Hypoxia-induced production of 12-hydroxyeicosanoids in the corneal epithelium: involvement of a cytochrome P-4504B1 isoform
Autor: | V, Mastyugin, E, Aversa, A, Bonazzi, C, Vafaes, P, Mieyal, M L, Schwartzman |
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Rok vydání: | 1999 |
Předmět: |
Male
DNA Complementary Transcription Genetic Molecular Sequence Data Polymerase Chain Reaction Mixed Function Oxygenases Organ Culture Techniques Cytochrome P-450 Enzyme System Sequence Homology Nucleic Acid Animals 12-Hydroxy-5 8 10 14-eicosatetraenoic Acid Clofibrate RNA Messenger Cloning Molecular Lung Arachidonic Acid Base Sequence Epithelium Corneal Stereoisomerism Cell Hypoxia Isoenzymes Phenobarbital Female Aryl Hydrocarbon Hydroxylases Rabbits Cytochrome P-450 CYP4A Sequence Alignment |
Zdroj: | The Journal of pharmacology and experimental therapeutics. 289(3) |
ISSN: | 0022-3565 |
Popis: | 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. |
Databáze: | OpenAIRE |
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