Cyclooxygenase-2-mediated metabolism of arachidonic acid to 15-oxo-eicosatetraenoic acid by rat intestinal epithelial cells

Hwa Lee Seon, Kannan Rangiah, Michelle V. Williams, Angela Y. Wehr, Raymond N. DuBois, Ian A. Blair

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

Rat intestinal epithelial cells mat permanently express the cyclooxygenase-2 (COX-2) gene (RIES cells) were used to investigate COX-2-mediated arachidonic acid (AA) metabolism. A targeted chiral lipidomics approach was employed to quantify AA metabolites that were secreted by the cells into the culture media. When intact RIES cells were treated with calcium ionophore A-23187 (1 μM) for 1 h, 11-(R)-hydroxyeicosatetraenoic acid (HETE) was the most abundant metabolite, followed by prostaglandin (PG) E2, 15-(S)-HETE, 15-oxo-eicosatetraenoic acid (ETE), and 15-(R)-HETE. Incubation for a further 23 h after the calcium ionophore was removed resulted in a substantial increase in PGE2 concentrations while HETE and 15-oxo-ETE concentrations decreased to almost undetectable levels. A similar metabolic profile was observed when RIES cells were treated with increasing concentrations of AA for 24 h. Incubation of the RIES cells with 10 μM AA revealed that maximal concentrations of 11-(R)-HETE, 15-(S)-HETE, and 15-oxo-ETE occurred after 10 min of incubation when the 15-(S)-HETE concentrations were approximately twice that of PGE2. There was a gradual decrease in the concentrations of HETE and 15-oxo-ETE over time, whereas PGE2 concentrations increased steadily until they reached a maximum after 24 h of incubation. The ratio of PGE2 to 15-(S)-HETE was then approximately 20:1. 15-(S)-HETE and 15-oxo-ETE concentrations declined in the cell media during prolonged incubations with pseudo-first-order rate constants of 0.0121 and 0.0073 min-1, respectively. 15-(S)-HETE was shown to undergo metabolism primarily to 15-oxo-ETE, which was further metabolized to a glutathione (GSH) adduct. The GSH adduct of 15-oxo-ETE was further metabolized in the extracellular milieu to a cysteinylglycine adduct. Thus, we have established for the first time that 15-oxo-ETE can be formed biosynthetically from AA, that 15-(S)-HETE is its immediate precursor, and that 15-oxo-ETE forms a GSH adduct. For ionophore-A-23187-stimulated cells and at early time points for AA-stimulated cells, 11-(R)-HETE was the major eicosanoid to be secreted into the media. Adding increasing concentrations of AA to cells in culture made it possible to estimate with surprising accuracy endogenous eicosanoid production using regression analyses. Thus, after 24 h in the absence of added AA, 11-(R)-HETE and 15-(R)-HETE were estimated to be present at concentrations close to the detection limit of our very sensitive assay. These data further highlight the importance of endogenous COX-2-mediated lipid peroxidation and illustrate the necessity to monitor eicosanoid formation from endogenous stores of AA in cell culture experiments.

Original languageEnglish (US)
Pages (from-to)1665-1675
Number of pages11
JournalChemical Research in Toxicology
Volume20
Issue number11
DOIs
StatePublished - Nov 2007
Externally publishedYes

Fingerprint

Arachidonic Acids
Keto Acids
Hydroxyeicosatetraenoic Acids
Cyclooxygenase 2
Arachidonic Acid
Metabolism
Rats
Epithelial Cells
Dinoprostone
Eicosanoids
Calcium Ionophores
Cell Culture Techniques
Calcimycin
Metabolites
cysteinylglycine
Metabolome
Ionophores

ASJC Scopus subject areas

  • Drug Discovery
  • Organic Chemistry
  • Chemistry(all)
  • Toxicology
  • Health, Toxicology and Mutagenesis

Cite this

Cyclooxygenase-2-mediated metabolism of arachidonic acid to 15-oxo-eicosatetraenoic acid by rat intestinal epithelial cells. / Seon, Hwa Lee; Rangiah, Kannan; Williams, Michelle V.; Wehr, Angela Y.; DuBois, Raymond N.; Blair, Ian A.

In: Chemical Research in Toxicology, Vol. 20, No. 11, 11.2007, p. 1665-1675.

Research output: Contribution to journalArticle

Seon, Hwa Lee ; Rangiah, Kannan ; Williams, Michelle V. ; Wehr, Angela Y. ; DuBois, Raymond N. ; Blair, Ian A. / Cyclooxygenase-2-mediated metabolism of arachidonic acid to 15-oxo-eicosatetraenoic acid by rat intestinal epithelial cells. In: Chemical Research in Toxicology. 2007 ; Vol. 20, No. 11. pp. 1665-1675.
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abstract = "Rat intestinal epithelial cells mat permanently express the cyclooxygenase-2 (COX-2) gene (RIES cells) were used to investigate COX-2-mediated arachidonic acid (AA) metabolism. A targeted chiral lipidomics approach was employed to quantify AA metabolites that were secreted by the cells into the culture media. When intact RIES cells were treated with calcium ionophore A-23187 (1 μM) for 1 h, 11-(R)-hydroxyeicosatetraenoic acid (HETE) was the most abundant metabolite, followed by prostaglandin (PG) E2, 15-(S)-HETE, 15-oxo-eicosatetraenoic acid (ETE), and 15-(R)-HETE. Incubation for a further 23 h after the calcium ionophore was removed resulted in a substantial increase in PGE2 concentrations while HETE and 15-oxo-ETE concentrations decreased to almost undetectable levels. A similar metabolic profile was observed when RIES cells were treated with increasing concentrations of AA for 24 h. Incubation of the RIES cells with 10 μM AA revealed that maximal concentrations of 11-(R)-HETE, 15-(S)-HETE, and 15-oxo-ETE occurred after 10 min of incubation when the 15-(S)-HETE concentrations were approximately twice that of PGE2. There was a gradual decrease in the concentrations of HETE and 15-oxo-ETE over time, whereas PGE2 concentrations increased steadily until they reached a maximum after 24 h of incubation. The ratio of PGE2 to 15-(S)-HETE was then approximately 20:1. 15-(S)-HETE and 15-oxo-ETE concentrations declined in the cell media during prolonged incubations with pseudo-first-order rate constants of 0.0121 and 0.0073 min-1, respectively. 15-(S)-HETE was shown to undergo metabolism primarily to 15-oxo-ETE, which was further metabolized to a glutathione (GSH) adduct. The GSH adduct of 15-oxo-ETE was further metabolized in the extracellular milieu to a cysteinylglycine adduct. Thus, we have established for the first time that 15-oxo-ETE can be formed biosynthetically from AA, that 15-(S)-HETE is its immediate precursor, and that 15-oxo-ETE forms a GSH adduct. For ionophore-A-23187-stimulated cells and at early time points for AA-stimulated cells, 11-(R)-HETE was the major eicosanoid to be secreted into the media. Adding increasing concentrations of AA to cells in culture made it possible to estimate with surprising accuracy endogenous eicosanoid production using regression analyses. Thus, after 24 h in the absence of added AA, 11-(R)-HETE and 15-(R)-HETE were estimated to be present at concentrations close to the detection limit of our very sensitive assay. These data further highlight the importance of endogenous COX-2-mediated lipid peroxidation and illustrate the necessity to monitor eicosanoid formation from endogenous stores of AA in cell culture experiments.",
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T1 - Cyclooxygenase-2-mediated metabolism of arachidonic acid to 15-oxo-eicosatetraenoic acid by rat intestinal epithelial cells

AU - Seon, Hwa Lee

AU - Rangiah, Kannan

AU - Williams, Michelle V.

AU - Wehr, Angela Y.

AU - DuBois, Raymond N.

AU - Blair, Ian A.

PY - 2007/11

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N2 - Rat intestinal epithelial cells mat permanently express the cyclooxygenase-2 (COX-2) gene (RIES cells) were used to investigate COX-2-mediated arachidonic acid (AA) metabolism. A targeted chiral lipidomics approach was employed to quantify AA metabolites that were secreted by the cells into the culture media. When intact RIES cells were treated with calcium ionophore A-23187 (1 μM) for 1 h, 11-(R)-hydroxyeicosatetraenoic acid (HETE) was the most abundant metabolite, followed by prostaglandin (PG) E2, 15-(S)-HETE, 15-oxo-eicosatetraenoic acid (ETE), and 15-(R)-HETE. Incubation for a further 23 h after the calcium ionophore was removed resulted in a substantial increase in PGE2 concentrations while HETE and 15-oxo-ETE concentrations decreased to almost undetectable levels. A similar metabolic profile was observed when RIES cells were treated with increasing concentrations of AA for 24 h. Incubation of the RIES cells with 10 μM AA revealed that maximal concentrations of 11-(R)-HETE, 15-(S)-HETE, and 15-oxo-ETE occurred after 10 min of incubation when the 15-(S)-HETE concentrations were approximately twice that of PGE2. There was a gradual decrease in the concentrations of HETE and 15-oxo-ETE over time, whereas PGE2 concentrations increased steadily until they reached a maximum after 24 h of incubation. The ratio of PGE2 to 15-(S)-HETE was then approximately 20:1. 15-(S)-HETE and 15-oxo-ETE concentrations declined in the cell media during prolonged incubations with pseudo-first-order rate constants of 0.0121 and 0.0073 min-1, respectively. 15-(S)-HETE was shown to undergo metabolism primarily to 15-oxo-ETE, which was further metabolized to a glutathione (GSH) adduct. The GSH adduct of 15-oxo-ETE was further metabolized in the extracellular milieu to a cysteinylglycine adduct. Thus, we have established for the first time that 15-oxo-ETE can be formed biosynthetically from AA, that 15-(S)-HETE is its immediate precursor, and that 15-oxo-ETE forms a GSH adduct. For ionophore-A-23187-stimulated cells and at early time points for AA-stimulated cells, 11-(R)-HETE was the major eicosanoid to be secreted into the media. Adding increasing concentrations of AA to cells in culture made it possible to estimate with surprising accuracy endogenous eicosanoid production using regression analyses. Thus, after 24 h in the absence of added AA, 11-(R)-HETE and 15-(R)-HETE were estimated to be present at concentrations close to the detection limit of our very sensitive assay. These data further highlight the importance of endogenous COX-2-mediated lipid peroxidation and illustrate the necessity to monitor eicosanoid formation from endogenous stores of AA in cell culture experiments.

AB - Rat intestinal epithelial cells mat permanently express the cyclooxygenase-2 (COX-2) gene (RIES cells) were used to investigate COX-2-mediated arachidonic acid (AA) metabolism. A targeted chiral lipidomics approach was employed to quantify AA metabolites that were secreted by the cells into the culture media. When intact RIES cells were treated with calcium ionophore A-23187 (1 μM) for 1 h, 11-(R)-hydroxyeicosatetraenoic acid (HETE) was the most abundant metabolite, followed by prostaglandin (PG) E2, 15-(S)-HETE, 15-oxo-eicosatetraenoic acid (ETE), and 15-(R)-HETE. Incubation for a further 23 h after the calcium ionophore was removed resulted in a substantial increase in PGE2 concentrations while HETE and 15-oxo-ETE concentrations decreased to almost undetectable levels. A similar metabolic profile was observed when RIES cells were treated with increasing concentrations of AA for 24 h. Incubation of the RIES cells with 10 μM AA revealed that maximal concentrations of 11-(R)-HETE, 15-(S)-HETE, and 15-oxo-ETE occurred after 10 min of incubation when the 15-(S)-HETE concentrations were approximately twice that of PGE2. There was a gradual decrease in the concentrations of HETE and 15-oxo-ETE over time, whereas PGE2 concentrations increased steadily until they reached a maximum after 24 h of incubation. The ratio of PGE2 to 15-(S)-HETE was then approximately 20:1. 15-(S)-HETE and 15-oxo-ETE concentrations declined in the cell media during prolonged incubations with pseudo-first-order rate constants of 0.0121 and 0.0073 min-1, respectively. 15-(S)-HETE was shown to undergo metabolism primarily to 15-oxo-ETE, which was further metabolized to a glutathione (GSH) adduct. The GSH adduct of 15-oxo-ETE was further metabolized in the extracellular milieu to a cysteinylglycine adduct. Thus, we have established for the first time that 15-oxo-ETE can be formed biosynthetically from AA, that 15-(S)-HETE is its immediate precursor, and that 15-oxo-ETE forms a GSH adduct. For ionophore-A-23187-stimulated cells and at early time points for AA-stimulated cells, 11-(R)-HETE was the major eicosanoid to be secreted into the media. Adding increasing concentrations of AA to cells in culture made it possible to estimate with surprising accuracy endogenous eicosanoid production using regression analyses. Thus, after 24 h in the absence of added AA, 11-(R)-HETE and 15-(R)-HETE were estimated to be present at concentrations close to the detection limit of our very sensitive assay. These data further highlight the importance of endogenous COX-2-mediated lipid peroxidation and illustrate the necessity to monitor eicosanoid formation from endogenous stores of AA in cell culture experiments.

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