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The first crystal structure of human cyclooxygenase-2, in the presence of a selective inhibitor, is similar to that of cyclooxygenase-1. The structure of the NSAID binding site is also well conserved, although there are differences in its overall size and shape which may be exploited for the further development of selective COX-2 inhibitors. A second COX-2 structure with a different bound inhibitor displays a new, open conformation at the bottom of the NSAID binding site, without significant changes in other regions of the COX-2 structure. These two COX-2 structures provide evidence for the flexible nature of cyclooxygenase, revealing details about how substrate and inhibitor may gain access to the cyclooxygenase active site from within the membrane.

Because of its structural similarity to polyunsaturated fatty acids, anandamide could serve as substrate for enzymes such as lipoxygenases and cyclooxygenases, which metabolize polyunsaturated fatty acids to potent bioactive metabolites. Here the ability of recombinant human cyclooxygenase-1 (hCOX-1) and cyclooxygenase-2 (hCOX-2) to metabolize anandamide was studied. Baculovirus-expressed and -purified hCOX-2, but not hCOX-1, effectively oxygenated anandamide. Reverse phase high pressure liquid chromatography analysis of the products derived from 1-14 C-labeled anandamide showed that the products formed are similar to those formed with arachidonic acid as substrate. The major prostanoid product derived from anandamide was determined by mass spectrometry to be prostaglandin E 2 ethanolamide. Incubation of anandamide with lysates and the intact cell line expressing COX-2 but not that of COX-1 produced prostaglandin E 2 ethanolamide. These results demonstrate the existence of a COX-2-mediated pathway for anandamide metabolism, and the metabolites formed represent a novel class of prostaglandins.Anandamide (arachidonoyl ethanolamide, AEA) 1 is a polyunsaturated fatty acyl amide that was identified from porcine brain lipids as an endogenous ligand for brain cannabinoid receptor (1). Although structurally different from cannabinoids, AEA by its ability to activate the central CB1 receptor displays pharmacological properties similar to cannabinoids (2, 3). In addition to its central action via the CB1 receptor, AEA displays potent immunomodulatory and anti-inflammatory activities by interacting with peripheral CB1 and/or CB2 receptors (4 -6).Free AEA is present in both central and peripheral tissues (see Ref. 7 for a review) and could interact with CB receptors to display some of its immunomodulatory and anti-inflammatory activities. In addition, AEA is also stored esterified to phosphatidylethanolamines and is released by the action of phospholipase D in response to various stimuli (7). The AEA thus released inside the cell could participate in signal transduction as a second messenger and display some of its immunomodulatory and anti-inflammatory activities independent of its interaction with the CB receptors. In fact, AEA has been shown to antagonize CB2 receptors, and it is not clear how this antagonism results in immunomodulatory activities observed in cells only expressing CB2 receptors (8). It is possible that a metabolite of AEA rather than AEA itself could account for all or some of these properties. Furthermore, because of its structural similarities to polyunsaturated fatty acids, endogenously released AEA could serve as substrate for lipoxygenases and cyclooxygenases (COX) that metabolize polyunsaturated fatty acids to potent bioactive molecules. It has been demonstrated that lipoxygenase could metabolize AEA, and the metabolites have potent biological activities (9, 10). However, it is not known whether COX can metabolize AEA. Arachidonic acid (AA) is the substrate for both COX-1 and COX-2. AEA is structur...

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The interaction of tumor necrosis factor a (TNF) with its two membrane-bound receptors initiates intracellular events in which arachidonic acid and its derivatives are involved. In HeLa cells, TNF treatment induces an arachidonic acid-selective, Ca2+-dependent cellular phospholipase A2 (cPLA2). By itself, TNF causes a modest increase in cPLA2 activity, but with the Ca2+ ionophore A23187 it provides a strong synergistic action. Within minutes in response to TNF, cPLA2 becomes phosphorylated and in the presence of Ca2+ produces a 3-to 4-fold increase in activity. TNF also increases cPLA2 mRNA and protein expression, an estimated 5-fold increase in an 8-hr period. This increase in cPLA2 activity occurs, therefore, in a biphasic time-dependent manner. Dexamethasone, known to antagonize the action of TNF, is here shown to inhibit TNF-induced gene expression and to prevent the second phase of increase in cPLA2 activation. Our results suggest that the cPLA2 activation may provide a regulatory function and may explain the proinflammatory action of TNF.Tumor necrosis factor alpha (TNF) is a multifunctional cytokine produced mainly by monocytes and macrophages (1). TNF is now considered to be one of the major "inflammatory" cytokines, playing an essential role as an immunostimulant to increase host defense mechanisms against infections (2). In this role, TNF induces the production of prostaglandins, leukotrienes, and platelet-activating factor, which serve as inflammatory mediators. Therefore, a variety of events are initiated through the interaction of TNF with its membrane-bound receptors. The presence of two divergent receptors (3-6) and the multiple effects of TNF suggest that various functions of TNF may be mediated by coupled or independent receptor-activated signal transduction pathways (7).Early events of TNF-mediated signal transduction appear to involve lipids as mediators (8-16). For example, TNF stimulates the release of arachidonic acid (AA) from a variety of cell types (11-13), and AA-depleted cells are less susceptible to the cytotoxic effects ofTNF (14). The induction ofthe protooncogene c-fos by TNF is mediated through lipoxygenase products of AA (15), which are also required for TNF-cycloheximide-induced cytotoxicity (16). Since AA metabolites such as prostaglandins and leukotrienes play an important role in inflammation (17, 18), the proinflammatory properties of TNF could be ascribed to its ability to release AA. Of the several enzymatic pathways responsible for the release of AA, which is esterified to the sn-2 position of phospholipids, phospholipase A2 is an important enzyme for this hydrolytic cleavage reaction (18,19). Thus, one possibility is that AA release by TNF is mediated through its effect on PLA2.Phospholipase A2 enzymes can be grouped into two classes based on their molecular weight and cellular distribution. The "'14-kDa small molecular weight granule-associated secretory PLA2 (sPLA2) enzymes are rich in disulfide bonds, require millimolar levels of Ca2+ for activity, but display...