Received for publication May 10, 2004.
Revised July 15, 2004.
Accepted for publication July 21, 2004.

Transfection and Functional Expression of CYP4A1 and CYP4A2 Using Bicistronic Vectors in Vascular Cells and Tissues

Abstract

20-hydroxyeicosatetraenoic acid (20-HETE), a CYP4A-derived arachidonic acid (AA) 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 EGFP and CYP4A1/4A2 were expressed in 80-90% of the cells. Western blot showed a 3-5-fold increase of CYP4A1 and CYP4A2 proteins in pIRES2-EGFP-4A1 and pIRES2-EGFP-4A2 transfected cells, respectively. Cells transfected with pIRES2-EGFP-4A1 and pIRES2-EGFP-4A2 catalyzed arachidonic acid -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 which was localized to the intima as well as to the 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 µmol/L, respectively, as compared to arteries transfected with pIRES2-EGFP (1.11±0.21 µmol/L; n=6, p<0.05). The increased vasoreactivity to phenylephrine was inhibited by DDMS, an inhibitor of CYP4A catalyzed reactions, suggesting that a product of CYP4A1 and CYP4A2 catalytic activity contributed to the increased constrictor responsiveness. The ability to monitor CYP expression by following EGFP immunofluorescence provides a useful tool for evaluating the consequences of increasing endogenous levels of 20-HETE, an important modulator of vascular function.

Key words: 20-HETE, acetylcholine, arachidonic acid, cytochrome P450, phenylephrine, vascular reactivity

This article has been cited by other articles:

				J.-S. Wang, H. Singh, F. Zhang, T. Ishizuka, H. Deng, R. Kemp, M. S. Wolin, T. H. Hintze, N. G. Abraham, A. Nasjletti, et al. Endothelial Dysfunction and Hypertension in Rats Transduced With CYP4A2 Adenovirus Circ. Res., April 14, 2006; 98(7): 962 - 969. [Abstract] [Full Text] [PDF]