Abstract
The corneal epithelium metabolizes arachidonic acid by a cytochrome P450-(CYP) mediated pathway to 12(R)hydroxy-5,8,10,14-eicosatrienoic acid [12(R)-HETE] and 12(R)hydroxy-5,8,14-eicosatrienoic acid [12(R)-HETrE]. Both metabolites possess potent inflammatory properties with 12(R)-HETrE being a powerful angiogenic factor and assume the role of inflammatory mediators in hypoxia- and chemical-induced injury in the cornea, in vivo. We developed an in vitro model of corneal organ culture to characterize the biochemical and molecular events involved in the increased synthesis of these metabolites. These cultured corneas exhibit epithelial cytochrome P450 CYP-dependent 12(R)-HETE and 12(R)-HETrE synthesis as indicated by chiral analysis and by the ability of CYP enzyme inhibitors to repress their synthesis. Hypoxia greatly and selectively stimulated the synthesis of 12(R)-HETE (7-fold over control normoxic conditions) and 12(R)-HETrE. The bacterial endotoxin, lipopolysaccharide, also increased the synthesis of these eicosanoids, substantiating the notion that this activity may function as an inflammatory pathway. These metabolites were detected in the culture medium by gas chromatography/mass spectroscopy (GC/MS) analysis and their levels significantly increased in hypoxia-treated corneas, further indicating their endogenous formation in response to injury. This in vitro model provides an excellent preparation for studying factors regulating the synthesis of these inflammatory eicosanoids and for isolating, identifying and characterizing the CYP protein responsible for their synthesis.
Footnotes
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Send reprint requests to: Dr. Michal Laniado Schwartzman, Department of Pharmacology, New York Medical College, Valhalla, NY 10595.
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↵1 This work was supported by National Institutes of Health Grants EY06513 (M.L.S.) and DK38226 (J.R.F.).
- Abbreviations:
- CYP
- cytochrome P450
- 12-HETE
- 12-hydroxy-5,8,10,14-eicosatrienoic acid
- 12-HETrE
- 12-hydroxy-5,8,14-eicosatrienoic acid
- 17-ODYA
- 17-octadecynoic acid
- CDC
- cinnamyl-3,4-dihydroxy-α-cyanocinnamate
- NCI-GC/MS
- negative chemical ionization-gas chromatography/mass spectroscopy
- RP-HPLC
- reverse-phase high performance liquid chromatography
- DMEM
- Dulbecco’s modified Eagle’s medium
- PMSF
- phenylmethanesulfonylfluoride
- PFB
- pentafluorobenzyl ester
- TMS
- trimethylsilyl ether
- LH
- lactalbumin enzymatic hydrolysate
- FBS
- fetal bovine serum
- IL
- interleukin
- GC/MS
- gas chromatography/mass spectroscopy
- LPS
- lipopolysaccharide
- Received March 23, 1998.
- Accepted June 23, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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