The rabbit pulmonary cytochrome P450 arachidonic acid metabolic pathway: characterization and significance.

Abstract

Abstractacid -dihydroxyeicosatrienoic acid * hydroxyeicosatetraenoic acid Cytochrome P450 metabolizes arachidonic acid to several unique and biologically active compounds in rabbit liver and kidney. Microsomal fractions prepared from rabbit lung homogenates metabolized arachidonic acid through cytochrome P450 pathways, yielding cis-epoxyeicosatrienoic acids (EETs) and their hydration products, vic-dihydroxyeicosatrienoic acids, mid-chain cis-trans conjugated dienols, and 19-and 20-hydroxyeicosatetraenoic acids. Inhibition studies using polyclonal antibodies prepared against purified CYP2B4 demonstrated 100% inhibition of arachidonic acid epoxide formation. Purified CYP2B4, reconstituted in the presence of NADPH-cytochrome P450 reductase and cytochrome b5, metabolized arachidonic acid, producing primarily EETs. EETs were detected in lung homogenate using gas chromatography/mass spectroscopy, providing evidence for the in vivo pulmonary cytochrome P450 epoxidation of arachidonic acid. Chiral analysis of these lung EETs demonstrated a preference for the 14(R),15(S)-, 11(S),12(R)-, and 8(S),9(R)-EET enantiomers. Both EETs and vic-dihydroxyeicosatrienoic acids were detected in bronchoalveolar lavage fluid. At micromolar concentrations, methylated 5,6-EET and 8,9-EET significantly relaxed histamine-contracted guinea pig hilar bronchi in vitro. In contrast, 20-hydroxyeicosatetraenoic acid caused contraction to near maximal tension. We conclude that CYP2B4, an abundant rabbit lung cytochrome P450 enzyme, is the primary constitutive pulmonary arachidonic acid epoxygenase and that these locally produced, biologically active eicosanoids may be involved in maintaining homeostasis within the lung. (J. Clin. Invest. 1995. 95:2150-2160

Identification, Functional Characterization, and Regulation of a New Cytochrome P450 Subfamily, the CYP2Ns

“…CYP2N1 and CYP2N2 metabolized arachidonic acid with catalytic turnovers of 1.1 and 0.21 nmol of product/nmol of P450/min, respectively. Although these rates are somewhat lower than those of mammalian P450s involved in metabolism of xenobiotic substrates, they are comparable to those reported for mammalian P450s involved in arachidonic acid metabolism including CYP1A2, CYP2B1, CYP2B4, CYP2B6, CYP2C8, CYP2C9, CYP2E1, CYP2G1, CYP2J2, CYP2J3, and CYP4A1 (21,22,51,53,(55)(56)(57). Importantly, the rates of arachidonic acid metabolism obtained in the current study for the recombinant CYP2Ns are comparable to that obtained for Fundulus liver microsomes and slightly higher than that obtained for recombinant CYP2P3, a recently described Fundulus P450 expressed in liver and intestine.…”

“…CYP2N1 and CYP2N2 also differ in the regiochemistry of HETE formation. Importantly, the regio-and stereochemical composition of CYP2N1 and CYP2N2 products are different from that of other P450 enzymes known to metabolize arachidonic acid including members of the CYP1A, CYP2B, CYP2C, CYP2E, CYP2J, and CYP4A subfamilies (21,22,(52)(53)(54). CYP2N1 and CYP2N2 metabolized arachidonic acid with catalytic turnovers of 1.1 and 0.21 nmol of product/nmol of P450/min, respectively.…”

“…Cultured Sf9 cells, grown in spinner flasks at a density of 1.5-2.0 ϫ 10 6 cells/ml, were infected with high titer viral stocks in the presence of 5 M hemin or 100 M ␦-aminolevulinic acid hydrochloride. Cells co-expressing recombinant CYP2N1 and CYPOR or recombinant CYP2N2 and CYPOR were harvested 72 h after infection, washed twice with phosphate-buffered saline, and used to prepare microsomal fractions by differential centrifugation at 4°C as described previously (22). The P450 content of the microsomes was determined spectrally according to the method of Omura and Sato (23) with a Shimadzu UV-3000 dual wavelength/ double-beam spectrophotometer (Shimadzu Scientific, Columbia, MD).…”

“…The abundance of CYP2Ns in these tissues together with their documented ability to catalyze arachidonic acid metabolism suggest that these P450s may have specialized endogenous functions, in addition to their potential involvement in metabolism of xenobiotic substrates. A number of studies indicate that P450-derived eicosanoids are endogenous constituents of mammalian hepatic and extrahepatic tissues (21,22,42,43,60) and possess potent biological activities in those tissues (8 -10, 21, 61-70). For example, in the liver, the EETs activate phosphorylase a, affect Ca 2ϩ flux, and may be involved in vasopressin-stimulated glycogenolysis (61,62).…”

Effects of ω-hydroxylase product on distal human pulmonary arteries

“…cytochrome P-450 -hydroxylase has been identified in various lung tissues (13,18,28,33,35), a key issue has been to demonstrate the endogenous involvement of 20-HETE in HPAs. In HPA tissue, it is relevant to analyze the expression of proteins, such the cytochrome P-450 isoforms that produce 20-HETE.…”