11,12-Epoxyeicosatrienoic Acid Attenuates Synthesis of Prostaglandin E 2 in Rat Monocytes Stimulated with Lipopolysaccharide WIESLAW KOZAK,* , † ,1 DAVID M. ARONOFF,‡ ,2 OLIVIER BOUTAUD,§ AND ANNA KOZAK* *Department of Physiology, Medical College of Georgia, Augusta, Georgia 30912; †Institute of General and Molecular Biology, University of Nicolaus Copernicus, 87-100 Torun, Poland; and Departments of ‡Medicine and §Pharmacology, Vanderbilt University, Nashville, Tennessee 37232 Cytochrome P-450 monooxygenase (epoxygenase)-derived arachidonic acid (AA) metabolites, including 11,12-epoxy- eicosatrienoic acid (11,12-EET), possess anti-inflammatory and antipyretic properties. Prostaglandin E 2 (PGE 2 ), a cyclooxygen- ase (COX)-derived metabolite of AA, is a well-defined mediator of fever and inflammation. We have tested the hypothesis that 11,12-EET attenuates synthesis of PGE 2 in monocytes, which are the cells that are indispensable for induction of fever and initiation of inflammation. Monocytes isolated from freshly col- lected rat blood were stimulated with lipopolysaccharide (LPS; 100 ng/2 × 10 5 cells) to induce COX-2 and stimulate generation of PGE 2 . SKF-525A, an inhibitor of epoxygenases, significantly augmented the lipopolysaccharide-provoked synthesis of PGE 2 in cell culture in a concentration-dependent manner. It did not affect, however, elevation of the expression of COX-2 protein in monocytes stimulated with LPS. 11,12-EET also did not affect the induction of COX-2 in monocytes incubated with lipopoly- saccharide. However, 11,12-EET suppressed, in a concentra- tion-dependent fashion, the generation of PGE 2 in incubates. Preincubation of a murine COX-2 preparation for 0–5 min with three concentrations of 11,12-EET (1, 5, and 10 μM) inhibited the oxygenation of [ 14 C]-labeled AA by the enzyme. The inhibitory effect of 11,12-EET on COX-2 was time-and-concentration- dependent, suggesting a mechanism-based inhibition. Based on these data, we conclude that 11,12-EET suppresses genera- tion of PGE 2 in monocytes via modulating the activity of COX-2. These data support the hypothesis that epoxygenase- derived AA metabolites constitute a negative feedback on the enhanced synthesis of prostaglandins upon inflammation. Exp Biol Med 228:786–794, 2003 Key words: eicosanoids; arachidonic acid; prostaglandins; cycloox- ygenase-2; epoxygenase; negative feedback; mononuclear cells; endotoxin I n addition to cyclooxygenases (COXs) and lipoxygen- ases (LOXs), the oxidative metabolism of arachidonic acid (AA) also includes multiple microsomal cyto- chrome P-450 enzymes collectively referred to as monooxy- genases (1). They convert free AA into three types of eico- sanoid products via reactions consisting of allylic oxidation, -terminal hydroxylation, and olefin epoxidation. The lat- ter, also referred to as the epoxygenase reaction, results in the production of four cis-epoxyeicosatrienoic acids (EETs): 5,6-, 8,9-, 11,12-, and 14,15-EET (2). In mammals, expression of monooxygenases and synthesis of EETs has been detected, among other tissues, in the brain, lungs, liver and kidney, and in cells such as astrocytes, hepatocytes, and endothelium (1, 3). Monocytes, the cells playing a key role in inflammation (4), contain various P-450s capable of EET biosynthesis (5, 6) and, in addition, they express specific high affinity receptors for EETs (7). The biology of COXs and LOXs has been extensively studied because their eicosanoid products play fundamental roles in health and disease and, increasingly, as therapeutic targeting agents (8). Inhibitors of COXs are the mainstays of current therapy aimed to modulate pain and inflammation and to control fever (9). It has been well documented that COX exists in two isoforms, COX-1 and COX-2 (10). In simplified terms, COX-1 is a constitutive housekeeping en- zyme, whereas COX-2 is inducible and can be markedly upregulated during stress, pain, inflammation, and fever. COXs are bifunctional enzymes that catalyze the oxygen- ation of AA to unstable prostaglandin G 2 (PGG 2 ), followed by the reduction of the lipid hydroperoxide PGG 2 to alcohol PGH 2 (11). PGH 2 serves as substrate for the respective syn- thases generating thromboxane (in platelets), prostacyclin (in endothelial cells), and prostaglandins such as PGE 2 (in many cell types, including monocytes). Induction of COX-2, e.g., by an injection of fever-inducing inflamma- This work was supported in part by the National Institutes of Health Grant GM 07569. 1 To whom requests for reprints should be addressed at Department of Physiology, Medical College of Georgia, 1120 Fifteenth Street, Augusta, GA 30912–3000. E-mail: wkozak@mail.mcg.edu 2 Present address and affiliation: Department of Medicine, University of Michigan Health System, Ann Arbor, MI 48109. Received October 18, 2002. Accepted January 28, 2003. 1535-3702/03/2287-0786$15.00 Copyright © 2003 by the Society for Experimental Biology and Medicine 786 11,12-EET ATTENUATES PGE 2 IN MONOCYTES