Whereas mPGES-2 and cytosolic PGE synthase are constitutively expressed in vivo, mPGES-1 is of particular interest because it has been shown to be the most potent (Tanikawa et al., 2002) among PGE synthases and is induced by various stimuli including inflammatory signals in various cells and tissues (Guay et al., 2004). by the presence of distal isomerases or by blocking the major metabolic outlet, which could determine the relative benefits and risks resulting from interdiction in nonrated-limiting components of PG synthesis pathways. Introduction Cyclooxygenase (COX) enzymes, also known as PGH2 synthases, catalyze the oxygenation of arachidonic acid (AA) to PGG2, followed by the reduction of PGG2 to PGH2, which serves Rabbit polyclonal to TIGD5 as a common substrate for various distal isomerases that generate five distinct primary PGs: PGE2, PGD2, PGF2, PGI2, and thromboxane A2 (TXA2), of which 6-keto-PGF1 and TXB2 are the main stable nonenzymatic products of PGI2 and TXA2, respectively (Fig. 1). These PGs consist of a series of extracellular and intracellular messengers that produce diverse physiologic effects on pain (Zeilhofer, 2007), inflammation and fever (McAdam et al., 2000), (S)-(+)-Flurbiprofen allergy (Pettipher et al., 2007), platelets (FitzGerald, 1991), cardiovascular system (Vane, 1983), cancer growth (Wang et al., 2007), renal function (Hbert et al., 2005), reproduction (Weems et al., 2006), and possibly Alzheimer’s disease (McGeer and McGeer, 1999). In many cases, different PGs have counter-regulatory effects. For example, in contrast to PGE2, PGD2 in the brain has (S)-(+)-Flurbiprofen a role in promoting sleep (Smyth et al., 2009). Furthermore, various PGs have the potential to both promote and counteract inflammatory processes in the body, especially in acute allergic inflammation. Thus, the exact physiologic or pathophysiologic response depends (S)-(+)-Flurbiprofen on the relative amounts of biologically active PG species. Open in a separate window Fig. 1. Scheme for the metabolism of AA to form different PGs. After the enzymatic conversion of PGH2 was reported (Christ-Hazelhof et al., 1976), each PG-specific isomerase was discovered and purified, including PGE synthase, PGD synthase, PGF synthase, PGI synthase, and TX synthase. Humans express three isoforms of PGE synthase: mPGES-1, mPGES-2, and cytosolic PGE synthase. Whereas mPGES-2 and cytosolic PGE synthase are constitutively expressed in vivo, mPGES-1 is of particular interest because it has been shown to be the most potent (Tanikawa et al., 2002) among PGE synthases and is induced by various stimuli including inflammatory signals in various cells and tissues (Guay et al., 2004). CAY10526 [4-(benzo[369 163 (retention time 1.6 min); TXB2, 369 169 (retention time 2.2 min); PGF2, 353 193 (retention time 2.7 min); PGE2 and PGD2 351 271 (retention times 3.2 and 3.7 min, respectively). The PGs formed abundant [M-H]? carboxylate ions during negative ion electrospray, which were fragmented using collision-induced dissociation with nitrogen as a collision gas. The collision energy (?24 to ?30 V) was optimized for each PG to maximize the formation of product ions for detection using selected reaction monitoring (SRM). Isomeric PGE2, PGD2 (Cao et al., 2008), and PGH2 were measured using a SRM transition of 351 to 271, and the SRM transition of 353 to 193 was selected for PGF2 (Dahl and van Breemen, 2010). The SRM transition of 369 to 163 was used for 6-keto-PGF1, and the transition of 369 to 169 was used for the measurement of TXB2. Likewise, the SRM of the transition of 355 to 275 was selected for the internal standards d4-PGE2 and d4-PGD2 (Cao et al., 2008). High-resolution negative ion electrospray tandem mass spectra of PGH2 and its metabolites were acquired using a Waters Synapt G1 quadrupole time of flight (TOF) (S)-(+)-Flurbiprofen hybrid tandem mass spectrometer with a Waters Alliance 2690 HPLC system or a Shimadzu ion trap-TOF mass spectrometer with a Prominence HPLC system. HPLC separations were carried out as described above except that the mobile phase consisted of an 11-min linear gradient from 33 to 90% acetonitrile in (S)-(+)-Flurbiprofen aqueous 0.1% formic acid. Cell Culture Assay. Although the in vitro assay provided information regarding biological mechanisms of action, the results might not necessarily reflect in vivo processes or even the situation within a cell. Therefore, the BMDM was used in which mPGES-1 and H-PGDS (L-PGDS) could be selectively.