Synthesis and Function of Cytochrome P450 4A1-Derived 20-Hydroxyeicosatetraenoic Acid in Rat Renal Arteries
20-hydroxyeicosatetraenoic acid (20-HETE) is the principal arachidonic acid metabolite in tubular and vascular structures of the rat kidney. In the tubules it inhibits sodium reabsorption, while in the renal microcirculation it is a vasoconstrictor and a regulator of myogenic tone. 20-HETE synthesis is catalyzed by the cytochrome P450 (CYP) 4A isoforms (4A1, 4A2, 4A3 and 4A8). Our studies indicated that despite the high homology, these isoforms display distinct catalytic properties. CYP4A1 is the low Km isoform and thus, by far, the most efficient 20-HETE synthesizing enzyme. Whereas CYP4A1 is solely an ω-hydroxylase, CYP4A2 and CYP4A3 also catalyze arachidonate 11,12-epoxidation. Systemic administration of CYP4A1 antisense oligodeoxynucleotides reduced the level of CYP4A proteins and 20-HETE synthesis in renal vessels by 50%, and decreased blood pressure in SHR from 137±3 to 121±4 mmHg (p < 0.05). Immunoblot analysis and inhibitor studies indicated that within the renal vasculature CYP4A1 is primarily localized to the interlobar arteries. Transfection of CYP4A1 cDNA, cloned into the expression plasmid pcDNA3.1, to renal interlobar arteries increased CYP4A immunoreactivity by 3-fold and 20-HETE synthesis from 92±10 to 239±71 pg 20-HETE/mg/h. CYP4A1-transfected arteries demonstrated a marked increase in the constrictor responses to phenylephrine (EC50 = 0.07±0.02 vs 0.37±0.04 μM). The increased constrictor responses to phenylephrine were greatly attenuated by DDMS, a selective inhibitor of 20-HETE synthesis (EC50from 0.07±0.01 to 0.74±0.11 μM) and by 20-HEDE, a specific 20-HETE antagonist (to 0.54±0.05 μM) without affecting the maximal response. The inhibitory effect of DDMS was reversed by addition of 20-HETE further substantiating the notion that increased endogenous levels of 20-HETE contributed to the increased vascular reactivity to phenylephrine in vessels expressing the CYP4A1 cDNA. Thus, 20-HETE of vascular origin serves as a stimulatory regulator of vascular responses to phenylephrine.