Competitive Enzymatic Interactions Determine the Relative Amounts of Prostaglandins E₂ and D₂

Abstract

Prostaglandins (PGs) are a family of cellular messengers exerting diverse homeostatic and pathophysiologic effects. Recently, several studies reported significant increases of PGI 2 and PGF 2␣ after the inhibition of microsomal PGE synthase-1 (mPGES-1) expression, which indicated that PGH 2 metabolism might be redistributed when the PGE 2 pathway is blocked. To address the determinants that govern the relative amounts of PGs, we developed an in vitro cell-free method, based on liquid chromatography-tandem mass spectrometry, to measure the exact amounts of these PGs formed in response to the addition of recombinant isomerases and their selective inhibitors. Our in vitro cell-free assay results were confirmed in cells using bone marrow-derived macrophage. Initially, we determined the in vitro stability of PGH 2 and noted that there was spontaneous nonenzymatic conversion to PGD 2 and PGE 2 . mPGES-1 markedly increased the conversion to PGE 2 and decreased conversion to PGD 2 . Reciprocally, the addition of hematopoietic or lipocalin PGD synthase resulted in a relative increase of PGD 2 and decrease of PGE 2 . A detailed titration study showed that the ratio of PGE 2 /PGD 2 was closely correlated with the ratio of PGE synthase/PGD synthase. Our redistribution results also provide the foundation for understanding how PGH 2 metabolism is redistributed 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.

Pivotal Role of Reactive Oxygen Species in Differential Regulation of Lipopolysaccharide-Induced Prostaglandins Production in Macrophages

“…Supernatant sample preparation was similar to what we previously described (Cao et al, 2008;Yu et al, 2011). Briefly, a 500-ml aliquot of supernatant was spiked together with d 4 -PGE 2 and d 4 -PGD 2 as surrogate standards.…”

“…The HPLC system was interfaced to a Thermo-Finnigan (San Jose, CA) TSQuantum triple quadruple mass spectrometer that was operated using negative ion electrospray. Isomeric PGD 2 and PGE 2 were measured using a SRM transition of m/z 351 to m/z 271, and the SRM transition of m/z 355 to m/z 275 was selected for the internal standards d 4 -PGE 2 and d 4 -PGD 2 (Cao et al, 2010(Cao et al, , 2011Yu et al, 2011).…”

“…For example, PGE 2 promotes tumor initiation, progression, and metastasis (Samuelsson et al, 2007), whereas PGD 2 triggers asthmatic responses (Matsuoka et al, 2000). Both PGE 2 and PGD 2 are converted from PGH 2 by various isoforms of PGE synthase (PGES) and PGDS (Samuelsson et al, 2007;Yu et al, 2011), respectively. We recently showed that BMDM express all three types of PGES including cytosolic PGES (cPGES), microsomal PGES-1 (mPGES-1), and mPGES-2, as well as H-PGDS (Xiao et al, 2012).…”

Microsomal Prostaglandin E Synthase Type 2 (mPGES2) Is a Glutathione-dependent Heme Protein, and Dithiothreitol Dissociates the Bound Heme to Produce Active Prostaglandin E2 Synthase in Vitro

“…Endoperoxide PGH 2 is a very unstable molecule, and its halflife at 37°C is known to be 5 min (19). It has been reported that PGH 2 converts non-enzymatically to various prostaglandins, including HHT and MDA (20,21).…”

A Simple and Highly Sensitive Quantitation of Eicosanoids in Biological Samples Using Nano-flow Liquid Chromatography/ Mass Spectrometry

“…A similar chromatogram was reported in other article. 24,31 We currently speculated that these peaks were derived from multi form TXB2 with the equilibrium state.…”