Kinetic profile of the rat CYP4A isoforms: arachidonic acid metabolism and isoform-specific inhibitors

Abstract

20-Hydroxyeicosatetraenoic acid (HETE), the cytochrome P-450 (CYP) 4A ω-hydroxylation product of arachidonic acid, has potent biological effects on renal tubular and vascular functions and on the control of arterial pressure. We have expressed high levels of the rat CYP4A1, -4A2, -4A3, and -4A8 cDNAs, using baculovirus and Sf 9 insect cells. Arachidonic acid ω- and ω-1-hydroxylations were catalyzed by three of the CYP4A isoforms; the highest catalytic efficiency of 947 nM−1 ⋅ min−1for CYP4A1 was followed by 72 and 22 nM−1 ⋅ min−1for CYP4A2 and CYP4A3, respectively. CYP4A2 and CYP4A3 exhibited an additional arachidonate 11,12-epoxidation activity, whereas CYP4A1 operated solely as an ω-hydroxylase. CYP4A8 did not catalyze arachidonic or linoleic acid but did have a detectable lauric acid ω-hydroxylation activity. The inhibitory activity of various acetylenic and olefinic fatty acid analogs revealed differences and indicated isoform-specific inhibition. These studies suggest that CYP4A1, despite its low expression in extrahepatic tissues, may constitute the major source of 20-HETE synthesis. Moreover, the ability of CYP4A2 and -4A3 to catalyze the formation of two opposing biologically active metabolites, 20-HETE and 11,12-epoxyeicosatrienoic acid, may be of great significance to the regulation of vascular tone.

Inhibition of Cytochrome P450ω-Hydroxylase

“…28 Although CYP4A1 and CYP4A3 can metabolize AA to 20-HETE and 11,12-EET, CYP4A1 specifically metabolizes AA to 20-HETE with greater catalytic activity. 32 Western blot analysis indicated immunoreactive bands associated with the primary antibodies against CYP4A1, CYP4A2, and CYP4F proteins from microsomes of heart tissue. As expected, the intensity of the immunoreactive bands was more intense from the membrane protein fractions 36 than from the cytosolic protein fractions.…”

“…It has been shown that CYP4A2 and CYP4A3 can produce 20-HETE as well as 11,12-EET (precursor of 11,12-DHET). 27,32 Thus, it is possible that DDMS also inhibits CYP4A2 and thus 11,12-EET (or 11,12-DHET) production. The extent of the contribution of 14,15-DHET in cardioprotection, if any, remains unknown.…”

Long-term modifications of blood pressure in normotensive and spontaneously hypertensive rats by gene delivery of rAAV-mediated cytochrome P450 arachidonic acid hydroxylase

“…Four isoforms of cytochrome p-450 in the 4A family (4A1, 4A2, 4A3 and 4A8) have been identified in rats to date [23,24]. All of the isoforms catalyze the ω-and ω-1 hydroxylation of arachidonic acid to produce 20-HETE [2,5,[25][26][27].…”

ω3 and ω6 CYP450 Eicosanoid Derivatives: Key Lipid Mediators in the Regulation of Pulmonary Hypertension

“…The CYP450-dependent EPA metabolites include the epoxyeicosatetraenoic acid regioisomers 5(6)-, 8(9)-, 11(12)-, 14(15)-and 17(18)-EpETE (Lauterbach et al, 2002). Specific CYP450 epoxygenase isoforms are involved in EPA metabolism which produce (17(18)-EpETE), and include CYP1A (Schwarz et al, 2004), CYP4A1, CYP4A3 (Nguyen et al, 1999, Lauterbach et al, 2002 and CYP4A12A. An additional potential source for 17(18)-EpETE are endothelial CYP450 isoforms of the 2C and 2J subfamilies that otherwise produce EET from AA.…”

“…Meanwhile, epoxyeicosatrienoic acid (EET) regioisomers have been shown to activate large conductance calcium-activated potassium channels in vascular smooth muscle cells and are considered as leading candidates for endothelium-derived hyperpolarizing factor (EDHF) in the coronary and systemic circulation (Zeldin et al, 1996, Fisslthaler et al, 1999, Node et al, 1999, Capdevila et al, 2000. Specific CYP450 epoxygenase isoforms are involved in EPA metabolism and produce 17(18)-epoxyeicosatetraenoic acid (17(18)-EpETE, see Fig.1B) (Lauterbach et al, 2002, Nguyen et al, 1999, Schwarz et al, 2004. Epoxy-docosapentaenoic acids are CYP450-dependent DHA metabolites (Fig.…”