Abstract
Prostaglandins (PGs) are a family of cellular messengers exerting diverse homeostatic and pathophysiologic effects. Recently, several studies reported significant increases of PGI2 and PGF2α after the inhibition of microsomal PGE synthase-1 (mPGES-1) expression, which indicated that PGH2 metabolism might be redistributed when the PGE2 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 PGH2 and noted that there was spontaneous nonenzymatic conversion to PGD2 and PGE2. mPGES-1 markedly increased the conversion to PGE2 and decreased conversion to PGD2. Reciprocally, the addition of hematopoietic or lipocalin PGD synthase resulted in a relative increase of PGD2 and decrease of PGE2. A detailed titration study showed that the ratio of PGE2/PGD2 was closely correlated with the ratio of PGE synthase/PGD synthase. Our redistribution results also provide the foundation for understanding how PGH2 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.
Footnotes
This work was supported by the National Institutes of Health Division of Intramural Research [Grants R01-HL075557, HL103643a, 5R01-HL083218, 3R01-HL083218-01A2S1]; the National Institutes of Health National Cancer Institute [Grant P01-CA048112]; the National Institutes of Health National Center for Complementary and Alternative Medicine [Grant P50-AT00155]; the Department of Veterans Affairs Merit Review [Grant 1I01BX000108]; and the University of Illinois, Chicago Faculty Scholarship Support Program.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.111.185405.
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ABBREVIATIONS:
- COX
- cyclooxygenase
- PG
- prostaglandin
- mPGES
- microsomal PGE synthase
- NSAID
- nonsteroidal anti-inflammatory drug
- LC-MS/MS
- liquid chromatography-tandem mass spectrometry
- BMDM
- bone marrow-derived macrophage
- H-PGDS
- hematopoietic PGD synthase
- L-PGDS
- lipocalin PGD synthase
- AA
- arachidonic acid
- TXA2
- thromboxane A2
- TXB2
- thromboxane B2
- LPS
- lipopolysaccharide
- HBSS
- Hanks' balanced salt solution
- FBS
- fetal bovine serum
- DMEM
- Dulbecco's modified Eagle's medium
- SRM
- selected reaction monitoring
- TOF
- time of flight
- HPLC
- high-performance liquid chromatography
- CAY10526
- 4-(benzo[b]thiophen-2-yl)-3-bromo-5-hydroxy-dihydro-furan-2(3H)-one
- CAY10589
- 2-[[4-[([1,1′-biphenyl]-4-ylmethyl)amino]-6-chloro-2-pyrimidinyl]thio]-octanoic acid
- HQL-79
- 4-(diphenylmethoxy)-1–3-(1H-tetrazol-5-yl)propyl-piperidine
- AT-56
- 4-(5H-dibenzo[a,d]cyclohepten-5-ylidene)-1-[4-(2H-tetrazol-5-yl)butyl]-piperidine.
- Received June 22, 2011.
- Accepted August 22, 2011.
- U.S. Government work not protected by U.S. copyright
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