Arachidonic Acid Epoxides Research Articles

Potassium channels in the peripheral microcirculation.

Vascular smooth muscle (VSM) cells, endothelial cells (EC), and pericytes that form the walls of vessels in the microcirculation express a diverse array of ion channels that play an important role in the function of these cells and the microcirculation in both health and disease. This brief review focuses on the K+ channels expressed in smooth muscle and endothelial cells in arterioles. Microvascular VSM cells express at least four different classes of K+ channels, including inward-rectifier K+ channels (Kin), ATP-sensitive K+ channels (KATP), voltage-gated K+ channels (Kv), and large conductance Ca2+-activated K+ channels (BKCa). VSM KIR participate in dilation induced by elevated extracellular K+ and may also be activated by C-type natriuretic peptide, a putative endothelium-derived hyperpolarizing factor (EDHF). Vasodilators acting through cAMP or cGMP signaling pathways in VSM may open KATP, Kv, and BKCa, causing membrane hyperpolarization and vasodilation. VSMBKc. may also be activated by epoxides of arachidonic acid (EETs) identified as EDHF in some systems. Conversely, vasoconstrictors may close KATP, Kv, and BKCa through protein kinase C, Rho-kinase, or c-Src pathways and contribute to VSM depolarization and vasoconstriction. At the same time Kv and BKCa act in a negative feedback manner to limit depolarization and prevent vasospasm. Microvascular EC express at least 5 classes of K+ channels, including small (sKCa) and intermediate(IKCa) conductance Ca2+-activated K+ channels, Kin, KATP, and Kv. Both sK and IK are opened by endothelium-dependent vasodilators that increase EC intracellular Ca2+ to cause membrane hyper-polarization that may be conducted through myoendothelial gap junctions to hyperpolarize and relax arteriolar VSM. KIR may serve to amplify sKCa- and IKCa-induced hyperpolarization and allow active transmission of hyperpolarization along EC through gap junctions. EC KIR channels may also be opened by elevated extracellular K+ and participate in K+-induced vasodilation. EC KATP channels may be activated by vasodilators as in VSM. Kv channels may provide a negative feedback mechanism to limit depolarization in some endothelial cells.

Cytochrome P450 4A Isoform Inhibitory Profile of N-Hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine (HET0016), a Selective Inhibitor of 20-HETE Synthesis

We examined the effect of N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine) (HET0016), an inhibitor of 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) synthesis on the omega-hydroxylation and epoxidation of arachidonic acid (AA) catalyzed by recombinant cytochrome P450 4A1 (CYP4A1), CYP4A2 and CYP4A3, and characterized the enzyme inhibitory profile of HET0016. The IC50 values of HET0016 for recombinant CYP4A1-, CYP4A2- and CYP4A3-catalyzed 20-HETE synthesis averaged 17.7 nM, 12.1 nM and 20.6 nM, respectively. The IC50 value for production of 11,12-epoxy-5,8,14-eicosatrienoic acid (11,12-EET) by CYP4A2 and 4A3 averaged 12.7 nM and 22.0 nM, respectively. The IC50 value for CYP2C11 activity was 611 nM which was much greater than that for CYP4As. The initial velocity study showed the Ki value of HET0016 for CYP4A1 was 19.5 nM and a plot of Vmax versus amount of recombinant CYP4A1 added shows HET0016 is an irreversible non-competitive inhibitor. These results indicate that HET0016 is a selective, non-competitive and irreversible inhibitor of CYP4A.

Polymorphisms in human soluble epoxide hydrolase.

Human soluble epoxide hydrolase (hsEH) metabolizes a variety of epoxides to the corresponding vicinal diols. Arachidonic and linoleic acid epoxides are thought to be endogenous substrates for hsEH. Enzyme activity in humans shows high interindividual variation (e.g., 500-fold in liver) suggesting the existence of regulatory and/or structural gene polymorphisms. We resequenced each of the 19 exons of the hsEH gene (EPHX2) from 72 persons representing black, Asian, and white populations. A variety of polymorphisms was found, six of which result in amino acid substitutions. Amino acid variants were localized on the crystal structure of the mouse sEH, resulting in the prediction that at least two of these (Arg287Gln and Arg103Cys) might significantly affect enzyme function. The six variants of the hsEH cDNA corresponding to each single polymorphism and one corresponding to a double polymorphism were then constructed by site-directed mutagenesis and expressed in insect cells. As predicted, Arg287Gln and the double mutant Arg287Gln/Arg103Cys showed decreased enzyme activity using trans-stilbene oxide, trans-diphenylpropene oxide, and 14,15-epoxyeicosatrienoic acid as substrates. Lys55Arg and Cys154Tyr mutants had elevated activity for all three substrates. Detailed kinetic studies revealed that the double mutant Arg287Gln/Arg103Cys showed significant differences in Km and Vmax. In addition, stability studies showed that the double mutant was less stable than wild-type protein when incubated at 37 degrees C. These results suggest that at least six hsEH variants exist in the human population and that at least four of these may influence hsEH-mediated metabolism of exogenous and endogenous epoxide substrates in vivo.

Structural refinement of inhibitors of urea-based soluble epoxide hydrolases

The soluble epoxide hydrolase (sEH) is involved in the metabolism of arachidonic, linoleic, and other fatty acid epoxides, endogenous chemical mediators that play an important role in blood pressure regulation and inflammation. 1,3-Disubstituted ureas, carbamates, and amides are new potent and stable inhibitors of sEH. However, the poor solubility of the lead compounds limits their use. Inhibitor structure–activity relationships were investigated to better define the structural requirements for inhibition and to identify points in the molecular topography that could accept polar groups without diminishing inhibition potency. Results indicate that lipophilicity is an important factor controlling inhibitor potency. Polar groups could be incorporated into one of the alkyl groups without loss of activity if they were placed at a sufficient distance from the urea function. The resulting compounds had a 2-fold higher water solubility. These findings will facilitate the rational design and optimization of sEH inhibitors with better physical properties.

Synthesis of arachidonic acid-derived lipoxygenase and cytochrome P450 products in the intact human lung vasculature.

Lipoxygenase (LO) and cytochrome P450 monooxygenase products of arachidonic acid (AA) have been implicated in a large number of vasoregulatory processes. In intact, blood-free, perfused and ventilated human lungs (n = 8), isolated during surgery for bronchial carcinoma, we analyzed leukotrienes (LTs), hydroxyeicosatetraenoic acids (HETEs), and epoxyeicosatrienoic acids (EETs) by sequential sampling of the recirculating buffer fluid. For the analysis we used multistep, solid-phase extraction, isocratic reversed-phase high-performance liquid chromatography, with elution of all metabolites within one run and photodiode array detection to obtain full UV spectra of eluting compounds. We detected no LT release in a 15-min baseline period, but the admixture of the calcium ionophore A23187 with the buffer fluid provoked the rapid appearance of all LTs. Some baseline release of 15-HETE was observed, and in response to A23187, maximum buffer concentrations were noted for 5-HETE, with 8-HETE, 9-HETE, 11-HETE, and 12-HETE being detected at lower levels. Marked baseline liberation of 11,12-EET and 8,9-EET was observed. In response to A23187, high oxirane buffer concentrations were registered, which far surpassed those of LTs and HETEs. The eicosanoid release was paralleled by a limited pulmonary artery pressor response and progressive vascular leakage. We conclude that ex-vivo-perfused human lungs release EETs > LTs > HETEs into the vascular compartment in response to inflammatory challenge. The marked oxirane synthesis in the lung vasculature may have major impact on lung vasoregulation when considering the possible function of these AA epoxides as endothelium-derived hyperpolarizing factors.

TCDD Induces CYP1A4 and CYP1A5 in Chick Liver and Kidney and Only CYP1A4, an Enzyme Lacking Arachidonic Acid Epoxygenase Activity, in Myocardium and Vascular Endothelium

The toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other Ah receptor ligands, species differences in sensitivity and the relationship of CYP1A induction to the toxicity, are poorly understood. Ah receptor ligands induce formation of CYP1A1 and 1A2 in mammals and of a different set of enzymes, CYP1A4 and 1A5, in chicks. We examined induction by TCDD of CYP1A4 and 1A5 mRNA and protein in chick embryo liver, heart, kidney, lung, intestine, bursa, spleen, thymus, brain, and muscle by in situ hybridization and immunohistochemistry and verified the histochemical findings by CYP-specific assays, 7-ethoxyresorufin deethylase for CYP1A4 and arachidonic acid epoxygenation for CYP1A5. CYP1A4 alone was extensively induced in the cardiovascular system, in cardiac myocytes, in perivascular cells having the same location as impulse-conducting Purkinje cells, and like CYP1A1, in vascular endothelium in every organ examined. Unlike mammalian CYP1A, CYP1A4 and 1A5 were both substantially induced in kidney proximal tubules as well as liver, and neither enzyme was induced in kidney glomeruli or lung or brain parenchymal cells. The findings demonstrate (a) a route for CYP1A4 to affect cardiac function, (b) that vascular endothelium is a major site of CYP1A induction across species, and (c) that CYP1A induced in heart or endothelial cells cannot affect cardiac or vascular function via generation of arachidonic acid epoxides because the CYP1A enzymes induced in those organs are not arachidonic acid epoxygenases. Further, the specificity of CYP1A induction sites and of the catalytically active enzymes induced at each site support a significant role for CYP1A induction in Ah receptor ligand toxicity and species differences in sensitivity.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Induces Hepatic Cytochrome P450-Dependent Arachidonic Acid Epoxygenation in Diverse Avian Orders: Regioisomer Selectivity and Immunochemical Comparison of the TCDD-Induced P450s to CYP1A4 and 1A5

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) markedly induced cytochrome P450 (CYP)-dependent arachidonic acid metabolism in liver microsomes from hatchlings of four avian species belonging to four different orders: chick, pigeon, cormorant, and great blue heron, increasing formation of arachidonic acid epoxides (EETs), monohydroxyeicosatetraenoic acids (HETEs), ω−1, and ω−2 OH arachidonic acid products by fivefold or more. Microsomes from TCDD-induced hatchling chicks had the highest activity and the least restricted EET regioselectivity. ω-OH arachidonic acid, the principal constitutive metabolite in chick and pigeon liver microsomes and a major product for cormorant and great blue heron was not induced by TCDD. Constitutive EET formation in avian liver microsomes was very low except in cormorant microsomes where 8,9-EET was generated almost exclusively. Western blots of liver microsomes using polyclonal antisera to chick embryo-derived CYP1A4 and 1A5 recognized two TCDD-induced bands in each of the species. The chick bands had the same molecular weights as CYP1A4 and 1A5 (55 and 55.5 kDa, respectively) but those of the other species differed. Immunopurified antiserum monospecific for CYP1A5 recognized a band in microsomes from all of the avian species, and monospecific antiserum for CYP1A4 recognized a band in microsomes from chick, pigeon, and great blue heron. AntiCYP1A4 and 1A5 IgG immunoinhibited TCDD-induced mixed function oxidase activity completely in chick and chick embryo microsomes and only partially in the other avian microsomes. The results demonstrate that (1) TCDD causes much greater induction of CYP-dependent arachidonic acid metabolism, and of arachidonic acid epoxygenation in particular, in avian than in mammalian species; (2) TCDD induces two CYP1A-related enzymes in birds as in mammals; (3) CYP1A enzymes in the birds other than chicks are not identical to CYP1A4 and 1A5 but share some enzymatic and immunochemical characteristics with them; (4) constitutive ω-OH arachidonic acid in all of the avian species and 8,9-EET in cormorant are formed by CYP enzymes unrelated to CYP1A; and (5) two distinct characteristics of avian CYP1A enzymes are the acquisition by avian CYP1A4-related P450 of unique epitope(s) and by CYP1A5-related P450 of unusual catalytic effectiveness for arachidonic acid epoxygenation.

The physiological role of P450-derived arachidonic acid metabolites

Arachidonic acid is metabolized to biologically active substances by three major enzyme systems including cyclooxygenases, lipoxygenases and cytochrome P450s. The third pathway, P450-dependent pathway, includes allylic oxidation, omega-hydroxylation, and epoxidation of arachidonic acid. Of these metabolites, the physiological role of 20-hydroxyeicosatetraenoic acid (20-HETE) produced by CYP4A isoforms has been extensively studied. 20-HETE affects ion transport, constricts blood vessels and participates in tubuloglomerular feed back. Increased production of 20-HETE is a major factor in elevating blood pressure in spontaneously hypertensive rats (SHR). We have found that CYP4A2 level in SHR is much higher than that of normotensive rat. Recently, factors of endothelial origin other than nitric oxide and prostaglandins were reported. Inhibitors of P450-dependent arachidonic acid metabolism greatly reduce the vasodilator effect and this factor is speculated to be an epoxide of arachidonic acid. We have isolated CYP2C23 from rat kidney and have found that it produces arachidonic acid epoxides. We have investigated changes in the CYP2C23 levels in physiological and pathophysiological conditions. Multiple pathways of arachidonic acid metabolism by P450 have been reported and the diverse properties of these metabolites and the wide distribution of the P450 system make them prime candidates for participation in regulatory mechanisms of the circulation and transporting epithelia.

Stimulation of prostaglandin G/H synthase-2 expression by arachidonic acid monoxygenase product, 14,15-epoxyeicosatrienoic acid

The relationship between arachidonic acid (AA) mobilization and transcription of immediate-early genes, particularly of prostaglandin G/H synthase-2 (PGHS-2), in intestinal crypt epithelial cells was analyzed. PGHS-2 mRNA and protein synthesis were stimulated by its own substrate, AA; actinomycin D, a transcription inhibitor, prevented the AA-induced increase in PGHS-2 mRNA. Eicosatetraynoic acid, an inhibitor of AA utilization, significantly reduced PGHS-2 mRNA synthesis elicited by AA. Inhibitors of cytochrome P450 monoxygenases, ketoconazole and miconazole, also prevented PGHS-2 mRNA synthesis in a dose-dependent manner. Phenyl chalcone oxide, an epoxide hydrolase inhibitor, potentiated AA-induced PGHS-2 mRNA synthesis. Of the four regioisomers of arachidonic acid epoxides, only 14,15-epoxyeicosatrienoic acid elicited the expression of PGHS-2 in intestinal crypt epithelial cells. This is the first direct evidence of stimulation of an immediate-early gene product, specifically PGHS-2, by an AA epoxygenase metabolite, 14,15-epoxyeicosatrienoic acid, as well as of a heterologous regulation of PGHS-2 synthesis by these monoxygenase products.

CYP2J subfamily cytochrome P450s in the gastrointestinal tract: expression, localization, and potential functional significance.

Our laboratory recently described a new human cytochrome P450 arachidonic acid epoxygenase (CYP2J2) and the corresponding rat homologue (CYP2J3), both of which were expressed in extrahepatic tissues. Northern analysis of RNA prepared from the human and rat intestine demonstrated that CYP2J2 and CYP2J3 mRNAs were expressed primarily in the small intestine and colon. In contrast, immunoblotting studies using a polyclonal antibody raised against recombinant CYP2J2 showed that CYP2J proteins were expressed throughout the gastrointestinal tract. Immunohistochemical staining of formalin-fixed, paraffin-embedded intestinal sections using anti-CYP2J2 IgG and avidin-biotin-peroxidase detection revealed that CYP2J proteins were present at high levels in nerve cells of autonomic ganglia, epithelial cells, intestinal smooth muscle cells, and vascular endothelium. The distribution of this immunoreactivity was confirmed by in situ hybridization using a CYP2J2-specific antisense RNA probe. Microsomal fractions prepared from human jejunum catalyzed the NADPH-dependent metabolism of arachidonic acid to epoxyeicosatrienoic acids as the principal reaction products. Direct evidence for the in vivo epoxidation of arachidonic acid by intestinal cytochrome P450 was provided by documenting, for the first time, the presence of epoxyeicosatrienoic acids in human jejunum by gas chromatography/mass spectrometry. We conclude that human and rat intestine contain an arachidonic acid epoxygenase belonging to the CYP2J subfamily that is localized to autonomic ganglion cells, epithelial cells, smooth muscle cells, and vascular endothelium. In addition to the known effects on intestinal vascular tone, we speculate that CYP2J products may be involved in the release of intestinal neuropeptides, control of intestinal motility, and/or modulation of intestinal fluid/electrolyte transport.

Lipid Peroxidation, CYP2E1 and Arachidonic Acid Metabolism in Alcoholic Liver Disease in Rats

The role of cytochrome P450 metabolism of fatty acids and lipid peroxidation in the alterations of the fatty acid composition of the liver and liver pathology was investigated. The CYP2E1 inhibitors partially prevented CYP2E1 induction by ethanol and completely blocked lipid peroxidation. However, the liver pathology induced by ethanol was only partially prevented as was the decrease in arachidonic acid in total liver lipid, triglycerides and cholesterol esters. This means that liver peroxidation induced by ethanol can not completely account for the liver pathology or the decrease in arachidonic acid caused by ethanol. Lauric acid ω-1 hydroxidation by the liver microsomes in vitro was increased by ethanol and partially blocked by CYP2E1 inhibitors. However, although ethanol feeding increased the total hydroxidation and epoxidation of arachidonic acid, these were not inhibited by CYP2E1 inhibitors. Thus the ethanol-induced arachidonic acid depletion is not likely due to CYP2E1 metabolism of arachidonic acid, since the severity of liver pathology correlated negatively with the decrease in arachidonic acid in the ethanol-fed rats. The increase in its metabolism by microsomes and decrease in synthesis may be an important mechanism of ethanol-induced liver injury.

Predominant Expression of an Arachidonate Epoxygenase in Islets of Langerhans Cells in Human and Rat Pancreas

Our laboratory recently described a new human cytochrome P450 arachidonic acid epoxygenase (CYP2J2) and the corresponding rat homolog (CYP2J3). Immunoblotting studies using a polyclonal antibody raised against recombinant human CYP2J2 confirmed CYP2J protein expression in human and rat pancreatic tissues. Immunohistochemical staining of formalin-fixed paraffin-embedded rat and human pancreas using the anti-CYP2J2 IgG and avidin-biotin-peroxidase detection revealed that CYP2J2 protein expression was highly localized to cells in the islets of Langerhans, with minimal staining in pancreatic exocrine cells. Colocalization studies using antibodies to the glucagon, insulin, somatostatin, and pancreatic polypeptide as markers for alpha-, beta-, delta-, and PP cells, respectively, showed that CYP2J protein expression was abundantly present in all four cell types, but was highest in the glucagon-producing alpha-cells. Direct evidence for the epoxidation of arachidonic acid by pancreatic cytochrome P450 was provided by documenting, for the first time, the presence of epoxyeicosatrienoic acids in vivo in human and rat pancreas by gas chromatography/mass spectrometry. Importantly, the levels of immunoreactive CYP2J2 in different human pancreatic tissues were highly correlated with endogenous epoxyeicosatrienoic acid concentrations. We conclude that human and rat pancreas contain an arachidonic acid epoxygenase belonging to the CYP2J subfamily that is highly localized to islet cells. These data together with previous work showing effects of epoxyeicosatrienoic acids in stimulating insulin and glucagon secretion from isolated rat pancreatic islets support the hypothesis that epoxygenase products may be involved in stimulus-secretion coupling in the pancreas.

An Active Site Substitution, F87V, Converts Cytochrome P450 BM-3 into a Regio- and Stereoselective (14S,15R)-Arachidonic Acid Epoxygenase

Cytochrome P450 BM-3 catalyzes the high turnover regio- and stereoselective metabolism of arachidonic and eicosapentaenoic acids. To map structural determinants of productive active site fatty acid binding, we mutated two amino acid residues, arginine 47 and phenylalanine 87, which flank the surface and heme ends of the enzyme's substrate access channel, respectively. Replacement of arginine 47 with glutamic acid resulted in a catalytically inactive mutant. Replacement of arginine 47 with alanine yielded a protein with reduced substrate binding affinity and arachidonate sp3 carbon hydroxylation activity (72% of control wild type). On the other hand, arachidonic and eicosapentaenoic acid epoxidation was significantly enhanced (154 and 137%, of control wild type, respectively). As with wild type, the alanine 47 mutant generated (18R)-hydroxyeicosatetraenoic, (14S,15R)-epoxyeicosatrienoic, and (17S,18R)-epoxyeicosatetraenoic acids nearly enantiomerically pure. Replacement of phenylalanine 87 with valine converted cytochrome P450 BM-3 into a regio- and stereoselective arachidonic acid epoxygenase ((14S,15R)-epoxyeicosatrienoic acid, 99% of total products). Conversely, metabolism of eicosapentaenoic acid by the valine 87 mutant yielded a mixture of (14S,15R)- and (17S,18R)-epoxyeicosatetraenoic acids (26 and 69% of total, 94 and 96% optical purity, respectively). Finally, replacement of phenylalanine 87 with tyrosine yielded an inactive protein. We propose that: (a) fatty acid oxidation by P450 BM-3 is incompatible with the presence of residues with negatively charged side chains at the surface opening of the substrate access channel or a polar aromatic side chain in the vicinity of the heme iron; (b) the high turnover regio- and stereoselective metabolism of arachidonic and eicosapentaenoic acids involves charge-dependent anchoring of the fatty acids at the mouth of the access channel by arginine 47, as well as steric gating of the heme-bound oxidant by phenylalanine 87; and (c) substrate binding coordinates, as opposed to oxygen chemistries, are the determining factors responsible for reaction rates, product chemistry, and, thus, catalytic outcome.

Bisallylic hydroxylation and epoxidation of polyunsaturated fatty acids by cytochrome P450.

Polyunsaturated fatty acids can be oxygenated by cytochrome P450 to hydroxy and epoxy fatty acids. Two major classes of hydroxy fatty acids are formed by hydroxylation of the omega-side chain and by hydroxylation of bisallylic methylene carbons. Bisallylic cytochrome P450-hydroxylases transform linoleic acid to 11-hydroxylinoleic acid, arachidonic acid to 13-hydroxyeicosa-5Z,8Z,11Z,14Z-tetraenoic acid, 10-hydroxyeicosa-5Z,8Z,11Z,14Z-tetraenoic acid and 7-hydroxyeicosa-5Z,8Z,11Z,14Z-tetraenoic acid and eicosapentaenoic acid to 16-hydroxyeicosa-5Z,8Z,11Z,14Z,17Z-pent aenoic acid, 13-hydroxyeicosa-5Z,8Z,11Z,14Z,17Z-pent aenoic acid and 10-hydroxyeicosa-5Z,8Z,11Z,14Z,17Z-pent aenoic acid as major metabolites. The bisallylic hydroxy fatty acids are chemically unstable and decompose rapidly to cis-trans conjugated hydroxy fatty acids during acidic extractive isolation. Bisallylic hydroxylase activity appears to be augmented in microsomes induced by the synthetic glucocorticoid dexamethasone and by some other agents, but the P450 gene families of these hydroxylases have yet to be determined. The fatty acid epoxides, which are formed by cytochrome P450, are chemically stable, but are hydrolyzed to diols by soluble epoxide hydrolases. Epoxidation of polyunsaturated fatty acids is a prominent pathway of metabolism in the liver and the renal cortex and epoxy-genase activity appears to be under homeostatic control in the kidney. Many arachidonate epoxygenases have been identified belonging to the CYP2C gene subfamily. Epoxygenases have also been found in the central nervous system, endocrine organs, the heart and endothelial cells. Epoxides of arachidonic acid have been found to exert pharmacological effects on many cells.

Inhibition of brain P-450 arachidonic acid epoxygenase decreases baseline cerebral blood flow.

Arachidonic acid (AA) is metabolized by the cytochrome P-450 (P-450) epoxygenase pathway to epoxyeicosatrienoic acids (EETs) in the brain parenchymal tissue and perivascular astrocytes. EETs dilate cerebral microvessels and enhance K+ current in cerebrovascular smooth muscle cells. In the current study, the effect of a subdural administration of miconazole, an inhibitor of P-450 epoxygenase, on microvascular perfusion of rat cerebral cortex was evaluated using laser-Doppler flowmetry (LDF) Baseline cerebral blood flow (CBF) decreased by 29.7 +/- 7.3% (n = 5) after administration of 20 microM miconazole into the subdural space for 30 min. Responses of CBF to sodium nitroprusside and 5-hydroxytryptamine were unaltered by miconazole treatment. Administration of vehicle alone in time-control experiments had no effect on CBF. In other experiments, the effects of miconazole on the metabolism of [14C]AA by cultured rat astrocytes and on nitric oxide synthase activity in homogenates of rat brain were examined. Miconazole inhibited conversion of AA to EETs by cultured astrocytes but had no effect on the conversion of L-arginine to L-citrulline by homogenates of rat brain. These results implicate endogenous P-450 epoxides of AA in the regulation of basal blood flow in cerebral microcirculation.

Molecular Cloning and Expression of CYP2J2, a Human Cytochrome P450 Arachidonic Acid Epoxygenase Highly Expressed in Heart

A cDNA encoding a human cytochrome P450 arachidonic acid epoxygenase was isolated from a human liver cDNA library. Sequence analysis revealed that this 1,876-base pair cDNA contained an open reading frame and encoded a new 502-amino acid protein designated CYP2J2. Blot hybridization analysis of RNA prepared from human tissues revealed that CYP2J2 was highly expressed in the heart. Recombinant CYP2J2 protein was prepared using the baculovirus expression system and purified to near electrophoretic homogeneity. The enzyme metabolized arachidonic acid predominantly via olefin epoxidation to all four regioisomeric cis-epoxyeicosatrienoic acids (catalytic turnover 65 pmol of product formed/nmol of cytochrome P450/min at 30 degrees C). Epoxidation of arachidonic acid by CYP2J2 at the 14,15-olefin was highly enantioselective for (14R, 15S)-epoxyeicosatrienoic acid (76% optical purity). Immunoblotting of microsomal fractions prepared from human tissues using a polyclonal antibody raised against the recombinant hemoprotein confirmed primary expression of CYP2J2 protein in human heart. The in vivo significance of CYP2J2 was suggested by documenting the presence of epoxyeicosatrienoic acids in the human heart using gas chromatography/mass spectroscopy. Importantly, the chirality of CYP2J2 products matched that of the epoxyeicosatrienoic acid enantiomers present, in vivo, in human heart. We propose that CYP2J2 is one of the enzymes responsible for epoxidation of endogenous arachidonic acid pools in human heart and that epoxyeicosatrienoic acids may, therefore, play important functional roles in cardiac physiology.

Human Umbilical Vein Endothelial Cells Express P450 2C8 mRNA: Cloning of Endothelial P450 Epoxygenase

Human umbilical vein endothelial cells (EC) metabolize arachidonic acid (AA) through three major pathways—cyclooxygenase, lipoxygenase and cytochrome P450 (P450) isozymes. Previously, we have shown that pathophysiological concentrations of native low density lipoprotein (n-LDL) increase EC P450-dependent epoxyeicosatrienoic acid (EET) production. The present study was designed to identify putative P450 isozymes involved in EC epoxidation of AA. Reverse transcription—polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) were employed to detect P450 2 family cDNA from EC mRNA. Degenerate primers complimenting 2 homologous regions from 6 different P450 2 families were designed to capture a 440 bp cDNA fragment corresponding to the heme binding region of P450 2 isozymes. RT-PCR of EC total RNA with these primers amplified a 440 bp fragment. After gel purification, the fragment was cloned, sequenced and found to share a high degree of identity with human liver P450 2C8 and 2C9. New pri...

Molecular Cloning, Expression and Characterization of an Endogenous Human Cytochrome P450 Arachidonic Acid Epoxygenase Isoform

A cDNA containing an open reading frame coding for a human cytochrome P450 arachidonic acid epoxygenase was isolated from a male human kidney cDNA library. Sequence analysis showed that, with few exceptions, this cDNA was nearly identical to the published sequence for human liver Cyp 2C8 (S. T. Okino et al., 1987, J. Biol. Chem. 262, 16072-16079) and encoded a polypeptide of 490 amino acids. Nucleic acid hybridization indicated that: (a) Cyp 2C8 and 2C10 were expressed at comparable levels in the human liver and (b) compared to Cyp 2C10, the steady state concentrations of Cyp 2C8 transcripts in the human kidney were substantially lower. The kidney 2C8 cDNA was cloned into a pBlue BacIII vector, expressed using a baculovirus/ Sf9 insect cell system, and the recombinant Cyp 2C8 protein was purified by a combination of hydrophobic and hydroxylapatite chromatography. Purified recombinant Cyp 2C8 and 2C10 were reconstituted in the presence of NADPH and NADPH-cytochrome P450 reductase and shown to metabolize arachidonic via olefin epoxidation with both proteins generating, almost exclusively, epoxygenase-derived products (94 and 90% of total products, respectively). Catalytic turnover (1.05 and 0.75 nmol of product/nmol of hemoprotein/min at 30°C for Cyp 2C8 and 2C10, respectively) was inhibited by the addition of purified cytochrome b 5. Metabolism by recombinant 2C8 was both regio- and enantioselective for 11( R), 12( S)- and 14( R), 15( S)-epoxyeicosatrienoic acids (82% optical purity, each). Compared to Cyp 2C8, arachidonic acid epoxidation by Cyp 2C10 was less regio- and stereoselective and generated mixtures of 8( S), 9( R)-, 11( S), 12( R)-, and 14( R), 15( S)-epoxyeicosatrienoic acids (with optical purities of 66, 69, 63%, respectively). Importantly, recombinant Cyp 2C8 and 2C10 epoxidized the arachidonic acid 11, 12-olefin with opposite enantiofacial selectivities. Only for Cyp 2C8 did the chirality of the products match that of the enantiomers present, in vivo in human kidney cortex (A. Karara et al. 1990, FEBS Lett. 268, 227-230). Hence, we propose that Cyp 2C8 is one of the human cytochrome P450 isoforms responsible for the metabolism of endogenous arachidonic acid pools.

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

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.

Epoxidation of arachidonic acid as an active-site probe of cytochrome P-450 2B isoforms

In the present study we determined the regioselectivity of arachidonic acid epoxidation by several members of the cytochrome P-450 2B subfamily, including rat P-450 2B1, 2B1-WM (an allelic variant of 2B1 expressed in Wistar-Munich rats), 2B2, and rabbit 2B4 and 2B5. The major products formed with all isoforms were the four regioisomeric epoxides, but each isoform produced a distinct distribution of the four epoxides. P-450 2B1 produced predominantly 14,15-epoxyeicosatrienoic acid (EET), while P-450 2B1-WM produced the 11,12-EET as the major product. P-450 2B2, 2B4, and 2B5 catalyzed the formation of all four epoxides in nearly equal amounts. The single Gly-478 → Ala substitution in the variant P-450 2B1-WM was sufficient to cause a dramatic change in the ratio of epoxides when compared with P-450 2B1. The Gly-478 → Ala mutation also changed the regioselective epoxidation of γ-linolenic acid at the three double bonds. Four site-directed mutants of P-450 2B1 were also evaluated. The mutations included two single mutants where Ile-ll4 was changed to either Val or Ala and two double mutants where the Ala-478 mutation was coupled with either Val or Ala at position 114. When Ile-ll4 was mutated to Val, the degree of epoxidation of arachidonic acid at all four double bonds was nearly equal. However, substitution of Ile-ll4 with Ala, resulted in a significant reduction in the degree of epoxidation at the 14,15- and 11,12-double bonds, and the 8,9- and 5,6-EETs were the major products. When Ala was introduced at position 478 in conjunction with Val at position 114 the regioselective epoxidation of the mutant enzyme more closely resembled P-450 2BI-WM in that 11,12-EET was the major metabolite. The double mutation with Ala at both positions 114 and 478 produced a unique distribution of epoxide products with 5,6-EET as the major metabolite. The results of these studies indicate that residues 114 and 478 in the P-450 2B subfamily are important for the orientation of fatty acids in the active site.