Document Detail

The effect of hypoxia on endogenous corneal epithelial eicosanoids.
MedLine Citation:
PMID:  10892859     Owner:  NLM     Status:  MEDLINE    
PURPOSE: Injury to the corneal epithelium increases arachidonic acid (AA) metabolism through the cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 pathways. The authors used the rabbit corneal organ culture model to demonstrate the effect of hypoxia on the endogenous formation of 12-hydroxy-5,8,11,14-eicosatetraenoic acid (12-HETE), 12-hydroxy-5,8,14-eicosatrienoic acid (12-HETrE), and prostaglandin (PG) E2 by the intact cornea in the absence of exogenously added cofactors or substrate. METHODS: Rabbit corneas were isolated and cultured for 24 hours in normoxia or hypoxia. After culture, PGE2 in media was quantitated by enzyme immunoassay. 12-HETE and 12-HETrE were extracted from culture media and corneal epithelium and quantitated by negative chemical ionization-gas chromatography-mass spectrometry. COX-1 and -2 protein expression in corneal epithelium was determined by Western blot. Acute (2 hours) COX activity in normoxia and hypoxia was determined as the conversion rate of [14C]AA to [14C]PGE2, quantitated through reverse-phase-high-performance liquid chromatography and radiodetection. RESULTS: In the media of cultured rabbit corneas, both 12-HETE and 12-HETrE were detected, with 12-HETrE levels being four times higher. Hypoxia did not significantly increase extracellular 12-HETE or 12-HETrE; however, it caused more than 90% inhibition of PGE2 synthesis. Intracellular 12-HETE and 12-HETrE were undetectable in normal corneas but increased to 7.7+/-1.3 and 2.2+/-0.4 ng/mg protein, respectively, after 24 hours in culture. Culture in hypoxia further increased intracellular 12-HETE threefold but had no additional effect on 12-HETrE. CONCLUSIONS: Hypoxia creates an environment in which epithelial COX activity is severely suppressed, whereas cytochrome P450-AA and/or 12-LOX metabolizing activity is maintained or enhanced. Additionally, the findings suggest that 12-HETE produced by the corneal epithelium acts intracellularly to promote corneal edema, whereas 12-HETrE acts in a paracrine manner to initiate an inflammatory cascade that can elicit neutrophil chemotaxis and neovascularization of the cornea.
P A Mieyal; A Bonazzi; H Jiang; M W Dunn; M L Schwartzman
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Investigative ophthalmology & visual science     Volume:  41     ISSN:  0146-0404     ISO Abbreviation:  Invest. Ophthalmol. Vis. Sci.     Publication Date:  2000 Jul 
Date Detail:
Created Date:  2000-07-31     Completed Date:  2000-07-31     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  7703701     Medline TA:  Invest Ophthalmol Vis Sci     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  2170-6     Citation Subset:  IM    
Department of Pharmacology, New York Medical College, Valhalla 10595, USA.
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MeSH Terms
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid / biosynthesis*
8,11,14-Eicosatrienoic Acid / analogs & derivatives*,  metabolism*
Anoxia / metabolism*
Blotting, Western
Chromatography, High Pressure Liquid
Culture Media
Cyclooxygenase 1
Cyclooxygenase 2
Dinoprostone / biosynthesis*
Epithelium, Corneal / metabolism*
Gas Chromatography-Mass Spectrometry
Immunoenzyme Techniques
Isoenzymes / metabolism
Organ Culture Techniques
Prostaglandin-Endoperoxide Synthases / metabolism
Grant Support
Reg. No./Substance:
0/Culture Media; 0/Isoenzymes; 117346-20-0/12-hydroxy-5,8,14-eicosatrienoic acid; 363-24-6/Dinoprostone; 59985-28-3/12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 7324-41-6/8,11,14-Eicosatrienoic Acid; EC 1; EC 2; EC Synthases

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