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, pIRES2-EGFP-4A1 and pIRES2-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 pIRES2-EGFP-4A1- or pIRES2-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 pIRES2-EGFP-4A1- and pIRES2-EGFP-4A2-transfected cells as compared with control pIRES2-transfected cells. Cells transfected with pIRES2-EGFP-4A1 and pIRES2-EGFP-4A2 catalyzed arachidonic acid omega-hydroxylation to 20-HETE at rates of 0.85 +/- 0.29 and 0.27 +/- 0.04 nmol/10(7) 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 pIRES2-EGFP-4A1 and pIRES2-EGFP-4A2 showed increased vasoreactivity displaying EC50 to phenylephrine of 0.24 +/- 0.07 and 0.11 +/- 0.03 microM, respectively, as compared with arteries transfected with pIRES2-EGFP (1.11 +/- 0.21 microM; 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 CYP4A1 cDNA, suggesting that the CYP4A product responsible for the sensitizing effect, presumably 20-HETE, is not of endothelial cell origin.
Wang
Ji-Shi
JS
Department of Pharmacology, New York Medical College, Valhalla, NY 10595, USA.
Zhang
Fan
F
Jiang
Miao
M
Wang
Mong-Heng
MH
Zand
Barbara A
BA
Abraham
Nader G
NG
Nasjletti
Alberto
A
Laniado-Schwartzman
Michal
M
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001158:Arteries / Q000201:enzymology / Q000378:metabolism; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics*; D000072470:Cytochrome P450 Family 4; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006893:Hydroxyeicosatetraenoic Acids / Q000096:biosynthesis; D007537:Isometric Contraction / Q000187:drug effects; D007850:Lauric Acids / Q000378:metabolism; D009119:Muscle Contraction / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D010656:Phenylephrine / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012079:Renal Circulation / Q000502:physiology; D014162:Transfection
D000316:Adrenergic alpha-Agonists; C465387:Cyp4a1protein, rat; D006893:Hydroxyeicosatetraenoic Acids; D007850:Lauric Acids; C030358:lauric acid; D049452:Green Fluorescent Proteins; D010656:Phenylephrine; D016718:Arachidonic Acid; C055987:20-hydroxy-5,8,11,14-eicosatetraenoic acid; D003577:Cytochrome P-450 Enzyme System; D000072470:Cytochrome P450 Family 4; C465388:cytochrome P-450 CYP4A2 (rat)