The role of SHP/REV-ERBα/CYP4A axis in the pathogenesis of alcohol-associated liver disease.

Alcohol-associated liver disease (ALD) represents a spectrum of histopathological changes, including alcoholic steatosis, steatohepatitis, and cirrhosis. One of the early responses to excessive alcohol consumption is lipid accumulation in the hepatocytes. Lipid ω-hydroxylation of medium- and long-chain fatty acid metabolized by the cytochrome P450 4A (CYP4A) family is an alternative pathway for fatty acid metabolism. The molecular mechanisms of CYP4A in ALD pathogenesis have not been elucidated. In this study, WT and Shp-/- mice were fed with a modified ethanol-binge, National Institute on Alcohol Abuse and Alcoholism model (10 days of ethanol feeding plus single binge). Liver tissues were collected every 6 hours for 24 hours and analyzed using RNA-Seq. The effects of REV-ERBα agonist (SR9009, 100 mg/kg/d) or CYP4A antagonist (HET0016, 5 mg/kg/d) in ethanol-fed mice were also evaluated. We found that hepatic Cyp4a10 and Cyp4a14 expression were significantly upregulated in WT mice, but not in Shp-/- mice, fed with ethanol. ChIP quantitative PCR and promoter assay revealed that REV-ERBα is the transcriptional repressor of Cyp4a10 and Cyp4a14. Rev-Erbα-/- hepatocytes had a marked induction of both Cyp4a genes and lipid accumulation. REV-ERBα agonist SR9009 or CYP4A antagonist HET0016 attenuated Cyp4a induction by ethanol and prevented alcohol-induced steatosis. Here, we have identified a role for the SHP/REV-ERBα/CYP4A axis in the pathogenesis of ALD. Our data also suggest REV-ERBα or CYP4A as the potential therapeutic targets for ALD.

Inhibition of COX-2, mPGES-1 and CYP4A by isoliquiritigenin blocks the angiogenic Akt signaling in glioma through ceRNA effect of miR-194-5p and lncRNA NEAT1.

BACKGROUND: Arachidonic acid (AA) metabolic enzymes including cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES-1) and cytochrome P450 (CYP) 4A11 play important roles in glioma angiogenesis. Thus, there is an urgent need to identify the underlying mechanisms and develop strategies to overcome them. METHODS: A homology model of human CYP4A11 was constructed using SYBYL-X 2.0. Structure-based virtual screening against COX-2, mPGES-1 and CYP4A11was performed using the Surflex-Dock of the SYBYL suite. The candidates were further evaluated their antiangiogenic activities in a zebrafish embryo and rabbit corneal angiogenesis model. Laser doppler analysis was used to measure tumor perfusion. The expression of CD31 and α-SMA was measured by immunofluorescence. Western blot was used to measure the expression of HIF-1, Akt and p-Akt. The gene expression of FGF-2, G-CSF, PDGF, TGF-ß, Tie-2, VEGF, lncRNA NEAT1 and miR-194-5p were determined using qPCR. The production of FGF-2, TGF-ß and VEGF were analyzed using ELISA. Bioinformatic analysis and luciferase reporter assays confirmed the interaction between lncRNA NEAT1 and miR-194-5p. RESULTS: The nearly 36,043 compounds from the Traditional Chinese Medicine (TCM) database were screened against COX-2, mPGES-1 and CYP4A11 3D models, and the 17 top flavonoids were identified. In zebrafish screening, isoliquiritigenin (ISL) exhibited the most potent antiangiogenic activities with the EC50 values of 5.9 µM. Conversely, the antiangiogenic effects of ISL in the zebrafish and rabbit corneal models were partly reversed by 20-hydroxyeicosatetraenoic acid (20-HETE) or prostaglandin E2 (PGE2). ISL normalized glioma vasculature and improved the efficacy of temozolomide therapy in the rat C6 glioma model. Inhibition of COX-2, mPGES-1 and CYP4A by ISL decreased FGF-2, TGF-ß and VEGF production in the C6 and U87 glioma cells with p-Akt downregulation, which was reversed by Akt overexpression. Furthermore, ISL downregulated lncRNA NEAT1 but upregulated miR-194-5p in the U87 glioma cell. Importantly, lncRNA NEAT1 overexpression reversed ISL-mediated increase in miR-194-5p expression, and thereby attenuated FGF-2, TGF-ß and VEGF production. CONCLUSIONS: Reprogramming COX-2, mPGES-1 and CYP4A mediated-AA metabolism in glioma by flavonoid ISL inhibits the angiogenic Akt- FGF-2/TGF-ß/VEGF signaling through ceRNA effect of miR-194-5p and lncRNA NEAT1, and may serve as a novel therapeutic strategy for human glioma.

A Specific Probe Substrate for Evaluation of CYP4A11 Activity in Human Tissue Microsomes and a Highly Selective CYP4A11 Inhibitor: Luciferin-4A and Epalrestat.

The specificity of cytochrome P450 4A11 (CYP4A11) against luciferin-4A O-demethylation in human liver microsomes (HLMs) and human renal microsomes (HRMs) and selectivity of CYP4A11 inhibition by epalrestat were investigated. Kinetic analysis of luciferin-4A O-demethylation yielded Vmax and S50 values of 39.7 pmol/min per milligram protein and 43.2 µM for HLMs (Hill coefficient 1.24) and 39.4 pmol/min per milligram protein and 33.8 µM for HRMs (Hill coefficient 1.34), respectively. Among the selective CYP inhibitors tested, HET0016 (CYP4 inhibitor) exclusively inhibited luciferin-4A O-demethylation by HLMs and HRMs. Furthermore, anti-CYP4A11 antibody nearly abolished the activity of both tissue microsomes. Luciferin-4A O-demethylase activity of HLMs was significantly correlated with lauric acid ω-hydroxylase activity, a marker of CYP4A11 activity (r = 0.904, P < 0.0001). Next, effects of epalrestat on CYP-mediated drug oxidations were examined. Epalrestat showed the most potent inhibition against CYP4A11 (IC50 = 1.82 µM) among the 17 recombinant enzymes tested. The inhibitory effect of epalrestat on CYP4A11 was at least 10-fold stronger than those on CYP4F2, CYP4F3B, and CYP4F12. For known CYP4 inhibitors, in contrast, HET0016 inhibited the activities of CYP4A11 and CYP4F2 (IC50 = 0.0137-0.0182 µM); 17-octadecynoic acid reduced activities of CYP4A11, CYP4F2, CYP4F3B, and CYP4F12 to a similar extent (IC50 = 5.70-17.7 µM). Epalrestat selectively and effectively inhibited the CYP4A11 activity of HLMs (IC50 = 0.913 µM) and HRMs (IC50 = 0.659 µM). These results indicated that luciferin-4A O-demethylase activity is a good CYP4A11 marker of HLMs and HRMs, and that epalrestat is a more selective CYP4A11 inhibitor compared with known CYP4 inhibitors.

Cytochrome P450 4A11 inhibition assays based on characterization of lauric acid metabolites.

This study was designed to characterize lauric acid metabolism to facilitate the establishment of cytochrome P450 4A11 (CYP4A11) inhibition assay. Three metabolites (2-, 11-, and 12-hydroxylauric acids) were identified in pooled human liver microsomes based on comparisons with authentic standards. Reaction phenotyping using 14 recombinant CYPs showed that ω-hydroxylation was mediated dominantly by CYP4A11 and marginally by CYP4F3B. CYP2B6 played an exclusive role in the formation of 2-hydroxylauric acid. The production of 11-hydroxylauric acid was mediated by CYP2E1, CYP2C9, CYP2B6, CYP1A2, CYP3A4, and CYP4A11. The IC50 values of HET0016, a well-known pan-CYP4 inhibitor, against the formation of 12-, 11-, and 2-hydroxylauric acid were 1.0, 1.0, and 0.009 µM, respectively. Among the 50 natural compounds examined, plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) inhibited the formation of 12-, 11-, and 2-hydroxylauric acid with IC50 values of 1.7, 2.3, and 2.7 µM, respectively. In the selectivity study, HET0016 inhibited CYP2B6 with an IC50 of 9.2 nM, as well as CYP1A2, CYP2C19, and CYP2E1 with IC50 values of 1-2 µM. Plumbagin inhibited all CYP enzymes tested with IC50 values of 1.7-3.0 µM. These methods can be used as tools to develop CYP4A11 inhibitors; simultaneous determination of the hydroxylauric acid metabolites provides further information on selectivity.

Inhibition of CYP4A by a novel flavonoid FLA-16 prolongs survival and normalizes tumor vasculature in glioma.

Glioblastomas rapidly become refractory to anti-VEGF therapies. We previously showed that cytochrome P450 (CYP) 4A-derived 20-hydroxyeicosatetraenoic acid (20-HETE) promotes angiogenesis. Here, we tested whether a novel flavonoid (FLA-16) prolongs survival and normalizes tumor vasculature in glioma through CYP4A inhibition. FLA-16 improved survival, reduced tumor burden, and normalized vasculature, accompanied with the decreased secretion of 20-HETE, VEGF and TGF-ß in tumor-associated macrophages (TAMs) and endothelial progenitor cells (EPCs) in C6 and U87 gliomas. FLA-16 attenuated vascular abnormalization induced by co-implantation of GL261 glioma cells with CYP4A10high macrophages or EPCs. Mechanistically, the conditional medium from TAMs and EPCs treated with FLA-16 enhanced the migration of pericyte cells, and decreased the proliferation and migration of endothelial cells, which were reversed by CYP4A overexpression or exogenous addition of 20-HETE, VEGF and TGF-ß. Furthermore, FLA-16 prevented crosstalk between TAMs and EPCs during angiogenesis. These results suggest that CYP4A inhibition by FLA-16 prolongs survival and normalizes vasculature in glioma through decreasing production of TAMs and EPCs-derived VEGF and TGF-ß. This may represent a potential therapeutic strategy to overcome resistance to anti-VEGF treatment by effects on vessels and immune cells.

Vascular endothelial over-expression of soluble epoxide hydrolase (Tie2-sEH) enhances adenosine A1 receptor-dependent contraction in mouse mesenteric arteries: role of ATP-sensitive K+ channels.

Soluble epoxide hydrolase (sEH) converts epoxyeicosatrienoic acids that are endothelium-derived hyperpolarizing factors into less active dihydroxyeicosatrienoic acids. Previously, we reported a decrease in adenosine A1 receptor (A1AR) protein levels in sEH knockout (sEH-/-) and an increase in sEH and A1AR protein levels in A2AAR-/- mice. Additionally, KATP channels are involved in adenosine receptor (AR)-dependent vascular relaxation. Thus, we hypothesize that a potential relationship may exist among sEH over-expression, A1AR upregulation, inactivation of KATP channels, and increased in vascular tone. We performed DMT myograph muscle tension measurements and western blot analysis in isolated mouse mesenteric arteries (MAs) from wild-type (WT) and endothelial over-expression of sEH (Tie2-sEH Tr) mice. Our data revealed that NECA (a non-selective adenosine receptors agonist)-induced relaxation was significantly reduced in Tie2-sEH Tr mice, and CCPA (A1AR agonist)-induced contraction was increased in Tie2-sEH Tr mice. A1AR-dependent contraction in Tie2-sEH Tr mice was significantly attenuated by pharmacological inhibition of CYP4A (HET0016, 10 µM), PKCα (GO6976, 1 µM), and ERK1/2 (PD58059, 1 µM). Our western blot analysis revealed significantly higher basal protein expression of CYP4A, A1AR, and reduced p-ERK in MAs of Tie2-sEH Tr mice. Notably, pinacidil (KATP channel opener)-induced relaxation was also significantly reduced in MAs of Tie2-sEH Tr mice. Furthermore, KATP channel-dependent relaxation in MAs was enhanced by inhibition of PKCα and ERK1/2 in WT but not Tie2-sEH Tr mice. In conclusion, our data suggest that over-expression of sEH enhances A1AR-dependent contraction and reduces KATP channel-dependent relaxation in MAs. These results suggest a possible interaction between sEH, A1AR, and KATP channels in regulating vascular tone.

Downregulation of COX-2 and CYP 4A signaling by isoliquiritigenin inhibits human breast cancer metastasis through preventing anoikis resistance, migration and invasion.

Flavonoids exert extensive in vitro anti-invasive and in vivo anti-metastatic activities. Anoikis resistance occurs at multiple key stages of the metastatic cascade. Here, we demonstrate that isoliquiritigenin (ISL), a flavonoid from Glycyrrhiza glabra, inhibits human breast cancer metastasis by preventing anoikis resistance, migration and invasion through downregulating cyclooxygenase (COX)-2 and cytochrome P450 (CYP) 4A signaling. ISL induced anoikis in MDA-MB-231 and BT-549 human breast cancer cells as evidenced by flow cytometry and the detection of caspase cleavage. Moreover, ISL inhibited the mRNA expression of phospholipase A2, COX-2 and CYP 4A and decreased the secretion of prostaglandin E2 (PGE2) and 20-hydroxyeicosatetraenoic acid (20-HETE) in detached MDA-MB-231 cells. In addition, it decreased the levels of phospho-PI3K (Tyr(458)), phospho-PDK (Ser(241)) and phospho-Akt (Thr(308)). Conversely, the exogenous addition of PGE2, WIT003 (a 20-HETE analog) and an EP4 agonist (CAY10580) or overexpression of constitutively active Akt reversed ISL-induced anoikis. ISL exerted the in vitro anti-migratory and anti-invasive activities, whereas the addition of PGE2, WIT003 and CAY10580 or overexpression of constitutively active Akt reversed the in vitro anti-migratory and anti-invasive activities of ISL in MDA-MB-231 cells. Notably, ISL inhibited the in vivo lung metastasis of MDA-MB-231 cells, together with decreased intratumoral levels of PGE2, 20-HETE and phospho-Akt (Thr(308)). In conclusion, ISL inhibits breast cancer metastasis by preventing anoikis resistance, migration and invasion via downregulating COX-2 and CYP 4A signaling. It suggests that ISL could be a promising multi-target agent for preventing breast cancer metastasis, and anoikis could represent a novel mechanism through which flavonoids may exert the anti-metastatic activities.

Combined therapy with COX-2 inhibitor and 20-HETE inhibitor reduces colon tumor growth and the adverse effects of ischemic stroke associated with COX-2 inhibition.

20-Hydroxyeicosatetraenoic acid (20-HETE), Cyp4a-derived eicosanoid, is a lipid mediator that promotes tumor growth, as well as causing detrimental effects in cerebral circulation. We determined whether concurrent inhibition of cyclooxygenase-2 (COX-2) and 20-HETE affects colon tumor growth and ischemic stroke outcomes. The expression of Cyp4a and COXs and production of 20-HETE and PGE2 were determined in murine colon carcinoma (MC38) cells. We then examined the effects of combined treatment with rofecoxib, a potent COX-2 inhibitor, and HET0016, a potent Cyp4a inhibitor, on the growth and proliferation of MC38 cells. Subsequently, we tested the effects of HET0016 plus rofecoxib in MC38 tumor and ischemic stroke models. Cyp4a and COXs are highly expressed in MC38 cells. Respectively, HET0016 and rofecoxib inhibited 20-HETE and PGE2 formation in MC38 cells. Moreover, rofecoxib combined with HET0016 had greater inhibitory effects on the growth and proliferation of MC38 cells than did rofecoxib alone. Importantly, rofecoxib combined with HET0016 provided greater inhibition on tumor growth than did rofecoxib alone in MC38 tumor-bearing mice. Prolonged treatment with rofecoxib selectively induced circulating 20-HETE levels and caused cerebrovascular damage after ischemic stroke, whereas therapy with rofecoxib and HET0016 attenuated 20-HETE levels and reduced rofecoxib-induced cerebrovascular damage and stroke outcomes during anti-tumor therapy. Thus these results demonstrate that combination therapy with rofecoxib and HET0016 provides a new treatment of colon tumor, which can not only enhance the anti-tumor efficacy of rofecoxib, but also reduce rofecoxib-induced cerebrovascular damage and stroke outcomes.

Inhibition of CYP4A reduces hepatic endoplasmic reticulum stress and features of diabetes in mice.

BACKGROUND & AIMS: Endoplasmic reticulum (ER) stress is implicated in the development of type 2 diabetes mellitus. ER stress activates the unfolded protein response pathway, which contributes to apoptosis and insulin resistance. We investigated the roles of cytochrome P450 4A (CYP4A) in the regulation of hepatic ER stress, insulin resistance, and the development of diabetes in mice. METHODS: We used mass spectrometry to compare levels of CYP450 proteins in livers from C57BL/6J and C57BL/KsJ-db/db (db/db) mice; findings were confirmed by immunoblot and real-time PCR analyses. To create a model of diet-induced diabetes, C57BL/6J mice were placed on high-fat diets. Mice were given intraperitoneal injections of an inhibitor (HET0016) or an inducer (clofibrate) of CYP4A, or tail injections of small hairpin RNAs against CYP4A messenger RNA; liver tissues were collected and analyzed for ER stress, insulin resistance, and apoptosis. The effect of HET0016 and CYP4A knockdown also were analyzed in HepG2 cells. RESULTS: Levels of the CYP4A isoforms were highly up-regulated in livers of db/db mice compared with C57BL/6J mice. Inhibition of CYP4A in db/db and mice on high-fat diets reduced features of diabetes such as insulin hypersecretion, hepatic steatosis, and increased glucose tolerance. CYP4A inhibition reduced levels of ER stress, insulin resistance, and apoptosis in the livers of diabetic mice; it also restored hepatic functions. Inversely, induction of CYP4A accelerated ER stress, insulin resistance, and apoptosis in livers of db/db mice. CONCLUSIONS: CYP4A proteins are up-regulated in livers of mice with genetically induced and diet-induced diabetes. Inhibition of CYP4A in mice reduces hepatic ER stress, apoptosis, insulin resistance, and steatosis. Strategies to reduce levels or activity of CYP4A proteins in liver might be developed for treatment of patients with type 2 diabetes.

Structural and mechanistic insight into alkane hydroxylation by Pseudomonas putida AlkB.

Pseudomonas putida GPo1 alkane hydroxylase (AlkB) is an integral membrane protein that catalyses the hydroxylation of medium-chain alkanes (C3-C12). 1-Octyne irreversibly inhibits this non-haem di-iron mono-oxygenase under turnover conditions, suggesting that it acts as a mechanism-based inactivator. Upon binding to the active site, 1-octyne is postulated to be oxidized to an oxirene that rapidly rearranges to a reactive ketene which covalently acylates nearby residues, resulting in enzyme inactivation. In analysis of inactivated AlkB by LC-MS/MS, several residues exhibited a mass increase of 126.1 Da, corresponding to the octanoyl moiety derived from oxidative activation of 1-octyne. Mutagenesis studies of conserved acylated residues showed that Lys18 plays a critical role in enzyme function, as a single-point mutation of Lys18 to alanine (K18A) completely abolished enzymatic activity. Finally, we present a computational 3D model structure of the transmembrane domain of AlkB, which revealed the overall packing arrangement of the transmembrane helices within the lipid bilayer and the location of the active site mapped by the 1-octyne modifications.

Transient postnatal fluoxetine leads to decreased brain arachidonic acid metabolism and cytochrome P450 4A in adult mice.

Fetal and perinatal exposure to selective serotonin (5-HT) reuptake inhibitors (SSRIs) has been reported to alter childhood behavior, while transient early exposure in rodents is reported to alter their behavior and decrease brain extracellular 5-HT in adulthood. Since 5-HT2A/2C receptor-mediated neurotransmission can involve G-protein coupled activation of cytosolic phospholipase A2 (cPLA2), releasing arachidonic acid (ARA) from synaptic membrane phospholipid, we hypothesized that transient postnatal exposure to fluoxetine would alter brain ARA metabolism in adult mice. Brain ARA incorporation coefficients k* and rates Jin were quantitatively imaged following intravenous [1-(14)C]ARA infusion of unanesthetized adult mice that had been injected daily with fluoxetine (10mg/kg i.p.) or saline during postnatal days P4-P21. Expression of brain ARA metabolic enzymes and other relevant markers also was measured. On neuroimaging, k* and Jin was decreased widely in early fluoxetine- compared to saline-treated adult mice. Of the enzymes measured, cPLA2 activity was unchanged, while Ca(2+)-independent iPLA2 activity was increased. There was a significant 74% reduced protein level of cytochrome P450 (CYP) 4A, which can convert ARA to 20-HETE. Reduced brain ARA metabolism in adult mice transiently exposed to postnatal fluoxetine, and a 74% reduction in CYP4A protein, suggest long-term effects independent of drug presence in brain ARA metabolism, and in CYP4A metabolites. These changes might contribute to reported altered behavior following early SSRI in rodents.

20-Hydroxyeicosatetraenoic acid inhibition attenuates balloon injury-induced neointima formation and vascular remodeling in rat carotid arteries.

20-Hydroxyeicosatetraenoic acid (20-HETE) contributes to the migration and proliferation of vascular smooth muscle cells (VSMC) in vitro, but there are few studies that address its effects on vascular remodeling in vivo. The present study determined whether inhibition of 20-HETE production attenuates intimal hyperplasia (IH) and vascular remodeling after balloon injury (BI). Sprague Dawley rats underwent BI of the common carotid artery and were treated with vehicle, 1-aminobenzotriazole (ABT, 50 mg/kg i.p. once daily), or HET0016 (N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine) (2 mg/kg s.c. twice daily) for 14 days. Fourteen days after BI and treatment, the animals underwent carotid angiography, and the arteries were harvested for morphometric, enzymatic and immunohistochemical analysis. There was a 96% reduction of angiographic stenosis in the rats treated with 1-ABT. There was a 61 and 66% reduction of the intima/media area ratios in the 1-ABT and HET0016 treated rats compared with the vehicle-treated group. 20-HETE levels were elevated in BI carotid arteries, and the levels were markedly suppressed in the groups treated with 1-ABT and HET0016 (P < 0.001). Immunostaining revealed that the expression of CYP4A enzyme was markedly increased in the neointima of BI arteries, and it colocalized with the expression of smooth muscle-specific actin, indicating increased proliferation of VSMC. An increase in the expression of CYP4A and the production of 20-HETE contributes to neointimal growth in BI rat carotid arteries. Systemic administration 1-ABT or HET0016 prevents the increase in 20-HETE levels and attenuates VSMC migration and proliferation, resulting in a marked reduction in IH and vascular remodeling after endothelial injury.

CYP4 enzymes as potential drug targets: focus on enzyme multiplicity, inducers and inhibitors, and therapeutic modulation of 20-hydroxyeicosatetraenoic acid (20-HETE) synthase and fatty acid ω-hydroxylase activities.

The Cytochrome P450 4 (CYP4) family of enzymes in humans is comprised of thirteen isozymes that typically catalyze the ω-oxidation of endogenous fatty acids and eicosanoids. Several CYP4 enzymes can biosynthesize 20- hydroxyeicosatetraenoic acid, or 20-HETE, an important signaling eicosanoid involved in regulation of vascular tone and kidney reabsorption. Additionally, accumulation of certain fatty acids is a hallmark of the rare genetic disorders, Refsum disease and X-ALD. Therefore, modulation of CYP4 enzyme activity, either by inhibition or induction, is a potential strategy for drug discovery. Here we review the substrate specificities, sites of expression, genetic regulation, and inhibition by exogenous chemicals of the human CYP4 enzymes, and discuss the targeting of CYP4 enzymes in the development of new treatments for hypertension, stroke, certain cancers and the fatty acid-linked orphan diseases.

Adenosine A1 receptors link to smooth muscle contraction via CYP4a, protein kinase C-α, and ERK1/2.

Adenosine A1 receptor (A1AR) activation contracts smooth muscle, although signaling mechanisms are not thoroughly understood. Activation of A1AR leads to metabolism of arachidonic acid, including the production of 20-hydroxyeicosatetraenoic acid (20-HETE) by cytochrome P4504a (CYP4a). The 20-HETE can activate protein kinase C-α (PKC-α), which crosstalks with extracellular signal-regulated kinase (ERK1/2) pathway. Both these pathways can regulate smooth muscle contraction, we tested the hypothesis that A1AR contracts smooth muscle through a pathway involving CYP4a, PKC-α, and ERK1/2. Experiments included isometric tension recordings of aortic contraction and Western blots of signaling molecules in wild type (WT) and A1AR knockout (A1KO) mice. Contraction to the A1-selective agonist 2-chloro-N cyclopentyladenosine (CCPA) was absent in A1KO mice aortae, indicating the contractile role of A1AR. Inhibition of CYP4a (HET0016) abolished 2-chloro-N cyclopentyladenosine-induced contraction in WT aortae, indicating a critical role for 20-HETE. Both WT and A1KO mice aortae contracted in response to exogenous 20-HETE. Inhibition of PKC-α (Gö6976) or ERK1/2 (PD98059) attenuated 20-HETE-induced contraction equally, suggesting that ERK1/2 is downstream of PKC-α. Contractions to exogenous 20-HETE were significantly less in A1KO mice; reduced protein levels of PKC-α, p-ERK1/2, and total ERK1/2 supported this observation. Our data indicate that A1AR mediates smooth muscle contraction via CYP4a and a PKC-α-ERK1/2 pathway.

Increase of 20-HETE synthase after brain ischemia in rats revealed by PET study with 11C-labeled 20-HETE synthase-specific inhibitor.

20-Hydroxyeicosatetraenoic acid (20-HETE), an arachidonic acid metabolite known to be produced after cerebral ischemia, has been implicated in ischemic and reperfusion injury by mediating vasoconstriction. To develop a positron emission tomography (PET) probe for 20-HETE synthase imaging, which might be useful for monitoring vasoconstrictive processes in patients with brain ischemia, we synthesized a (11)C-labeled specific 20-HETE synthase inhibitor, N'(4-dimethylaminohexyloxy)phenyl imidazole ([(11)C]TROA). Autoradiographic study showed that [(11)C]TROA has high-specific binding in the kidney and liver consistent with the previously reported distribution of 20-HETE synthase. Using transient middle cerebral artery occlusion in rats, PET study showed significant increases in the binding of [(11)C]TROA in the ipsilateral hemisphere of rat brains after 7 and 10 days, which was blocked by co-injection of excess amounts of TROA (10 mg/kg). The increased [(11)C]TROA binding on the ipsilateral side returned to basal levels within 14 days. In addition, quantitative real-time PCR revealed that increased expression of 20-HETE synthase was only shown on the ipsilateral side on day 7. These results indicate that [(11)C]TROA might be a useful PET probe for imaging of 20-HETE synthase in patients with cerebral ischemia.

Predicting oxidation sites with order of occurrence among multiple sites for CYP4A-mediated reactions.

To predict CYP4A-mediated reactions, we developed a two-dimensional template scoring system based on published data. The system predicts the order of occurrence among multiple oxidation sites, as well as the regioselectivity. The template has a linearly arranged honeycomb shape and an adjacent area. Molecules are overlaid on the template with the locations of the atoms restricted to the corners of hexagonal blocks. The overlaid conformers are then checked to determine whether they reside within the template area, and their position occupancy and position function scores are calculated. The position occupancy score is determined based on occupation of the respective positions on the template. The functional and steric properties are reflected in the position function score. The sum of these scores is compared among possible conformers, and the conformer with the highest total score is predicted to be preferentially metabolized. In the present study, prediction of sites of CYP4A-mediated oxidation and classification into substrates and non-substrates were performed for collected compounds, and agreement between predicted and experimental data exceeded 95% for substrates and non-substrates. The template scoring system can be easily linked to databases of two-dimensional chemical structures, and thus this system may be useful for drug development and studies of drug metabolism.

Salt modulates vascular response through adenosine A(2A) receptor in eNOS-null mice: role of CYP450 epoxygenase and soluble epoxide hydrolase.

High salt (HS) intake can change the arterial tone in mice, and the nitric oxide (NO) acts as a mediator to some of the receptors mediated vascular response. The main aim of this study was to explore the mechanism behind adenosine-induced vascular response in HS-fed eNOS(+/+) and eNOS(-/-) mice The modulation of vascular response by HS was examined using aortas from mice (eNOS(+/+) and eNOS(-/-)) fed 4% (HS) or 0.45% (NS) NaCl-diet through acetylcholine (ACh), NECA (adenosine-analog), CGS 21680 (A(2A) AR-agonist), MS-PPOH (CYP epoxygenase-blocker; 10(-5) M), AUDA (sEH-blocker; 10(-5) M), and DDMS (CYP4A-blocker; 10(-5) M). ACh-response was greater in HS-eNOS(+/+) (+59.3 ± 6.3%) versus NS-eNOS(+/+) (+33.3 ± 8.0%; P < 0.05). However, there was no response in both HS-eNOS(-/-) and NS-eNOS(-/-). NECA-response was greater in HS-eNOS(-/-) (+37.4 ± 3.2%) versus NS-eNOS(-/-) (+7.4.0 ± 3.8%; P < 0.05). CGS 21680-response was also greater in HS-eNOS(-/-) (+45.4 ± 5.2%) versus NS-eNOS(-/-)(+5.1 ± 5.0%; P < 0.05). In HS-eNOS(-/-), the CGS 21680-response was reduced by MS-PPOH (+7.3 ± 3.2%; P < 0.05). In NS-eNOS(-/-), the CGS 21680-response was increased by AUDA (+38.2 ± 3.3%; P < 0.05) and DDMS (+30.1 ± 4.1%; P < 0.05). Compared to NS, HS increased CYP2J2 in eNOS(+/+) (35%; P < 0.05) and eNOS(-/-) (61%; P < 0.05), but decreased sEH in eNOS(+/+) (74%; P < 0.05) and eNOS(-/-) (40%; P < 0.05). Similarly, CYP4A decreased in HS-eNOS(+/+) (35%; P < 0.05) and HS-eNOS(-/-) (34%; P < 0.05). These data suggest that NS causes reduced-vasodilation in both eNOS(+/+) and eNOS(-/-) via sEH and CYP4A. However, HS triggers possible A(2A)AR-induced relaxation through CYP epoxygenase in both eNOS(+/+) and eNOS(-/-).

Modulation by cytochrome P450-4A ω-hydroxylase enzymes of adrenergic vasoconstriction and response to reduced PO2 in mesenteric resistance arteries of Dahl salt-sensitive rats.

OBJECTIVE: This study evaluated the contribution of the 20-HETE/cytochrome P450-4A ω-hydroxylase (CYP4A) system to the early development of salt-induced vascular changes in Dahl salt-sensitive (SS) rats. METHODS: CYP4A expression and 20-HETE production were evaluated and responses to norepinephrine, endothelin, and reduced PO2 were determined by video microscopy in isolated mesenteric resistance arteries from SS rats fed high salt (HS; 4% NaCl) diet for three days vs. low salt (LS; 0.4% NaCl) controls. RESULTS: CYP4A enzyme inhibition with dibromododecenyl methylsulfimide (DDMS) selectively reduced norepinephrine sensitivity and restored impaired vasodilation in response to reduced PO2 in SS rats fed HS diet. In the presence of DDMS, vasodilatation to reduced PO2 was eliminated by indomethacin and unaffected by l-NAME in rats fed LS diet, and eliminated by l-NAME and unaffected by indomethacin in rats fed HS diet. The 20-HETE agonist WIT003 restored norepinephrine sensitivity in DDMS-treated arteries of HS-fed rats. HS diet increased vascular 20-HETE production and CYP4A protein levels by ∼24% and ∼31%, respectively, although these differences were not significant. CONCLUSIONS: These findings support the hypothesis that the 20-HETE/CYP4A system modulates vessel responses to norepinephrine and vascular relaxation to reduced PO2 in mesenteric resistance arteries of SS rats fed HS diet.

Onset of pulmonary ventilation in fetal sheep produces pial arteriolar constriction dependent on cytochrome p450 omega-hydroxylase activity.

With the onset of ventilation at birth, cerebral blood flow decreases as oxygenation increases, but the mechanism of cerebral vasoconstriction is unknown. Cytochrome P-450 omega-hydroxylase activity metabolizes arachidonic acid to 20-HETE, a potent vasoconstrictor, in a physiologically relevant O(2)-dependent manner. We tested the hypothesis that the omega-hydroxylase inhibitor, 17-octadecynoic acid (17-ODYA), reduces cerebral vasoconstriction during in utero ventilation with O(2) in fetal sheep. In anesthetized pregnant sheep near term, the fetal head was exposed with the rest of the body remaining in utero. Pial arteriolar diameter was measured by intravital microscopy through a closed cranial window superfused with vehicle or 17-ODYA. Mechanical ventilation of the fetal lungs with a high O(2) mixture to increase arterial Po(2) from approximately 20 to approximately 90 Torr markedly decreased pial arteriolar diameter by 24 + or - 3% (+ or - SE) without a change in arterial pressure. In contrast, superfusion of 17-ODYA completely blocked the decrease in diameter (2 + or - 3%) with increased oxygenation. Vasoconstriction to hypocapnia was intact after returning to the baseline intrauterine oxygenation state, thereby indicating that the effect of 17-ODYA was selective for increased oxygenation. In cerebral arteries isolated from fetal sheep, increasing oxygenation increased 20-HETE production. We conclude that cytochrome P-450 omega-hydroxylase activity makes an important contribution to cerebral vasoconstriction associated with the onset of ventilation at birth.

The role of ethanol metabolism in development of alcoholic steatohepatitis in the rat.

The importance of ethanol metabolism in the development of alcoholic liver disease remains controversial. The present study examined the effects of selective inhibition of the cytochrome P450 enzyme CYP2E1 compared with the inhibition of overall ethanol metabolism on the development of alcoholic steatohepatitis. Adult male Sprague-Dawley rats were fed via total enteral nutrition for 45 days with or without 10-12g/kg/d ethanol. Some groups were given 200mg/kg/d of the CYP2E1 inhibitor diallyl sulfide (DAS). Other groups were treated with 164mg/kg/d of the alcohol dehydrogenase (ADH) inhibitor 4-methylpyrazole (4-MP) and dosed at 2-3g/kg/d ethanol to maintain similar average urine ethanol concentrations. Liver pathology scores and levels of apoptosis were elevated by ethanol (P<.05) but did not differ significantly on cotreatment with DAS or 4-MP. However, liver triglycerides were lower when ethanol-fed rats were treated with DAS or 4-MP (P<.05). Serum alanine aminotransferase values were significantly lower in ethanol-fed 4-MP-treated rats indicating reduced necrosis. Hepatic oxidative stress and the endoplasmic reticulum (ER) stress marker tribbles-related protein 3 were increased after ethanol (P<.05); further increased by DAS but partly attenuated by 4-MP. Both DAS and 4-MP reversed ethanol increases in the cytokine, tumor necrosis factor-alpha (TNF-alpha), and the chemokine CXCL-2 (P<.05). However, neither inhibitors prevented ethanol suppression of interleukins IL-4 or IL-12. Moreover, neither inhibitors prevented ethanol increases in tumor growth factor-beta mRNA. Ethanol and DAS additively induced hepatic hyperplasia (P<.05). These data suggest that a significant proportion of hepatic injury after ethanol exposure is independent of alcohol metabolism. Ethanol metabolism by CYP2E1 may be linked in part to triglyceride accumulation, to induction of TNF-alpha, and to chemokine production. Ethanol metabolism by ADH may be linked in part to oxidative and ER stress and necrotic injury.