Human umbilical vein endothelial cells express P450 2C8 mRNA : cloning of endothelial P450 epoxygenase

Human umbilical vein endothelial cells (EC) metabolize arachidonic acid (AA) through three major pathways—cyclooxygenase, lipoxygenase and cytochrome P450 (P450) isozymes. Previously, we have shown that pathophysiological concentrations of native low density lipoprotein (n-LDL) increase EC P450-dependent epoxyeicosatrienoic acid (EET) production. The present study was designed to identify putative P450 isozymes involved in EC epoxidation of AA. Reverse transcription—polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) were employed to detect P450 2 family cDNA from EC mRNA. Degenerate primers complimenting 2 homologous regions from 6 different P450 2 families were designed to capture a 440 bp cDNA fragment corresponding to the heme binding region of P450 2 isozymes. RT-PCR of EC total RNA with these primers amplified a 440 bp fragment. After gel purification, the fragment was cloned, sequenced and found to share a high degree of identity with human liver P450 2C8 and 2C9. New pri...