Synthesis of Prostaglandin E2 Ethanolamide from Anandamide by Cyclooxygenase-2

Abstract

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...

Competition and allostery govern substrate selectivity of cyclooxygenase-2

“…Specifically, the active site of COX-2 is larger than that of COX-1, allowing COX-2 to oxygenate bulkier amide and ester analogs of AA that are poor COX-1 substrates. Among these COX-2-selective substrates are the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonoylethanolamide (3)(4)(5). COX-2-dependent oxygenation of these substrates leads to the biosynthesis of prostaglandin glyceryl esters (PG-Gs) and prostaglandin ethanolamides (prostamides), respectively (3,4).…”

“…Among these COX-2-selective substrates are the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonoylethanolamide (3)(4)(5). COX-2-dependent oxygenation of these substrates leads to the biosynthesis of prostaglandin glyceryl esters (PG-Gs) and prostaglandin ethanolamides (prostamides), respectively (3,4). In vitro, COX-2 uses 2-AG and AA with similar kinetic efficiencies (3); however, PG-G production in intact cells is much lower than would be expected based on the relative amounts of cellular AA and 2-AG (6).…”

Altered GABAergic neurotransmission is associated with increased kainate-induced seizure in prostaglandin-endoperoxide synthase-2 deficient mice

“…Specifically, the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) was decreased in the CA1 pyramidal neurons of PTGS-2 -/-mice, suggesting an alteration of GABAergic function. Augmented susceptibility to excitotoxicity was not mediated by endocannabinoid CB1 receptor signaling due to AA metabolites catabolized by PTGS-2, but not PTGS-1 [30][31][32]70], that are known to alter neuronal excitability [3,28].…”

Upregulation of orphan nuclear receptor Nur77 following PGF2α, Bimatoprost, and Butaprost treatments. Essential role of a protein kinase C pathway involved in EP2 receptor activated Nur77 gene transcription

“…In contrast to prostaglandin F 2a , Prostamides (prostaglandin ethanolamides) were recently identified as a new class of compounds that were formed from anandamide via metabolic transformation sequentially catalyzed by cyclooxygenase-2 (Yu et al, 1997). Although their physiological actions have not been fully investigated, a synthetic prostamide analog (Bimatoprost) has been shown to be very potent in reducing IOP by increasing both uveosceral and trabecular outflow of aqueous humor (Woodward et al, 2001).…”