identi®ed as a novel orally active and highly selective cyclo-oxygenase-2 (COX-2) inhibitor. 2 In CHO cells stably transfected with human COX isozymes, DFU inhibited the arachidonic aciddependent production of prostaglandin E 2 (PGE 2 ) with at least a 1,000 fold selectivity for COX-2 (IC 50 =41+14 nM) over COX-1 (IC 50 450 mM). Indomethacin was a potent inhibitor of both COX-1 (IC 50 =18+3 nM) and COX-2 (IC 50 =26+6 nM) under the same assay conditions. The large increase in selectivity of DFU over indomethacin was also observed in COX-1 mediated production of thromboxane B 2 (TXB 2 ) by Ca 2+ ionophore-challenged human platelets (IC 50 450 mM and 4.1+1.7 nM, respectively). 3 DFU caused a time-dependent inhibition of puri®ed recombinant human COX-2 with a K i value of 140+68 mM for the initial reversible binding to enzyme and a k 2 value of 0.11+0.06 s 71 for the ®rst order rate constant for formation of a tightly bound enzyme-inhibitor complex. Comparable values of 62+26 mM and 0.06+0.01 s 71 , respectively, were obtained for indomethacin. The enzyme-inhibitor complex was found to have a 1 : 1 stoichiometry and to dissociate only very slowly (t 1/2 =1 ± 3 h) with recovery of intact inhibitor and active enzyme. The time-dependent inhibition by DFU was decreased by co-incubation with arachidonic acid under non-turnover conditions, consistent with reversible competitive inhibition at the COX active site. 4 Inhibition of puri®ed recombinant human COX-1 by DFU was very weak and observed only at low concentrations of substrate (IC 50 =63+5 mM at 0.1 mM arachidonic acid). In contrast to COX-2, inhibition was time-independent and rapidly reversible. These data are consistent with a reversible competitive inhibition of COX-1. 5 DFU inhibited lipopolysaccharide (LPS)-induced PGE 2 production (COX-2) in a human whole blood assay with a potency (IC 50 =0.28+0.04 mM) similar to indomethacin (IC 50 =0.68+0.17 mM). In contrast, DFU was at least 500 times less potent (IC 50 497 mM) than indomethacin at inhibiting coagulationinduced TXB 2 production (COX-1) (IC 50 =0.19+0.02 mM). 6 In a sensitive assay with U937 cell microsomes at a low arachidonic acid concentration (0.1 mM), DFU inhibited COX-1 with an IC 50 value of 13+2 mM as compared to 20+1 nM for indomethacin. CGP 28238, etodolac and SC-58125 were about 10 times more potent inhibitors of COX-1 than DFU. The order of potency of various inhibitors was diclofenac4indomethacin*naproxen4nimesulide* meloxicam*piroxicam4NS-398*SC-576664SC-581254CGP 28238*etodolac4L-745,3374DFU. 7 DFU inhibited dose-dependently both the carrageenan-induced rat paw oedema (ED 50 of 1.1 mg kg 71 vs 2.0 mg kg 71 for indomethacin) and hyperalgesia (ED 50 of 0.95 mg kg 71 vs 1.5 mg kg 71 for indomethacin). The compound was also e ective at reversing LPS-induced pyrexia in rats (ED 50 =0.76 mg kg 71 vs 1.1 mg kg 71 for indomethacin). 8 In a sensitive model in which 51 Cr faecal excretion was used to assess the integrity of the gastrointestinal tract in rats, no signi®cant e ect was detected after oral...

SummaryThe intracellular distribution of the enzyme 5-lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) in resting and ionophore-activated human leukocytes has been determined using immuno-electronmicroscopic labeling of ultrathin frozen sections and subcellular fractionation techniques. 5-LO is a 78-kD protein that catalyzes the conversion ofarachidonic acid to leukotrienes. FLAP is an 18-kD membrane bound protein that is essential for leukotriene synthesis in cells. In response to ionophore stimulation, 5-LO translocates from a soluble to a sedimentable fraction of cell homogenates. In activated leukocytes, both FLAP and 5-LO were localized in the lumen of the nuclear envelope. Neither protein could be detected in any other cell compartment or along the plasma membrane. In resting cells, the FLAP distribution was identical to that observed in activated cells . In addition, subcellular fractionation techniques showed >83% of immunoblot-detectable FLAP protein and -64% of the FLAP ligand binding activity was found in the nuclear membrane fraction. A fractionation control demonstrated that a plasma membrane marker, detected by a monoclonal antibody PMN13F6, was not detectable in the nuclear membrane fraction . In contrast to FLAP, 5-LO in resting cells could not be visualized along the nuclear envelope. Except for weak labeling of the euchromatin region of the nucleus, 5-LO could not be readily detected in any other cellular compartment . These results demonstrate that the nuclear envelope is the intracellular site at which 5-LO and FLAP act to metabolize arachidonic acid, and that ionophore activation of neutrophils and monocytes results in the translocation of 5-LO from a nonsedimentable location to the nuclear envelope .eukotrienes are products of arachidonic acid metabolism that are produced by leukocytes and that have a variety of effects on the immune system (1) . The enzyme 5-lipoxygenase (5-LO, 1 EC 1.13 .11.12) catalyzes two key steps in the leukotriene biosynthetic pathway (2, 3). These steps are the oxygenation ofarachidonate to 5-(S)-hydroperoxy-6,8, 11,14-eicosatetraenoic acid (5-HPETE), followed by its dehydrogenation, which results in the formation of the unstable epoxide LTA4. In neutrophils the enzyme LTA4 hydrolase then converts LTA4 to LTB4, which is a potent chemotactic and activating factor for neutrophils and eosinophils (4-6). In eo-1 Abbreviations used in this paper. FLAP, 5-lipoxygenase-activating protein; GAM, goat anti-mouse IgG; GAR, goat anti-rabbit IgG ; 5-HPETE, 5-(S)-hydroperoxy-6,8,11,14-eicosatetraenoic acid; 5-LO, 5-lipoxygenase; LSM, lymphocyte separation medium ; PLP, paraformaldehyde-lysineperiodate . sinophils and mast cells, LTA4 can also be converted to LTC4, LTD4, and LTE4, all of which stimulate contraction of vascular and bronchial smooth muscle (7,8), resulting in effects on local circulation and bronchoconstriction . These potent biological effects implicate leukotrienes in a number of hypersensitivity and inflammatory disorders, including asthma and in...

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Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible protein recently shown to be an important source of inflammatory PGE 2 . Here we have used mPGES-1 wild type, heterozygote, and null mice to assess the impact of reduction or absence mPGES-1 protein on the production of PGE 2 and other prostaglandins in lipopolysaccharide (LPS)-treated macrophages and mice. Thioglycollate-elicited peritoneal macrophages with mPGES-1 deficiency were found to lose their ability to produce PGE 2 upon LPS stimulation. Resident mPGES-1 ؊/؊ peritoneal macrophages exhibited severely impaired PGE 2 -releasing activity but retained some LPS-inducible PGE 2 production capacity. Both macrophage types showed a 50% decrease in PGE 2 production with removal of one copy of the mPGES-1 gene. In vivo, mPGES-1 deletion abolished the LPS-stimulated production of PGE 2 in spleen, kidney, and brain. Surprisingly, lack of mPGES-1 activity resulted in an 80 -90% decrease in basal, cyclooxygenase-1 (COX-1)-dependent PGE 2 production in stomach and spleen, and a 50% reduction in brain and kidney. Other prostaglandins (thromboxane B 2 , PGD 2 , PGF 2␣ , and 6-keto-PGF 1␣ ) were significantly elevated in stomachs of mPGES-1-null mice but not in other tissues. Examination of mRNA for several terminal prostaglandin synthases did not reveal changes in expression levels associated with mPGES-1 deficiency, indicating that gastric prostaglandin changes may be due to shunting of cyclooxygenase products to other terminal synthases. These data demonstrate for the first time a dual role for mPGES-1 in both inflammatory and COX-1-mediated PGE 2 production and suggest an interdependence of prostanoid production with tissue-specific alterations of prostaglandin levels in the absence of mPGES-1. Prostaglandins (PG)1 are lipid metabolites of arachidonic acid that are synthesized by a two-step reaction catalyzed by a cyclooxygenase and a terminal prostaglandin synthase. The cyclooxygenase product PGH 2 serves as common precursor to all five major prostanoids (TXA 2 , PGE 2 , PGD 2 , PGI 2 , and PGF 2␣ ). The physiological roles of PG are both diverse and complex, with effects on kidney ion transport, vascular homeostasis, gastrointestinal protection and motility, pregnancy and parturition, sleep, and immune function (1-3). In particular, the primary mediators of pain and inflammation are PGE 2 and PGI 2 (4), whereas pyresis is mediated by PGE 2 through the EP3 receptor (5).Two major cyclooxygenase isoforms are known, each with distinct roles. Expression patterns suggest that the constitutively expressed COX-1 plays housekeeping functions, whereas the inducible COX-2 is implicated in inflammatory processes. Exceptions to this paradigm have been uncovered with observations of constitutive COX-2 expression in several neuronal structures of the brain and in the kidney (6 -9). Thus, although COX-2 plays a pivotal role in inflammation, it also serves housekeeping functions. Indeed, the phenotype of the COX-2 null mice is indicative of COX-2 roles during kidney dev...

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