Hypoxia-Induced Production of 12-Hydroxyeicosanoids in the Corneal Epithelium: Involvement of a Cytochrome P-4504B1 Isoform

• Published in: The Journal of pharmacology and experimental therapeutics Vol. 289; no. 3; pp. 1611 - 1619
• Main Authors: Mastyugin, V, Aversa, E, Bonazzi, A, Vafaes, C, Mieyal, P, Schwartzman, M L
• Format: Journal Article
• Language: English
• Published: United States American Society for Pharmacology and Experimental Therapeutics 01.06.1999
• Subjects: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid - biosynthesis; 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid - metabolism; Animals; Arachidonic Acid - metabolism; Aryl Hydrocarbon Hydroxylases; Base Sequence; Cell Hypoxia - physiology; Clofibrate - pharmacology; Cloning, Molecular; Cytochrome P-450 CYP4A; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; DNA, Complementary; Epithelium, Corneal - drug effects; Epithelium, Corneal - metabolism; Female; Isoenzymes - genetics; Isoenzymes - metabolism; Lung - enzymology; Male; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; Molecular Sequence Data; Organ Culture Techniques; Phenobarbital - pharmacology; Polymerase Chain Reaction; Rabbits; RNA, Messenger - metabolism; Sequence Alignment; Sequence Homology, Nucleic Acid; Stereoisomerism; Transcription, Genetic
• ISSN: 0022-3565; 1521-0103

The corneal epithelium metabolizes arachidonic acid by a cytochrome P-450 (CYP)-mediated activity to 12-hydroxy-5,8,11,14-eicosatetraenoic acid (12( R )-HETE) and 12-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 they assume the role of inflammatory mediators in hypoxia- and chemical-induced injury in the cornea in vivo and in vitro. We used a model of corneal organ culture that exhibits hypoxia-induced epithelial CYP-dependent 12( R )-HETE and 12( R )-HETrE synthesis for isolating, identifying, and characterizing the CYP protein responsible for these eicosanoid syntheses. Northern analysis revealed the presence of a CYP4A-hybridizable mRNA, the levels of which were increased after hypoxia. Reverse transcription-polymerase chain reaction analysis with primers specific for the CYP4A family led to the isolation of a 671-base pair fragment with a 98.8% sequence homology to the rabbit lung CYP4B1 isoform, of which the levels in the corneal epithelium were greatly increased under hypoxic conditions. Moreover, phenobarbital, an inducer of hepatic CYP4B1 in the rabbit, also induced 12-HETE and 12-HETrE synthesis. Antibodies against CYP4B1, but not against CYP4A1, inhibited hypoxia-, clofibrate-, and phenobarbital-induced 12-HETE and 12-HETrE synthesis. These results suggest the involvement of a CYP4B1 isoform in the corneal epithelial synthesis of these eicosanoids in response to hypoxia.