Transfection and functional expression of CYP4A1 and CYP4A2 using bicistronic vectors in vascular cells and tissues

20-hydroxyeicosatetraenoic acid (20-HETE), a CYP4A-derived arachidonic acid metabolite, is a potent vasoconstrictor and a modulator of vascular reactivity. We have shown that CYP4A1 and CYP4A2 are the major CYP4A isoforms expressed in the rat renal microcirculation. In the present study, we constructed two bicistronic vectors, plRES2-EGFP-4A1 and plRES2-EGFP-4A2, and examined their functional efficacy in COS-1 and vascular smooth muscle (A7r5) cells and in microdissected rat interlobar arteries. Immunocytochemistry coupled with fluorescence microscopy of plRES2-EGFP-4A1- or plRES2-EGFP-4A2-transfected COS-1 and A7r5 cells indicated that both enhanced green fluorescence protein (EGFP) and CYP4A1/4A2 were expressed in 80 to 90% of the cells. Western blot analysis showed a 3- to 5-fold increase of CYP4A1 and CYP4A2 proteins in plRES2-EGFP-4A1- and plRES2-EGFP-4A2-transfected cells as compared with control plRES2-transfected cells. Cells transfected with plRES2-EGFP-4A1 and plRES2-EGFP-4A2 catalyzed arachidonic acid ω-hydroxylation to 20-HETE at rates of 0.85 ± 0.29 and 0.27 ± 0.04 nmol/10⁷ cells/h, respectively. Transfection of interlobar arteries with either plasmid yielded EGFP immunofluorescence that was localized to the intima, media, and adventitia. Arteries transfected with plRES2-EGFP-4A1 and plRES2-EGFP-4A2 showed increased vasoreactivity displaying EC₅₀ to phenylephrine of 0.24 ± 0.07 and 0.11 ± 0.03 μM, respectively, as compared with arteries transfected with plRES2-EGFP (1.11 ± 0.21 μM; n = 6, p < 0.05). The increased vasoreactivity to phenylephrine was inhibited by N-methylsulfonyl-12, 12-dibromododec-11-enamide, an inhibitor of CYP4A-catalyzed reactions, suggesting that a product of CYP4A1 and CYP4A2 catalytic activity contributed to the increased constrictor responsiveness. Removal of the endothelium did not prevent the sensitization to phenylephrine in vessels transfected with the plasmid containing the CYP4A1c cDNA, suggesting that the CYP4A product responsible for the sensitizing effect, presumably 20-HETE, is not of endothelial cell origin.