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(2α) 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.

Fig. 1.

Scheme for the metabolism of AA to form different PGs.

Fig. 2.

Negative ion electrospray LC-MS/MS SRM chromatograms showing the detection of 1 μM PGE₂, PGD₂, PGF_2α, 6-keto-PGF_1α, and TXB₂ standards in methanol/water (50:50, v/v). SRM transitions: 6-keto-PGF_1α, m/z 369 → 163 (retention time 1.6 min); TXB₂, m/z 369 → 169 (retention time 2.2 min); PGF_2α, m/z 353 → 193 (retention time 2.7 min); PGE₂ and PGD₂ m/z 351 → 271 (retention times 3.2 and 3.7 min, respectively).

Fig. 3.

Ovine COX-1, human COX-2, mPGES-1, H-PGDS, and L-PGDS were incubated at 37°C for 10 min with substrate PGH₂ (A) or AA (B). PG formation (%) represents the sum of all PGs that were detected relative to the theoretical maximum based on the substrate PGH₂ or AA. All values are the mean ± S.D. (n = 3). *, p < 0.05, significance differences in PG formation compared with no enzyme.

Fig. 4.

Negative ion electrospray SRM chromatograms of the transition m/z 351 to m/z 271 showing the detection of 2 μM PGH₂ standard in methanol/water (50:50, v/v) at pH 7 and 4°C and its nonenzymatic derivatives using an 11-min linear gradient from 33 to 90% acetonitrile in aqueous 0.1% formic acid at a flow rate of 200 μl/min. PGE₂ and PGD₂ (retention times 4.5 and 4.8 min, respectively) were identified by comparison with standards. PGH₂ (retention time 7.7 min) was identified by monitoring its level over 160 min at pH 7 and 4°C. The remaining two peaks (retention times 5.7 and 6.3 min) remain unidentified but were not (by comparison with standards) PGI₂, 15-keto PGF_2α, or 13,14-dihydro-15-keto PGE₂.

Fig. 5.

Levels of PGE₂ (A), PGD₂ (B), PGH₂ (E), and two unknown compounds (C and D) formed over 160 min from an initial concentration of 2 μM PGH₂ in methanol/water (50:50, v/v) at pH 7 and 4°C. The formation of PGE₂ and PGD₂ was constant during the first 50 min and reached a plateau by 70 min (A and B), whereas the amount of PGH₂ continued to decrease over 160 min (E). The curves representing the unknown compounds had inflection points suggesting that these are unstable compounds.

Fig. 6.

PGH₂ decomposition curve in Tris·HCl buffer at pH 8 and 37°C (time points 1, 2, 3, 5, 7, 10, 15, 25, and 40 min). Based on this curve, the half-life of PGH₂ was determined to be 5 min.

Fig. 7.

PG formation from PGH₂ under different conditions. The initial concentration of PGH₂ was 2 μM, and the GSH and H₂O₂ concentrations were 1 mM for H-PGDS and L-PGDS or 2.5 mM for mPGES-1. mPGES-1, H-PGDS, and L-PGDS are shown in units/μl; CAY10526, CAY10589, HQL-79, and AT-56 are shown in μM. All values are mean ± S.D. (n = 3).

Fig. 8.

The effects on PGE₂ and PGD₂ formation from 2 μM PGH₂ of different ratios of mPGES-1 and H-PGDS (A) or mPGES-1 and L-PGDS (B). PGE₂ formation increased when the proportion of mPGES-1 was increased, and PGD₂ formation increased when the proportion of H-PGDS or L-PGDS was increased. All values are expressed as the mean ± S.D. (n = 3). *, p < 0.05, significant differences in PGE₂ formation compared with mPGES-1 (0.3 unit/μl)/H-PGDS (0.01 units/μl) (A) and mPGES-1 (0.3 unit/μl)/L-PGDS (0.1 unit/μl) (B). #, p < 0.05, significant differences in PGD₂ formation compared with mPGES-1 (0.3 unit/μl)/H-PGDS (0.01 unit/μl) (A) and mPGES-1 (0.3 unit/μl)/L-PGDS (0.1 unit/μl) (B).

Fig. 9.

The consequences of adding different concentrations of the H-PGDS-selective inhibitor HQL-79 (A) or L-PGDS-selective inhibitor AT-56 (B) to a system containing equivalent volumes of mPGES-1 (3 units/μl) and H-PGDS (0.1 unit/μl) or L-PGDS (1 unit/μl) incubated with 2 μM substrate PGH₂. All values are expressed as mean ± S.D. (n = 3). *, p < 0.05, significant differences in PGE₂ formation compared without addition of HQL-79 (A) or without addition of AT-56 (B). PGE₂ formation increased, whereas PGD₂ formation decreased with increasing concentrations of HQL-79 or AT-56. #, p < 0.05, significant differences in PGD₂ formation compared without addition of HQL-79 (A) or without addition of AT-56 (B).

Fig. 10.

A, the effect of the H-PGDS-selective inhibitor HQL-79 on the LPS-stimulated production of PGE₂ and PGD₂ from BMDM. Values are the mean ± S.D. (n = 3). *, p < 0.05, significant differences in PGE₂ formation compared with no HQL-79 addition; #, p < 0.05, significant differences in PGD₂ formation compared with no HQL-79 addition. PGE₂ formation increased and PGD₂ formation decreased with increasing concentrations of HQL-79. B, examples of LC-MS/MS analyses of PGE₂ and PGD₂ extracted from BMDM after treatment without or with 100 μM HQL-79.