids located in the oxygenase active site that control the stereochemistry at C-15. The most crucial determinant is Ser-530, the residue that is acetylated by aspirin. In COX-2, site-directed mutagenesis of Ser-530 to methionine, threonine, or valine produced highly active enzymes that formed 82-95% 15R-configuration prostaglandins; these have the opposite stereochemistry at C-15 to the natural products. In COX-1, the corresponding Ser-530 mutations inactivated the enzyme. The second residue, Val-349, exerts a more subtle influence. When Val-349 was replaced by isoleucine, the mutant COX-1 and COX-2 enzymes formed 41 and 65% 15R-prostaglandins, respectively. This change was highly specific for isoleucine, as mutations of Val-349 to alanine, leucine, asparagine, or threonine did not alter or only slightly altered (< or =13%) the S-configuration at C-15. These results establish a previously unrecognized role for Ser-530 and Val-349 in maintaining the correct S stereochemistry of the carbon-15 hydroxyl group during prostaglandin synthesis. The findings may also explain the absolute conservation of Ser-530, the target of aspirin, throughout the families of cyclooxygenase enzymes.     

The basis of prostaglandin synthesis in coral: molecular cloning and expression of a cyclooxygenase from the Arctic soft coral Gersemia fruticosa

In vertebrates, the synthesis of prostaglandin hormones is catalyzed by cyclooxygenase (COX)-1, a constitutively expressed enzyme with physiological functions, and COX-2, induced in inflammation and cancer. Prostaglandins have been detected in high concentrations in certain corals, and previous evidence suggested their biosynthesis through a lipoxygenase-allene oxide pathway. Here we describe the discovery of an ancestor of cyclooxygenases that is responsible for prostaglandin biosynthesis in coral. Using a homology-based polymerase chain reaction cloning strategy, the cDNA encoding a polypeptide with approximately 50% amino acid identity to both mammalian COX-1 and COX-2 was cloned and sequenced from the Arctic soft coral Gersemia fruticosa. Nearly all the amino acids essential for substrate binding and catalysis as determined in the mammalian enzymes are represented in coral COX: the arachidonate-binding Arg(120) and Tyr(355) are present, as are the heme-coordinating His(207) and His(388); the catalytic Tyr(385); and the target of aspirin attack, Ser(530). A key amino acid that determines the sensitivity to selective COX-2 inhibitors (Ile(523) in COX-1 and Val(523) in COX-2) is present in coral COX as isoleucine. The conserved Glu(524), implicated in the binding of certain COX inhibitors, is represented as alanine. Expression of the G. fruticosa cDNA afforded a functional cyclooxygenase that converted exogenous arachidonic acid to prostaglandins. The biosynthesis was inhibited by indomethacin, whereas the selective COX-2 inhibitor nimesulide was ineffective. We conclude that the cyclooxygenase occurs widely in the animal kingdom and that vertebrate COX-1 and COX-2 are evolutionary derivatives of the invertebrate precursor.     

Investigating substrate promiscuity in cyclooxygenase-2: the role of Arg-120 and residues lining the hydrophobic groove

The cyclooxygenases (COX-1 and COX-2) generate prostaglandin H(2) from arachidonic acid (AA). In its catalytically productive conformation, AA binds within the cyclooxygenase channel with its carboxylate near Arg-120 and Tyr-355 and ω-end located within a hydrophobic groove above Ser-530. Although AA is the preferred substrate for both isoforms, COX-2 can oxygenate a broad spectrum of substrates. Mutational analyses have established that an interaction of the carboxylate of AA with Arg-120 is required for high affinity binding by COX-1 but not COX-2, suggesting that hydrophobic interactions between the ω-end of substrates and cyclooxygenase channel residues play a significant role in COX-2-mediated oxygenation. We used structure-function analyses to investigate the role that Arg-120 and residues lining the hydrophobic groove play in the binding and oxygenation of substrates by murine (mu) COX-2. Mutations to individual amino acids within the hydrophobic groove exhibited decreased rates of oxygenation toward AA with little effect on binding. R120A muCOX-2 oxygenated 18-carbon ω-6 and ω-3 substrates albeit at reduced rates, indicating that an interaction with Arg-120 is not required for catalysis. Structural determinations of Co(3+)-protoporphyrin IX-reconstituted muCOX-2 with α-linolenic acid and G533V muCOX-2 with AA indicate that proper bisallylic carbon alignment is the major determinant for efficient substrate oxygenation by COX-2. Overall, these findings implicate Arg-120 and hydrophobic groove residues as determinants that govern proper alignment of the bisallylic carbon below Tyr-385 for catalysis in COX-2 and confirm nuances between COX isoforms that explain substrate promiscuity.     

Nutritionally essential fatty acids and biologically indispensable cyclooxygenases

The study of cyclooxygenases (COXs), targets of aspirin and related drugs, is rooted in the discovery of essential fatty acids (EFAs). There are two COXs that convert EFAs, primarily arachidonic acid, to prostaglandins. Each COX is involved with distinct biologies. COX-1 expression is constitutive while COX-2 is inducible. The two COXs might have evolved partly to permit prostaglandin formation at different tissue sites. However, COX-2 is sometimes induced in cells already expressing COX-1, and in these instances, COX-2 functions while COX-1 is latent. This can occur because of unique biochemical properties of COX-2 that enable cells to form prostaglandins when arachidonic acid comprises a small fraction of available fatty acids and the concentrations of peroxides that are necessary for COX to function are low.     

An ELISA method to measure inhibition of the COX enzymes

The cyclooxygenase (COX) reaction can be monitored by measurement of oxygen consumption, peroxidase co-substrate oxidation or prostaglandin (PG) detection. This protocol describes a procedure measuring cyclooxygenase activity by quantifying PGE2 produced by enzymatic conversion of arachidonic acid, in the presence or absence of potential inhibitors. This high-throughput method has the advantage that it directly measures cyclooxygenase activity and requires little enzyme. The first part of the assay consists of incubating arachidonic acid, cyclooxygenase and the test samples to generate prostaglandins. The second part uses an ELISA method to quantify the amount of PGE2 produced by the enzymatic reaction. The isolation of COX-1 and COX-2 enzymes is also described. This protocol can be completed in approximately 23 h, including 16-h and 4-h incubation phases. This does not include enzyme preparation (3 h for COX-1 and 24 h for COX-2) or preparation of ELISA plates (23 h, including incubation).     

Cyclooxygenase-1 signaling is required for vascular tube formation during development

Prostaglandin endoperoxide synthases (PTGS), commonly referred to as cyclooxygenases (COX-1 and COX-2), catalyze the key step in the synthesis of biologically active prostaglandins (PGs), the conversion of arachidonic acid (AA) into prostaglandin H2 (PGH2). Although COX and prostaglandins have been implicated in a wide variety of physiologic processes, an evaluation of the role of prostaglandins in early mammalian development has been difficult due to the maternal contribution of prostaglandins from the uterus: COX null mouse embryos develop normally during embryogenesis. Here, we verify that inhibition of COX-1 results in zebrafish gastrulation arrest and shows that COX-1 expression becomes restricted to the posterior mesoderm during somitogenesis and to posterior mesoderm organs at pharyngula stage. Inhibition of COX-1 signaling after gastrulation results in defective vascular tube formation and shortened intersomitic vessels in the posterior body region. These defects are rescued completely by PGE(2) treatment or, to a lesser extent, by PGF(2alpha), but not by other prostaglandins, such as PGI(2), TxB(2), or PGD(2). Functional knockdown of COX-1 using antisense morpholino oligonucleotide translation interference also results in posterior vessel defect in addition to enlarged posterior nephric duct, phenocopying the defects caused by inhibition of COX-1 activity. Together, we provide the first evidence that COX-1 signaling is required for development of posterior mesoderm organs, specifically in the vascular tube formation and posterior nephric duct development.     

Relationship of prostaglandin secretion by rabbit alveolar macrophages to phagocytosis and lysosomal enzyme release

The phospholipids of rabbit alveolar macrophages were pulse-labelled with [(14)C]-arachidonic acid, and the subsequent release of labelled prostaglandins was measured. Resting macrophages released measurable amounts of arachidonic acid, the prostaglandins E(2), D(2) and F(2alpha) and 6-oxoprostaglandin F(1alpha). Phagocytosis of zymosan increased the release of arachidonic acid and prostaglandins to 2.5 times the control value. In contrast, phagocytosis of inert latex particles had no effect on prostaglandin release. Indomethacin inhibited the release of prostaglandin, and, at high doses (20mug/ml), increased arachidonic acid release. Analysis of the cellular lipids showed that after zymosan stimulation the proportion of label was decreased in phosphatidylcholine, but not in other phospholipids or neutral lipids. Cytochalasin B, at a dose of 2mug/ml, inhibited the phagocytosis induced by zymosan but increased prostaglandin synthesis to 3.4 times the control. These data suggest that the stimulation of prostaglandin synthesis by zymosan is not dependent on phagocytosis. Exposure to zymosan also resulted in the release of the lysosomal enzyme, acid phosphatase. Furthermore, cytochalasin B augmented the zymosan-stimulated release of acid phosphatase at the same dose that stimulated prostaglandin synthesis. However, indomethacin, at a dose that completely inhibited prostaglandin synthesis, failed to block the lysosomal enzyme release. Thus despite some parallels between the release of prostaglandins and lysosomal enzymes, endogenous prostaglandins do not appear to mediate the release of lysosomal enzymes. The prostaglandins released from the macrophages may function as humoral substances affecting other cells.     

Prostaglandin H Synthase-2-catalyzed Oxygenation of 2-Arachidonoylglycerol Is More Sensitive to Peroxide Tone than Oxygenation of Arachidonic Acid

The endocannabinoid, 2-arachidonoylglycerol (2-AG), is a selective substrate for the inducible isoform of prostaglandin H synthase (PGHS), PGHS-2. Its turnover leads to the formation of glyceryl esters of prostaglandins (PG-Gs), a subset of which elicits agonism at unique, as yet unidentified, receptors. The k_cat/K_m values for oxygenation of arachidonic acid (AA) and 2-AG by PGHS-2 are very similar, but the sensitivities of the two substrates to peroxide-dependent activation have not been compared. 15-Hydroperoxy derivatives of AA and 2-AG were found to be comparable in their ability to serve as substrates for the peroxidase activities of PGHS-2, PGHS-1, and glutathione peroxidase (GPx). They also were comparable in the activation of AA oxygenation by cyanide-inhibited PGHS-2. However, oxygenation of 2-AG was significantly suppressed relative to AA by the presence of GPx and GSH. Furthermore, 2-AG oxygenation by peroxidase-deficient H388YmPGHS-2 was much less efficient than AA oxygenation. Wild-type rates of 2-AG oxygenation were restored by treatment of H388YmPGHS-2 with hydroperoxide derivatives of AA or 2-AG. RNAi silencing of phospholipid hydroperoxide-specific GPx (GPx4) in NIH/3T3 cells led to increases in cellular peroxidation and in the levels of the isoprostane product, 8-epi-PGF_2α. GPx4 silencing led to 2–4-fold increases in PG-G formation but no change in PG formation. Thus, cellular peroxide tone may be an important determinant of the extent of endocannabinoid oxygenation by PGHS-2.