Cytochrome P450 Arachidonic Acid Research Articles

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.

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.

Analysis of responses to bradykinin in the rat hindquarters vascular bed.

The mechanism by which bradykinin (BK) decreases vascular resistance was investigated in the hindquarters vascular bed of the rat. Under conditions of controlled blood flow, BK produced dose-related decreases in hindquarters perfusion pressure when injected into the perfusion circuit in doses of 0.1-1.0 pg. Responses to BK were reproducible with respect to time, and HOE-140 (D-Arg, [Hyp3,Thi5,D-Tic7,Oic8] -BK), a kinin B2 receptor antagonist, decreased hindquarters vasodilator responses to the peptide. HOE-140 had no significant effect on responses to vasodilator agents, which act by a variety of pharmacologically distinct mechanisms. Nitric oxide synthase inhibitors N(omega)-nitro-L-arginine methyl ester or N(omega)-nitro-L-arginine benzyl ester did not decrease responses to BK in doses that decreased hindquarters vasodilator responses to acetylcholine. The cyclooxygenase inhibitors meclofenamate and indomethacin had no effect on responses to BK in doses that attenuated vasodilator responses to the prostaglandin precursor, arachidonic acid. Clotrimazole or 5,8,11,14-eicosatetraynoic acid (ETYA), cytochrome P-450 arachidonic acid metabolism inhibitors, in doses that attenuated vasodilator responses to arachidonic acid, had no effect on responses to BK. Glybenclamide or U-37883A (4-morpholinecarboximidine-N-1-adamantyl-N'-cyclohexyl), K+-ATP channel antagonists, in doses that attenuated responses to lemakalim, had no significant effect on responses to BK. Finally, des-Arg9-BK, a reported kinin B1 receptor agonist, decreased hindquarters perfusion pressure when injected in doses of 3-30 microg, and responses to the B1 agonist were attenuated by HOE-140. The observation that des-Arg9-BK, in high doses, induces modest HOE-140-sensitive responses suggests that kinin B1 receptors are not normally expressed in the hindquarters vascular bed of the rat. The present results indicate that BK dilates the hindquarters vascular bed by a kinin B2 receptor mechanism that is independent of the release of nitric oxide, cyclooxygenase, or P-450 metabolites of arachidonic acid or the activation of K+-ATP channels.

Overexpression of the Heme Oxygenase Gene in Renal Cell Carcinoma

Heme oxygenase (HO) activity has been implicated in the regulation of renal function and cell growth in normal and disease states. Expression of HO genes has been shown to regulate important hemoprotein(s) such as cytochrome P450. In the present study, HO activity was measured in samples of human adenocarcinoma, juxtatumor, and normal renal tissues. The samples were histologically examined to verify the malignant and normal nature. HO activity was 4-fold higher in the adenocarcinoma than in either normal or juxtatumor tissues. We designed a reverse transcriptase-polymerase chain reaction (RT-PCR) method to assess the presence of HO-1 and HO-2 mRNA in biopsy samples of various human renal tissues. Total RNA from renal samples was reverse transcribed and amplified simultaneously by PCR using specific primers for HO-1 and HO-2. Results show that both HO-1 and HO-2 mRNAs were expressed in all renal tissues examined and that HO-1 appeared to be amplified more than HO-2. Northern blot analysis revealed that HO-1 mRNA was elevated by several-fold in adenocarcinoma compared with juxtatumor or normal tissues. In contrast, no differences in HO-2 mRNA levels were observed using either RT-PCR or Northern blot. Cytochrome P450 arachidonic acid epoxygenase and omega-hydroxylase activities were markedly reduced in the tumor tissues, whereas, in the juxtatumor tissue, cytochrome P450 omega-hydroxylase activity was significantly increased. Northern blot analysis using cytochrome P450 cDNA probe 4A2 cDNA for the omega-hydroxylase gene family revealed that mRNA levels for omega-hydroxylase transcripts were significantly decreased in the adenocarcinoma compared with juxtatumor. The decrease in cytochrome P450 4All mRNA levels correlated with a decrease in the arachidonic acid omega-hydroxylation metabolite, 20-HETE. The production of 20-HETE was significantly higher in juxtatumor in agreement with omega-hydroxylase mRNA. Higher levels of HO-1 may be a contributing factor for the undetectable levels of cytochrome P450 arachidonic acid metabolites, 20-HETE, in the adenocarcinoma. Our results suggest that increased generation of mitogenic activities by omega-hydroxylase and 20-HETE in the juxtatumor may be a contributing factor in the development and growth of neoplastic tissues, and the induction of HO in the tumor tissue may be an attempt to limit oxidative injury caused by the cytochrome P450 metabolites and other oxidative stress.

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.

Cytochrome P450 4A expression and arachidonic acid omega-hydroxylation in the kidney of the spontaneously hypertensive rat.

20-Hydroxyeicosatetraenoic acid (20-HETE) is a major arachidonate metabolite in the kidney of the spontaneously hypertensive rat (SHR). The increase in its synthesis has been associated with the elevation of blood pressure in the SHR. The omega-hydroxylation of arachidonic acid is an activity associated with members of the CYP4A gene family which, in the rat, comprises three major isoforms: 4A1, 4A2 and 4A3. 20-HETE displays potent and diverse biological activities which can affect pro- and anti-hypertensive mechanisms dependent upon where, when and by which isoform it has been produced. Therefore, it is important to identify and characterize its biosynthetic system. We compared CYP4A mRNA and protein expression to patterns of 20-HETE synthesis in the SHR kidney. The reverse transcription/polymerase chain reaction (RT/PCR) technique was used to amplify CYP4A mRNA in microdissected nephron segments. Southern blot hybridization of PCR products obtained from nephron segments with the CYP4A1 cDNA probe demonstrated strong signals in S2 and S3 segments of the proximal tubule. Immunoblots of nephron segments using a polyclonal anti-rat liver CYP4A1 antibody which cross-reacts with CYP4A2 and CYP4A3, and 14C-arachidonic acid metabolism, confirmed that arachidonic acid omega-hydroxylation, i.e., 14C-20HETE formation, and CYP4A proteins were also localized mainly in the S2 and S3 segments. Correlation also existed between the age-dependent increase in arachidonate omega-hydroxylation in the kidney and CYP4A mRNA levels as measured by Northern hybridization of total RNA using the CYP4A1 cDNA probe. Immunoblot analysis revealed that at 7 weeks, where 20-HETE production is at its maximum, all three proteins are expressed. CYP4A3 and 4A1 immunoreactive proteins appeared at 3 weeks, showed maximum levels at 5 and 7 weeks, respectively, and gradually decreased to lower levels at 13 and 20 weeks, whereas CYP4A2 levels were undetectable at 3, 5 and 7 weeks but appeared at 13-20 weeks. Additional immunoblots indicated that renal cortical CYP4A1 protein levels were higher in SHR compared to Sprague-Dawley and Wistar-Kyoto rats. The increased levels of CYP4A1-immunoreactive band at 7 weeks corresponded to the maximal activity of arachidonate omega-hydroxylation. Thus, CYP4A1 might play a significant role in contributing to the increased cortical/proximal production of 20-HETE seen in 7-week-old SHR. However, given the high homology among members of the CYP4A gene family and the lack of specific tools to discern among these isoforms, additional studies have to be carried out to substantiate our findings.

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.

Arachidonic acid metabolism modulates vasopressin-induced renal vasoconstriction.

Previous studies have shown that cytochrome P450-Arachidonic Acid (P450-AA) metabolites modify the vascular tone of several vessels and that vasopressin (AVP) stimulates P450-AA metabolism. Thus, in the present study, we decided to investigate if the vasoconstrictor effect of AVP is related to activation of P450-AA metabolism. We used the isolated perfused kidney of a rat, to test this hypothesis. Bolus injection of AVP (5.5, 11, 22 and 45 ng) increased the perfusion pressure of the isolated kidney of a rat by 66 +/- 2, 87 +/- 4, 110 +/- 2 and 130 +/- 3 mmHg respectively. This AVP-induced vasoconstriction was significantly reduced by inhibition of AA metabolism with ETYA, or 7 ethoxyresorsorufin (7ER). Furthermore, in vivo induction of P450 system with dexamethasone, enhanced the AVP-induced vasoconstrictor effect. Conversely, depletion of P450 system with SnCl2 diminished the vasoconstrictor response to AVP. Measurement of P450-14cAA metabolites in the renal effluent, showed the presence of 3 radioactive peaks. The % of the recovered radioactivity was 0.12 +/- 0.05%, 0.11 +/- 0.03% and 1.13 +/- 0.5% and corresponded to Dihydroxyeicosatrienoic acids (DHTs), Hydroxyeicosatetraenoic acids (HETEs) and Epoxyeicosatrienoic acids (EETs) respectively, when kidneys were stimulated by AVP (300 ng) the % recovered were 0.34 +/- 0.01%, 0.38 +/- 0.01% and 3.11 +/- 0.7% for the DHTs, HETEs and EETs respectively. Treatment with dexamethasone or SnCl2 potentiated or inhibited the AVP-dependent release of the P450-AA metabolites. In conclusion, we suggest that AVP stimulates AA metabolism via P450 pathway in the kidney and that these AA metabolites participate in the vasoconstrictor effect of AVP in the renal circulation.

Experimental and/or genetically controlled alterations of the renal microsomal cytochrome P450 epoxygenase induce hypertension in rats fed a high salt diet.

Excess dietary salt induces a cytochrome P450 arachidonic acid epoxygenase isoform in rat kidneys (Capdevila, J. H., S. Wei, J. Yang, A. Karara, H. R. Jacobson, J. R. Falck, F. P. Guengerich, and R. N. Dubois. 1992. J. Biol. Chem. 267:21720-21726). Treatment of rats on a high salt diet with the epoxygenase inhibitor, clotrimazole, produces significant increases in mean arterial blood pressure (122 +/- 2 and 145 +/- 4 mmHg for salt and salt- and clotrimazole-treated rats, respectively). The salt- and clotrimazole-dependent hypertension is accompanied by reductions in the urinary excretion of epoxygenase metabolites and by a selective inhibition of the renal microsomal epoxygenase reaction. The prohypertensive effects of clotrimazole are readily reversed when either the salt or clotrimazole treatment is discontinued. The indication that a salt-inducible renal epoxygenase protects against hypertension, are supported by studies with the Dahl rat model of genetic salt-sensitive hypertension. Dahl resistant animals responded to excess dietary salt by inducing the activity of their kidney microsomal epoxygenase(s) (0.102 +/- 0.01 and 0.240 +/- 0.04 nmol of products formed/min per mg of microsomal protein for control and salt-treated rats, respectively). Despite severe hypertension during excess dietary salt intake (200 +/- 20 mmHg), Dahl salt-sensitive rats demonstrated no increase in renal epoxygenase activity. These studies indicate that acquired or inherited abnormalities in renal epoxygenase activities and/or regulation can be related to salt-sensitive hypertension in rodents. Studies on the human renal epoxygenase and its relationship to salt hypertension may prove useful.

Cytochrome P450 arachidonic acid omega-hydroxylation in the proximal tubule of the rat kidney.

20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) is a major cytochrome P450-dependent arachidonate metabolite in the rat kidney. In the present study we characterized the formation of 20-HETE in the proximal tubule, the nephron segment with the highest concentration of cytochrome P450 activities, including P450 arachidonic acid metabolism. Freshly isolated tubules showed a basal formation of 20-HETE, implying that it is an endogenous constituent of the proximal tubule. Conversion of exogenous arachidonic acid to 20-HETE in proximal tubule homogenates was enzymatic and NADPH-dependent (i.e., 0 and 65.5 +/- 1.1 pmol/mg/min in the absence and presence of NADPH, respectively). That its formation was not affected by indomethacin but inhibited following preincubation with 17-ODYA (17-octadecynoic acid) and 7-ER (7-ethoxyresorufin) suggested that a P450 monooxygenase activity was involved in its synthesis. This was further strengthened by the demonstration that antibody raised against the rat cytochrome P450 4A1, a major fatty acid omega-hydroxylase isozyme, inhibited 20-HETE formation, suggesting the involvement of a P450 4A1 or P450 4A1-like activity in this reaction. Pretreatment of rats with clofibrate and dexamethasone, inducers of the P450 4A gene family, yielded a twofold increase in the proximal tubular synthesis of 20-HETE as well as an increase in P450 4A1 mRNA. These results, together with previous demonstrations that 20-HETE vasoconstricts isolated blood vessels, namely, renal microvessels, and affects tubular ion transport, suggest a role for 20-HETE in the regulation of renal vascular tone and transport functions and further stress the importance of understanding the regulation of 20-HETE synthesis in the kidney.

Tin-Mediated Heme Oxygenase Gene Activation and Cytochrome P450 Arachidonate Hydroxylase Inhibition in Spontaneously Hypertensive Rats

The effect of SnCl2 on the transcription of the heme oxygenase gene in spontaneously hypertensive rats was examined using cDNA for the rat heme oxygenase (HO-1). An increase in renal HO-1 mRNA levels was observed in response to SnCl2 treatment. Quantitative evaluation by scanning densitometry demonstrated a maximal increase in HO-1 mRNA 24-fold over control at 8 hours after SnCl2 administration. Nuclear runoff assay using isolated renal nuclei from SnCl2-treated rats revealed an active HO-1 gene transcription. Transcription of HO-1 in rat kidney was greatly increased within 3 hours of administration of SnCl2, as evidenced by the level of [alpha 32P]UTP incorporation into nuclear RNA. As a consequence of activation of the HO-1 gene transcription, renal enzyme activity increased eightfold at 16 hours after SnCl2, and reached maximal activity of 16-fold over control at 32 hours after injection. No significant change in cytochrome P450 fatty acid omega-hydroxylase (P450 4A) mRNA was observed after SnCl2 administration. Cytochrome P450-arachidonic acid omega/omega-1 hydroxylase(s) activity (formation of 20- and 19-HETE) was significantly reduced 24 hours after SnCl2 administration and remained lower than the control level 48 and 72 hours after injection. In addition, blood pressure was reduced from 151 +/- 2.5 mm Hg to 133 +/- 2.3 mm Hg after 48 hours of SnCl2 treatment. The reduction in blood pressure preceded natriuresis. It is concluded that SnCl2 induces activation of the HO-1 gene, which is followed by elevation in enzyme activity and a decrease in cytochrome P450-arachidonic acid omega-hydroxylase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning, expression, and enzymatic characterization of the rat kidney cytochrome P-450 arachidonic acid epoxygenase

A cDNA containing an open reading frame coding for the rat kidney cytochrome P-450 arachidonic acid epoxygenase was isolated from a male rat kidney cDNA library. Sequence analysis showed that with the exception of 11 nucleotides, this cDNA is identical with the published sequence for rat liver cytochrome 2C23 and encodes a polypeptide of 494 amino acids. Nucleic acid blot hybridization indicated that the levels of expression of the corresponding mRNA are high in rat kidney and liver and are undetectable in brain and heart. The cDNA coding region was cloned into a pCMV2 vector and expressed in COS-1 cells. The recombinant microsomal protein catalyzed the NADPH-dependent metabolism of arachidonic acid to a mixture of 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids as the only oxygenation products. The enantiofacial selectivity of the recombinant protein was nearly identical with that reported for the kidney microsomal enzyme and generated 8(R),9(S)-, 11(R),12(S)-, and 14(S),15(R) with optical purities of 95, 85, and 75%, respectively. On the basis of mRNA abundance and the close similarities between the regio- and stereochemical selectivity of the recombinant and kidney microsomal proteins, we concluded that cytochrome P-450 2C23 is the predominant enzyme isoform responsible for arachidonic acid epoxidation in the rat kidney.

General anesthetics inhibit cytochrome P450 monooxygenases and arachidonic acid metabolism.

Identification of a specific biomolecular target appropriately sensitive to a wide array of anesthetics has been elusive. At concentrations close to their respective ED50's for anesthesia in man or other species, 18 compounds, differing in potencies up to 66,000 fold, inhibited cytochrome P450 mediated metabolism of aminopyrine, a synthetic substrate, and arachidonic acid (AA), an endogenous substrate, in isolated liver microsomes. There was a highly significant correlation for both substrates between the absolute concentrations required for anesthesia (EC50) and for inhibition of P450 activity (Ki or IC50). The mean Ki/EC50 ratio was 0.97 for inhibition of aminopyrine demethylase. The mean IC50/EC50 ratios were 0.42 and 0.64 for inhibition of two AA-derived products and 2.8 for a third; a mean ratio of 1.4 for inhibition of overall AA metabolism suggests interaction of general anesthetics with a composite of P450 isozymes. The universal cytochrome P450 monooxygenases, in conjunction with other lipid oxygenases (cyclooxygenases and lipoxygenases) participate in the second messenger AA cascade. In nerve cells the sensitivity of these enzymes to hydrophobic neurodepressant drugs may underlie the state of general anesthesia: reversible disruption of intracellular and intercellular signalling without impairment of enzymes vital to cell respiration.

Cytochrome P-450 arachidonic acid epoxygenase. Regulatory control of the renal epoxygenase by dietary salt loading.

The rat kidney microsomal epoxygenase catalyzed the asymmetric epoxidation of arachidonic acid to generate as major products: 8(R),9(S)-, 11(R),12(S)- and 14(S),15(R)-epoxyeicosatrienoic acids with optical purities of 97, 88, and 70%, respectively. Inhibition studies utilizing a panel of polyclonal antibodies to several rat liver cytochrome P-450 isoforms, indicated that the renal epoxygenase(s) belongs to the cytochrome P-450 2C gene family. Dietary salt, administered either as a 2-2.5% (w/v) solution in the drinking water or as a modified solid diet containing 8% NaCl (w/w), resulted in marked and selective increases in the renal microsomal epoxygenase activity (416 and 260% of controls, for the liquid and solid forms of NaCl, respectively) with no significant changes in the microsomal omega/omega-1 oxygenase or in the hepatic arachidonic acid monooxygenase reaction. Immunoblotting studies demonstrated that dietary salt induced marked increases in the concentration of a cytochrome P-450 isoform(s) recognized by polyclonal antibodies raised against human liver cytochrome P-450 2C10 or rat liver cytochrome P-450 2C11. Comparisons of the stereochemical selectivity of the induced and non-induced microsomal epoxygenase(s) with that of purified rat liver cytochrome P-450 2C11 suggest that the salt-induced protein(s) is catalytically and structurally different from liver cytochrome P-450 2C11. The in vivo significance of dietary salt in regulating the activities of the kidney endogenous arachidonic acid epoxygenase was established by the demonstration of a salt-induced 10-20-fold increase in the urinary output of epoxygenase metabolites. These results, in conjunction with published evidence demonstrating the potent biological activities of its metabolites, suggest a role for the epoxygenase in the renal response to dietary salt.

Beta-naphthoflavone induction of a cytochrome P-450 arachidonic acid epoxygenase in chick embryo liver distinct from the aryl hydrocarbon hydroxylase and from phenobarbital-induced arachidonate epoxygenase.

Cytochrome P-450-mediated arachidonic acid metabolism in chick embryo liver microsomes was increased by both Ah receptor-dependent (2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and beta-naphthoflavone) and independent (phenobarbital) P-450 inducers. Arachidonic acid epoxides and monohydroxyeicosatetraenoic acids were increased 9-12-fold. omega-1-OH arachidonic acid was also significantly increased by TCDD and beta-naphthoflavone while omega-OH arachidonic acid, the main metabolite in uninduced livers, was decreased by all three agents. The P-450s catalyzing the enhanced arachidonate metabolism in beta-naphthoflavone- and phenobarbital-treated liver were investigated in reconstituted systems containing wholly or partially purified P-450s. beta-Naphthoflavone induced formation of a 55-kDa P-450 selective for arachidonate metabolism and for epoxygenation in particular. This P-450 was purified (beta NFAA). It was found to be distinct from a 54.5-kDa beta-naphthoflavone-induced P-450 catalyzing aryl hydrocarbon hydroxylase and 7-ethoxyresorufin deethylase (designated NF1). Mean turnover numbers for arachidonate epoxygenase, aryl hydrocarbon hydroxylase, and 7-ethoxyresorufin deethylase were 11.2, 0.56, and 0.04, respectively, for reconstituted beta NFAA and 0.33, 11.8, and 2.4 for NF1. beta NFAA and NF1 also differed in chromatography elution characteristics and N-terminal amino acid sequences. Both were low spin, with carbon monoxide binding peaks at 448 nm. The phenobarbital-induced arachidonate epoxygenation was catalyzed by P-450 fractions containing the main 48- and 49-kDa phenobarbital-induced P-450s; fractions in which the 49-kDa P-450 predominated were the most active. Turnover numbers for arachidonic acid epoxygenation were not correlated with those for aminopyrine demethylation or 7-ethoxycoumarin deethylation for P-450s from phenobarbital-treated livers or with aryl hydrocarbon hydroxylase, 7-ethoxyresorufin deethylase, or 7-ethoxycoumarin deethylase for P-450s from beta-naphthoflavone-treated livers. Also, different P-450s catalyzed the epoxygenation and the omega-hydroxylation of arachidonic acid in both beta-naphthoflavone- and phenobarbital-treated livers. The findings support a physiologic role for P-450-induced arachidonate metabolism and provide a basis for a possible link between TCDD's induction of P-450 and alterations of cellular homeostasis.

Ocular and renal cytochrome P450 arachidonic acid metabolism: Hormonal and pathological regulation

Ocular and renal cytochrome P450 arachidonic acid metabolism: Hormonal and pathological regulation

Purification and properties of a cytochrome P450 arachidonic acid epoxygenase from rabbit renal cortex.

A rabbit cytochrome P450 which catalyzes the epoxidation of arachidonic acid to two of the four possible regioisomeric epoxyeicosatrienoic acid metabolites was purified from renal cortex. A small amount of the unresolved omega/omega-1 hydroxylated eicosatetraenoic acid products were also produced. The enzyme had a specific content of 8.4 nmol of P450/mg of protein and exhibited a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after silver staining. Sequencing revealed a single NH2-terminal amino acid sequence with the first 20 residues identical to rabbit cytochrome P450 2C2. We suggest this enzyme be termed P450 2CAA (for arachidonic acid) until the complete sequence and substrate selectivity are established. Purified P450 2CAA was in the low spin state as evidenced by an absorption maximum at 415 nm; the reduced-carbonyl complex exhibited a maximum at 451 nm. The specific activity for metabolism of 7 microM arachidonic acid was 1.1 nmol of product formed/min/nmol of P450. About 75% of the metabolites were two of the four possible epoxyeicosatrienoic acids identified as the 11,12- and 14,15-epoxyeicosatrienoic acids by coelution with synthetic and commercial standards on reversed and normal-phase high pressure liquid chromatographic separations. The ratio of the 11,12- to 14,15-epoxyeicosatrienoic acids was 1.5:1. The purified enzyme exhibited no significant activity toward 7-ethoxyresorufin or progesterone, but demethylated aminopyrine and benzphetamine. Other fatty acids were also substrates for the enzyme. Oleic, linoleic, and lauric acids, all at about 10 microM, were metabolized at rates of 0.32, 0.72, and 0.73 nmol/min/nmol of P450, respectively. Monoclonal antibody that cross-reacts with P450 2C2 inhibited 63% of the microsomal epoxidation activity from renal cortex microsomes from phenobarbital-treated rabbits. The production of the epoxide metabolites of arachidonic acid suggests that P450 2CAA may have a significant role in arachidonic acid-mediated intra- and intercellular signalling pathways.

Age-related changes in renal cytochrome P-450 arachidonic acid metabolism in spontaneously hypertensive rats

We recently demonstrated that renal synthesis of cytochrome P-450-dependent arachidonic acid (AA) metabolites is increased in spontaneously hypertensive rats (SHR) during the rapid elevation of blood pressure. In this study, the chemical identity of these metabolites is described, and the structural analysis together with differential susceptibility to antibodies suggested that they are derived from at least two different cytochrome P-450 isozymes: 1) the epoxygenase that metabolizes AA mainly to 11,12-epoxyeicosatrienoic acid (EET), which is further hydrolyzed to 11,12-dihydroxyeicosatrienoic acid (DHT) and 2) omega/omega-1 hydroxylase(s) that generate the 20-hydroxyeicosatetraenoic acid (HETE) and 19-HETE, respectively. Their production and release from the isolated kidney was activated by arginine vasopressin and inhibited by cytochrome P-450 enzyme inhibitors. The formation of these metabolites in SHR or WKY cortical microsomes was age dependent. The production rates of EET, DHT, and 19-HETE increased from fetal to 9 wk of age by 3-, 6- and 4-fold, respectively, whereas that of 20-HETE increased by 27-fold. The omega/omega-1 hydroxylase activities were significantly higher in SHR, whereas epoxygenase activity (sum of EET and DHT production) demonstrated no differences between the two strains at any age group tested, although the amount of EET vs. DHT in a given age was significantly different. Since these metabolites have a wide and contrasting spectrum of biological and renal effects (vasodilation and vasoconstriction, inhibition and stimulation of Na(+)-K(+)-ATPase), their relative production rates at a given age may influence not only renal hemodynamics and salt and water balance but also pro- and antihypertensive mechanisms in SHR.

Modulation of erythropoiesis by novel human bone marrow cytochrome P450- dependent metabolites of arachidonic acid

In the hematopoietic system the adherent stromal cells produce cytokines necessary for proliferation and differentiation of hematopoietic cells. In the present study, we showed the ability of adherent stromal cells to generate novel metabolites of arachidonic acid via the NADPH-cytochrome P450-dependent monooxygenase system. These metabolites were recovered in the incubation media, suggesting their release from cells. The formation of arachidonic acid metabolites was inhibited by 7-ethoxyresorufin and SKF-525A, but not by indomethacin or BW-755C. By using two-step high-pressure liquid chromatography (HPLC), bone marrow-adherent stromal cells and incubation media showed the presence of metabolites in a peak eluted at 19 to 20 minutes. The isolated HPLC peak was used to measure its effect on colony-forming unit-erythroid (CFU-E) growth and compare it with that of synthetic cytochrome P450 arachidonate metabolites, 19- and 20- hydroxyeicosatetraenoic (HETE) acid. These bone marrow cytochrome P450 arachidonic acid metabolites at picomolar concentration potentiated erythropoietin (Epo)-induced CFU-E growth by fourfold to sixfold. Addition of 19- and 20-HETE to the bone marrow culture resulted in a potentiating effect on CFU-E number in a dose-dependent manner. 20-HETE was much more potent in stimulating CFU-E growth than 19-HETE at a similar concentration of 10(-11) mol/L. The potentiating effect of 20- HETE resulted in a shifting to the left of the dose-response curve to Epo. To substantiate the finding of an active NADPH-dependent cytochrome P450-metabolizing system, we further examined the ability of adherent cells to metabolize exogenous pharmacologic compounds such as benzo(a)pyrene, a substrate for the heme-cytochrome P450 system, aryl hydrocarbon hydroxylase. The adherent stromal cytochrome P450 metabolizes benzo(a)pyrene at comparable levels to blood vessel endothelial cells. These novel observations underscore the importance of adherent stromal cytochrome P450 to metabolize endogenous substrates, including arachidonic acid, to compounds that may interact in a paracrine manner with Epodependent hematopoietic cells.