Postmenopausal hypertension: role of 20-HETE

Blood pressure (BP) increases after menopause. However, the mechanisms responsible have not been elucidated. In this study we tested the hypothesis that 20-hydroxyeicosatetraenoic acids (20-HETE), produced by cytochrome P-450 (CYP450) ω-hydroxylase, contributes to the hypertension in a model of postmenopausal hypertension, aged female spontaneously hypertensive rats (PMR). 1-Aminobenzotriazole, a nonselective inhibitor of arachidonic acid metabolism, for 7 days, reduced BP in PMR but had no effect in young females. Acute intravenous infusion of HET-0016, a specific inhibitor of 20-HETE, over 3 h, also reduced BP in PMR. CYP4A isoform mRNA expression showed no difference in renal CYP4A1 or CYP4A3 but increases in CYP4A2 and decreases in CYP4A8. CYP4A protein expression was decreased in kidney of PMR compared with young females. Endogenous 20-HETE was significantly higher in cerebral vessels of PMR than young females (YF) but was significantly lower in renal vessels of PMR. Omega-hydroxylase activity in cerebral vessels was also higher in PMR but was similar in kidney vessels in both groups. In renal microsomal preparations, endogenous 20-HETE was not different in PMR and young females, but ω-hydroxylase activity was significantly lower in PMR than YF. The data with blockers suggest that 20-HETE contributes to postmenopausal hypertension in SHR. The data also suggest that cerebral production of 20-HETE may be increased and renal tubular production may be decreased in PMR, thus both contributing to their elevated BP.     

The Cytochrome P450 Epoxygenase Pathway Regulates the Hepatic Inflammatory Response in Fatty Liver Disease

Fatty liver disease is an emerging public health problem without effective therapies, and chronic hepatic inflammation is a key pathologic mediator in its progression. Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid to biologically active epoxyeicosatrienoic acids (EETs), which have potent anti-inflammatory effects. Although promoting the effects of EETs elicits anti-inflammatory and protective effects in the cardiovascular system, the contribution of CYP-derived EETs to the regulation of fatty liver disease-associated inflammation and injury is unknown. Using the atherogenic diet model of non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH), our studies demonstrated that induction of fatty liver disease significantly and preferentially suppresses hepatic CYP epoxygenase expression and activity, and both hepatic and circulating levels of EETs in mice. Furthermore, mice with targeted disruption of Ephx2 (the gene encoding soluble epoxide hydrolase) exhibited restored hepatic and circulating EET levels and a significantly attenuated induction of hepatic inflammation and injury. Collectively, these data suggest that suppression of hepatic CYP-mediated EET biosynthesis is an important pathological consequence of fatty liver disease-associated inflammation, and that the CYP epoxygenase pathway is a central regulator of the hepatic inflammatory response in NAFLD/NASH. Future studies investigating the utility of therapeutic strategies that promote the effects of CYP-derived EETs in NAFLD/NASH are warranted.     

PPAR-alpha activator fenofibrate increases renal CYP-derived eicosanoid synthesis and improves endothelial dilator function in obese Zucker rats

Previous studies have shown that the synthesis of renal cytochrome P-450 (CYP)-derived eicosanoids is downregulated in genetic or high-fat diet-induced obese rats. Experiments were designed to determine whether fenofibrate, a peroxisome proliferator-activated receptor (PPAR)-alpha agonist, would induce renal eicosanoid synthesis and improve endothelial function in obese Zucker rats. Administration of fenofibrate (150 mg.kg(-1).day(-1) for 4 wk) significantly reduced plasma insulin, triglyceride, and total cholesterol levels in obese Zucker rats. CYP2C11 and CYP2C23 proteins were downregulated in renal vessels of obese Zucker rats. Consequently, renal vascular epoxygenase activity decreased by 15% in obese Zucker rats compared with lean controls. Chronic fenofibrate treatment significantly increased renal cortical and vascular CYP2C11 and CYP2C23 protein levels in obese Zucker rats, whereas it had no effect on epoxygenase protein and activity in lean Zucker rats. Renal cortical and vascular epoxygenase activities were consequently increased by 54% and 18%, respectively, in fenofibrate-treated obese rats. In addition, acetylcholine (1 microM)-induced vasodilation was significantly reduced in obese Zucker kidneys (37% +/- 11%) compared with lean controls (67% +/- 9%). Chronic fenofibrate administration increased afferent arteriolar responses to 1 microM of acetylcholine in obese Zucker rats (69% +/- 4%). Inhibition of the epoxygenase pathway with 6-(2-propargyloxyphenyl)hexanoic acid attenuated afferent arteriolar diameter responses to acetylcholine to a greater extent in lean compared with obese Zucker rats. These results demonstrate that the PPAR-alpha agonist fenofibrate increased renal CYP-derived eicosanoids and restored endothelial dilator function in obese Zucker rats.     

Renal Ischemia/Reperfusion Injury in Soluble Epoxide Hydrolase-Deficient Mice

Aim

20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs) are cytochrome P450 (CYP)-dependent eicosanoids that play opposite roles in the regulation of vascular tone, inflammation, and apoptosis. 20-HETE aggravates, whereas EETs ameliorate ischemia/reperfusion (I/R)-induced organ damage. EETs are rapidly metabolized to dihydroxyeicosatrienoic acids (DHETs) by the soluble epoxide hydrolase (sEH). We hypothesized that sEH gene (EPHX2) deletion would increase endogenous EET levels and thereby protect against I/R-induced acute kidney injury (AKI).

Methods

Kidney damage was evaluated in male wildtype (WT) and sEH-knockout (KO)-mice that underwent 22-min renal ischemia followed by two days of reperfusion. CYP-eicosanoids were analyzed by liquid chromatography tandem mass spectrometry.

Results

Contrary to our initial hypothesis, renal function declined more severely in sEH-KO mice as indicated by higher serum creatinine and urea levels. The sEH-KO-mice also featured stronger tubular lesion scores, tubular apoptosis, and inflammatory cell infiltration. Plasma and renal EET/DHET-ratios were higher in sEH-KO than WT mice, thus confirming the expected metabolic consequences of sEH deficiency. However, CYP-eicosanoid profiling also revealed that renal, but not plasma and hepatic, 20-HETE levels were significantly increased in sEH-KO compared to WT mice. In line with this finding, renal expression of Cyp4a12a, the murine 20-HETE-generating CYP-enzyme, was up-regulated both at the mRNA and protein level, and Cyp4a12a immunostaining was more intense in the renal arterioles of sEH-KO compared with WT mice.

Conclusion

These results indicate that the potential beneficial effects of reducing EET degradation were obliterated by a thus far unknown mechanism leading to kidney-specific up-regulation of 20-HETE formation in sEH-KO-mice.     

Disturbed ratio of renal 20-HETE/EETs is involved in androgen-induced hypertension in cytochrome P450 4F2 transgenic mice

We have previously established a cytochrome P450 4F2 (CYP4F2) transgenic mouse model. The present study elucidated the molecular foundation of hypertension by androgen-induction in this model. The renal expression of CYP4F2 in transgenic mice was highly expressed and strongly induced with 5α-dihydrotestosterone (DHT) treatment determined by Western blot. DHT also increased the renal arachidonic acid ω-hydroxylation and urinary 20-hydroxyeicosatetraenoic acid (20-HETE) excretion (P<0.01), and furthermore elevated the systolic blood pressure by 10 and 22 mm Hg (P<0.05) in female and castrated male transgenic mice, respectively. HET0016 completely eliminated the androgen-induced effects (P<0.01). Endogenous Cyp4a ω-hydroxylases, evaluated by real-time quantitative PCR, were significantly suppressed in transgenic mice (P<0.05). Importantly, transgenic mice with increased 20-HETE showed decreased epoxyeicosatrienoic acids (EETs) and increased dihydroxyeicosatetraenoic acids determined by liquid chromatography-tandem mass spectrometry, contributing to significantly raised ratio of 20-HETE/EETs in the urine and kidney homogenate (P<0.01). These data demonstrate that the androgen aggravated hypertension possibly through an altered ratio of 20-HETE/EETs in CYP4F2 transgenic mice.     

Multiple arachidonic acid metabolites inhibit sodium-dependent phosphate transport in OK cells

The cytochrome P450-dependent monoxygenase pathway represents a major route for the metabolism of arachidonic acid (AA) in the kidney. In turn, AA metabolites have been shown to affect renal electrolyte metabolism, including sodium transport. Specifically AA, 20-HETE and 12-HETE inhibit sodium-dependent (Na+-Pi) uptake into renal culture cells, and both 12-HETE and 14,15 EET have been shown to reduce renin release from renal cortical slices. Since the bulk of Pi transport occurs in the proximal tubule (PT), and the PT is a major site of AA metabolism, we studied the effect of AA and several of its metabolites on Na+-Pi uptake into PT-like opossum kidney (OK) cells. Incubation of OK cells in AA (10(-8) M) resulted in 17% inhibition of Pi uptake. Three metabolites of omega-hydroxylation of AA induced significant decreases in Pi uptake: 19R-HETE (10(-8) M) by 36% (P=0.008), 19S-HETE (10(-8) M) by 24% (P=0.002) and 20-COOH-AA (10(-8) M), a metabolite of 20-HETE, by 25% (P<0.0001). 14,15 EET (10(-8) M), a breakdown product of AA by the epoxygenase pathway, had the greatest effect on Pi uptake in OK cells. It decreased Pi uptake by 47% (P < 0.0001). Addition of the P450 inhibitor, 7-ER (10(-8) M), to OK cells resulted in a significant stimulation (28%) of Pi uptake (P=0.016). These results indicate that these AA metabolites have a significant inhibitory effect on Na+-Pi uptake in OK cells.     

Hypoxic pulmonary vasoconstriction is modified by P-450 metabolites

20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P-450 4A (CYP4A) metabolite of arachidonic acid (AA) in human and rabbit lung microsomes and is a dilator of isolated human pulmonary arteries (PA). However, little is known regarding the contribution of P-450 metabolites to pulmonary vascular tone. We examined 1) the effect of two mechanistically distinct omega- and omega1-hydroxylase inhibitors on perfusion pressures in isolated rabbit lungs ventilated with normoxic or hypoxic gases, 2) changes in rabbit PA ring tone elicited by 20-HETE or omega- and omega1-hydroxylase inhibitors, and 3) expression of CYP4A protein in lung tissue. A modest increase in perfusion pressure (55 +/- 11% above normoxic conditions) was observed in isolated perfused lungs during ventilation with hypoxic gas (FI(O(2)) = 0.05). Inhibitors of 20-HETE synthesis, 17-oxydecanoic acid (17-ODYA) or N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), increased baseline perfusion pressure above that of vehicle and amplified hypoxia-induced increases in perfusion pressures by 92 +/- 11% and 105 +/- 11% over baseline pressures, respectively. 20-HETE relaxed phenylephrine (PE)-constricted PA rings. Treatment with 17-ODYA enhanced PE-induced contraction of PA rings, consistent with inhibition of a product that promotes arterial relaxation, whereas 6-(20-propargyloxyphenyl)hexanoic acid (PPOH), an epoxygenase inhibitor, blunted contraction to PE. Conversion of AA into 20-HETE was blocked by 17-ODYA, DDMS, and hypoxia. CYP4A immunospecific protein confirms expression of CYP4A in male rabbit lung tissue. Our data suggest that endogenously produced 20-HETE could modify rabbit pulmonary vascular tone, particularly under hypoxic conditions.     

Activation of the abl oncogene in murine and human leukemias

Deuterium-labelled standards of four regionally isomeric epoxyeicosatrienoic acids (EETs) and their hydrolysis products, the dihydroxyeicosatrienoic acids (DHETs), have been prepared and analyzed by capillary column gas chromatography (GC)-negative ion (NI)-methane chemical ionization (MCI)-mass spectrometry (MS) as the pentafluorobenzyl esters. As little as 40 pg of these compounds were readily visualized by these methods, and the deuterium-labelled standards were used in a stable isotope dilution mass spectrometric assay which was linear from near the detection limit over several orders of magnitude. NADPH-dependent synthesis of both EETs and DHETs from arachidonate by hepatic microsomal cytochrome P-450-mono-oxygenase activity was demonstrable with these methods and was significantly suppressed by the compound BW755C (500 microM), but not by eicosa-5,8,11,14-tetraynoic acid (ETYA, 20 microM) or by nordihydroguaiaretic acid (NDGA, 50 microM). All three compounds suppress glucose-induced insulin secretion and 12-hydroxyeicosatetraenoic acid (12-HETE) synthesis by isolated pancreatic islets with similar concentration dependence. Microsomes derived from isolated pancreatic islets synthesized less than 3% of the EET and DHET compounds as a comparable amount of hepatic microsomes. Intact islets synthesized less than 3% by mass of the EET and DHET compounds compared to the mass of 12-HETE produced by the islets. Islets also failed to convert 3H-labelled arachidonate to 3H-labelled EETs or DHETs under conditions where conversion to [3H]12-HETE and to [3H]prostaglandin E2 (but not to [3H]leukotriene C4, D4, or E4) was clearly demonstrable. Neither exogenous EETs nor leukotriene C4 stimulated insulin secretion from the isolated islets or reversed the suppression of glucose-induced secretion by the lipoxygenase inhibitor BW755C. The cytochrome P-450-monooxygenase inhibitor, metyrapone (50 microM), did not influence insulin secretion from the isolated islets under conditions where the lipoxygenase inhibitor, NDGA, suppressed glucose-induced secretion. These observations argue against the recently suggested hypothesis that EETs derived from arachidonate by monooxygenase action participate in glucose-induced insulin secretion by isolated pancreatic islets.     

Functional characterization of cytochrome P450-derived epoxyeicosatrienoic acids in adipogenesis and obesity

Adipogenesis plays a critical role in the initiation and progression of obesity. Although cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) have emerged as a potential therapeutic target for cardiometabolic disease, the functional contribution of EETs to adipogenesis and the pathogenesis of obesity remain poorly understood. Our studies demonstrated that induction of adipogenesis in differentiated 3T3-L1 cells (in vitro) and obesity-associated adipose expansion in high-fat diet (HFD)-fed mice (in vivo) significantly dysregulate the CYP epoxygenase pathway and evoke a marked suppression of adipose-derived EET levels. Subsequent in vitro experiments demonstrated that exogenous EET analog administration elicits potent anti-adipogenic effects via inhibition of the early phase of adipogenesis. Furthermore, EET analog administration to mice significantly mitigated HFD-induced weight gain, adipose tissue expansion, pro-adipogenic gene expression, and glucose intolerance. Collectively, these findings suggest that suppression of EET bioavailability in adipose tissue is a key pathological consequence of obesity, and strategies that promote the protective effects of EETs in adipose tissue offer enormous therapeutic potential for obesity and its downstream pathological consequences.     

Cytochrome P-450 epoxygenases protect endothelial cells from apoptosis induced by tumor necrosis factor-alpha via MAPK and PI3K/Akt signaling pathways

Endothelial cells play a vital role in the maintenance of cardiovascular homeostasis. Epoxyeicosatrienoic acids (EETs), cytochrome P-450 (CYP) epoxygenase metabolites of arachidonic acid in endothelial cells, possess potent and diverse biological effects within the vasculature. We evaluated the effects of overexpression of CYP epoxygenases on tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in bovine aortic endothelial cells. CYP epoxygenase overexpression significantly increased endothelial cell viability and inhibited TNF-alpha induction of endothelial cell apoptosis as evaluated by morphological analysis of nuclear condensation, DNA laddering, and fluorescent-activated cell sorting (FACS) analysis. CYP epoxygenase overexpression also significantly inhibited caspase-3 activity and downregulation of Bcl-2 expression induced by TNF-alpha. The antiapoptotic effects of CYP epoxygenase overexpression were significantly attenuated by inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK signaling pathways; however, inhibition of endothelial nitric oxide synthase activity had no effect. Furthermore, CYP epoxygenase overexpression significantly attenuated the extent of TNF-alpha-induced ERK1/2 dephosphorylation in a time-dependent manner and significantly increased PI3K expression and Akt phosphorylation in both the presence and absence of TNF-alpha. Collectively, these results suggest that CYP epoxygenase overexpression, which is known to increase EET biosynthesis, significantly protects endothelial cells from apoptosis induced by TNF-alpha. This effect is mediated, at least in part, through inhibition of ERK dephosphorylation and activation of PI3K/Akt signaling.     

Role of CYP eicosanoids in the regulation of pharyngeal pumping and food uptake in Caenorhabditis elegans

Cytochrome P450 (CYP)-dependent eicosanoids comprise epoxy- and hydroxy-metabolites of long-chain PUFAs (LC-PUFAs). In mammals, CYP eicosanoids contribute to the regulation of cardiovascular and renal function. Caenorhabditis elegans produces a large set of CYP eicosanoids; however, their role in worm’s physiology is widely unknown. Mutant strains deficient in LC-PUFA/eicosanoid biosynthesis displayed reduced pharyngeal pumping frequencies. This impairment was rescued by long-term eicosapentaenoic and/or arachidonic acid supplementation, but not with a nonmetabolizable LC-PUFA analog. Short-term treatment with 17,18-epoxyeicosatetraenoic acid (17,18-EEQ), the most abundant CYP eicosanoid in C. elegans, was as effective as long-term LC-PUFA supplementation in the mutant strains. In contrast, 20-HETE caused decreased pumping frequencies. The opposite effects of 17,18-EEQ and 20-HETE were mirrored by the actions of neurohormones. 17,18-EEQ mimicked the stimulating effect of serotonin when added to starved worms, whereas 20-HETE shared the inhibitory effect of octopamine in the presence of abundant food. In wild-type worms, serotonin increased free 17,18-EEQ levels, whereas octopamine selectively induced the synthesis of hydroxy-metabolites. These results suggest that CYP eicosanoids may serve as second messengers in the regulation of pharyngeal pumping and food uptake in C. elegans.     

20-Hydroxyeicosatetraenoic acid synthesis is increased in human neutrophils and platelets by angiotensin II and endothelin-1

The cytochrome P-450 arachidonic acid metabolite 20-HETE is central to the regulation of vascular tone, renal function, and blood pressure and is synthesized in the rat kidney in response to angiotensin II (ANG II) and endothelin-1 (ET-1). There are very few studies examining the cellular synthesis of 20-HETE in humans. We aimed to measure human neutrophil and platelet 20-HETE levels under basal conditions and after ANG II, ET-1, and calcium ionophore (CaI). 20-HETE was measured in human platelets and neutrophils after saline (control), CaI (2.5 μg/ml), and ANG II or ET-1 (10 nmol/l-1 μmol/l) incubations. The effect of cells, which were preincubated with the ω-hydroxylase inhibitor N-hydroxy-N'-(4-butyl-2-methylphenyl) (HET0016, 10 nM), ANG II types 1 or 2 (AT(1) or AT(2)) receptor inhibition with irbesartan (1 μmol/l) or PD-123319 (1 μmol/l), or endothelin receptor subtypes A or B (ET(A) or ET(B)) receptor inhibition with BQ-123 or BQ-778 (100 nmol/l), was studied. Neutrophil and platelet content and release of 20-HETE was significantly increased by CaI and blocked by the ω-hydroxylase inhibitor HET0016. ANG II and ET-1 significantly increased neutrophil and platelet content and release of 20-HETE. ANG II increased 20-HETE via the AT(2) receptor. ET-1 increased 20-HETE through the ET(B) receptor in platelets and both the ET(A) and ET(B) receptors in neutrophils. These studies show that human platelets and neutrophils synthesize 20-HETE in response to ANG II and ET-1. 20-HETE synthesis in both cell types was predominantly mediated via the AT(2) and ET(B) receptors. Stimulation via these receptor pathways has generally been thought to be cardioprotective and requires further studies in clinical situations associated with low-grade inflammation or where ANG II and ET-1 are elevated to clarify the role of 20-HETE.     

Simultaneous resolution of underivatized regioisomers and stereoisomers of arachidonate epoxides by capillary electrophoresis

cis-Epoxyeicosatrienoic acids (EETs) and their hydrolysis products (threo-DHETs) have been proposed to be endothelial-dependent hyperpolarizing factors (EDHFs) which upregulate blood flow when tissue perfusion is impaired. Various EET regioisomers and enantiomers are formed from arachidonate by inducible cytochrome P450 epoxygenase isoforms, and tissue EET profiles may vary with diet, time, and disease. Because EET actions and metabolism may be regio- and stereospecific, convenient methods to measure profiles of EET isomers in tissues are needed. In the current studies, we describe two simple capillary electrophoretic methods for resolving EETs. The first method involves capillary electrophoresis with a mixture of neutral and anionic beta-cyclodextrins, which in one step baseline-resolves underivatized EET regioisomers and their enantiomers. Low picogram amounts of EET enantiomers were identified based on migration times and UV spectra. The method was also used to assess the antipode purity of EET standards, and to determine murine hepatic levels of EET enantiomers. The second method involves capillary electrochromatography, which also baseline-resolves underivatized EET and DHET regioisomers in one step. We conclude that in EET assays the major advantages of capillary electrophoresis over reversed-phase HPLC are improved peak efficiency, sensitivity, and resolution, plus precise coelution of deuterated and nondeuterated EETs.     

Determination of cytochrome P450 metabolites of arachidonic acid in coronary venous plasma during ischemia and reperfusion in dogs

Arachidonic acid (AA) can be metabolized by cytochrome P450 enzymes to many biologically active compounds including 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs), their corresponding dihydroxyeicosatrienoic acids (DHETs), as well as 19- and 20-hydroxyeicosatetraenoic acids (HETEs). These eicosanoids are potent regulators of vascular tone. However, their role in the ischemic myocardium has not been well investigated. In this study, we used a gas chromatographic-mass spectrometric technique to analyze total EETs, DHETs, and 20-HETE released into coronary venous plasma during coronary artery occlusion and reperfusion in anesthetized dogs. Pentafluorobenzyl esters (PFB-esters) of EETs and PFB-esters/trimethylsilyl ethers (TMS-ethers) of DHETs and 20-HETE were detected in the negative ion chemical ionization (NICI) using methane as a reagent gas. Under the conditions used, all four regioisomers of EET eluted from the capillary gas chromatographic column at similar retention times while four regioisomers of DHETs and 20-HETE eluted separately. The detection limits in plasma samples are 5 pg for total EETs, 40 pg for DHET, and 15 pg for 20-HETE. 14,15-DHET is the major regioisomer detected in the plasma samples while other regioisomers of DHETs are probably present at too low a concentration for detection. During the first 5 to 15 min of coronary occlusion, a slight decrease in the concentration of EETs, 14,15-DHET, and 20-HETE from the control values was observed in coronary venous plasma. At 60 min of occlusion, their concentrations significantly increased and remained elevated during 5 to 60 min of reperfusion. The concentrations decreased at 120 min of reperfusion. The NICI GC-MS was successfully used as a sensitive technique to determine cP450 metabolites of AA in plasma during prolonged occlusion-reperfusion periods. Furthermore, the results indicate that these metabolites may play a role in mediating ischemic-reperfusion injury.     

Induction of cytochrome P450 4A14 contributes to angiotensin II-induced renal fibrosis in mice

Angiotensin II (AngII) plays an important role in the pathogenesis of hypertension and associated renal injuries. To elucidate the molecular mechanism by which AngII induces renal damage, we found that AngII infusion significantly induced CYP4A14 expression in renal proximal tubule cells (RPTCs) with marked increases in blood pressure and proteinuria. Renal production of the major CYP4A metabolite, 20-HETE, was also significantly increased in the AngII-treated mice. Compared to wild-type (WT) mice, CYP4A14 knockout (CYP4A14^?/?) mice exhibited significantly lower levels of blood pressure, renal 20-HETE production, proteinuria and renal fibrosis following AngII infusion. Furthermore, AngII-induced renal expression of profibrotic genes and proinflammatory genes was significantly attenuated in CYP4A14^?/? mice. In vitro studies using cultured RPTCs demonstrated that AngII significantly induced CYP4A14 expression and 20-HETE production via the MAPK signaling pathway. AngII treatment increased TGF-β and collagen expression, which was attenuated by the CYP4A inhibitor, TS-011. Moreover, 20-HETE treatment potently induced CYP4A14 expression and TGF-β and collagen levels. Collectively, these findings suggest that attenuated renal fibrosis in AngII-treated CYP4A14^?/? mice may result from both reduced systemic blood pressure and renal 20-HETE production. Therefore, CYP4A14 may represent a useful target for the treatment of AngII-associated renal damage.     

Cytochromes P450 from family 4 are the main omega hydroxylating enzymes in humans: CYP4F3B is the prominent player in PUFA metabolism

Human CYP450 omega-hydroxylases of the CYP4 family are known to convert arachidonic acid (AA) to its metabolite 20-hydroxyeicosatetraenoic acid (20-HETE). This study deals with hydroxylations of four PUFAs, eicosatrienoic acid (ETA), AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) by either human recombinant CYP4s enzymes or human liver microsomal preparations. CYP4F3A and CYP4F3B were the most efficient omega-hydroxylases of these PUFAs. Moreover, the differences in the number of unsaturations of ETA, AA, and EPA allowed us to demonstrate a rise in the metabolic rate of hydroxylation when the double bond in 14-15 or 17-18 was missing. With the CYP4F enzymes, the main pathway was always the omega-hydroxylation of PUFAs, whereas it was the (omega-1)-hydroxylation with CYP1A1, CYP2C19, and CYP2E1. Finally, we demonstrated that the omega9 and omega3 PUFAs (ETA, EPA, and DHA) could all be used as alternative substrates in AA metabolism by human CYP4F2 and -4F3B. Thus, they decreased the ability of these enzymes to convert AA to 20-HETE. However, although ETA was the most hydroxylated substrate, EPA and DHA were the most potent inhibitors of the conversion of AA to 20-HETE. These findings suggest that some physiological effects of omega3 FAs could partly result from a shift in the generation of active hydroxylated metabolites of AA through a CYP-mediated catalysis.     

Role of NO and cytochrome P-450-derived eicosanoids in ET-1-induced changes in intrarenal hemodynamics in rats

Endothelin-1 (ET-1) produces potent renal effects that we have previously shown to be dependent on cytochrome P-450 (CYP450) metabolites of aracidonic acid (24) This study evaluated the role of these metabolites in the effects produced by ET-1 on renal blood flow (RBF), cortical blood flow (CBF), medullary blood flow (MBF), and mean arterial blood pressure (MBP). ET-1 (20-200 pmol/kg) increased MBP, renal vascular resistance (RVR), and MBF but reduced CBF and RBF in a dose-dependent manner. The decreases in CBF and RBF, and increases in MBP and RVR were blunted by BMS-182874, an ET(A) receptor antagonist or BQ-788, an ET(B) receptor antagonist. Similarly, indomethacin, an inhibitor of cyclooxygenase activity, or 12,12-dibromododecenoic acid (DBDD), a CYP450-dependent inhibitor of production of 20-hydroxyeicosatetraenoic acid (20-HETE), blunted these effects. ET-3 elicited dose-related reduction in CBF and increase in MBF. Indomethacin accentuated the reduction in CBF and attenuated the increase in MBF, as did DBDD. ET-1-induced increase in MBF was attenuated by BQ-788, N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthesis, indomethacin, or DBDD. DBDD inhibited the hemodynamic effects of L-NAME. Miconazole, the inhibitor of CYP450-dependent epoxygenase activity, was without effect. These results indicate that hemodynamic changes produced by ET-1 are mediated by vasoconstrictor prostanoids and/or prostanoid-like substances, possibly, 20-HETE via activation of ET(A) and ET(B) receptors. However, the increase in MBF is mediated by vasodilator prostanoids or by NO via ET(B) receptor activation.     

Arachidonic acid epoxygenase and 12(S)-lipoxygenase: evidence of their concerted involvement in ductus arteriosus constriction to oxygen

Oxygen promotes closure of the ductus arteriosus at birth. We have previously presented a scheme for oxygen action with a cytochrome P450 (CYP450) hemoprotein and endothelin-1 (ET-1) being, respectively, sensor and effector, and a hypothetical monooxygenase product serving as a coupling link. We have also found in the vessel arachidonic acid (AA) 12(S)-lipoxygenase (12-lipoxygenase) undergoing upregulation at birth. Here, we examined the feasibility of a sensor-to-effector messenger originating from AA monooxygenase and 12-lipoxygenase pathways. The epoxygenase inhibitor, N-methylsulfonyl-6-(2-)hexanamide, suppressed the tonic contraction of ductus to oxygen. A similar effect was obtained with 12-lipoxygenase inhibitors baicalein and PD 146176. By contrast, none of the inhibitors modified the endothelin-1 contraction. Furthermore, an AA ω-hydroxylation product, 20-hydroxyeicosatetraenoic acid (20-HETE), reportedly responsible for oxygen contraction in the systemic microvasculature, had no such effect on the ductus. We conclude that AA epoxygenase and 12-lipoxygenase jointly produce a hitherto uncharacterized compound acting as oxygen messenger in the ductus.     

Endothelium‐specific CYP2J2 overexpression attenuates age‐related insulin resistance

Ample evidences demonstrate that cytochrome P450 epoxygenase‐derived epoxyeicosatrienoic acids (EETs) exert diverse biological activities, which include potent vasodilatory, anti‐inflammatory, and cardiovascular protective effects. In this study, we investigated the effects of endothelium‐specific CYP2J2 overexpression on age‐related insulin resistance and metabolic dysfunction. Endothelium‐specific targeting of the human CYP epoxygenase, CYP2J2, transgenic mice (Tie2‐CYP2J2‐Tr mice) was utilized. The effects of endothelium‐specific CYP2J2 overexpression on aging‐associated obesity, inflammation, and peripheral insulin resistance were evaluated by assessing metabolic parameters in young (3 months old) and aged (16 months old) adult male Tie2‐CYP2J2‐Tr mice. Decreased insulin sensitivity and attenuated insulin signaling in aged skeletal muscle, adipose tissue, and liver were observed in aged adult male mice, and moreover, these effects were partly inhibited in 16‐month‐old CYP2J2‐Tr mice. In addition, CYP2J2 overexpression‐mediated insulin sensitization in aged mice was associated with the amelioration of inflammatory state. Notably, the aging‐associated increases in fat mass and adipocyte size were only observed in 16‐month‐old wild‐type mice, and CYP2J2 overexpression markedly prevented the increase in fat mass and adipocyte size in aged Tie2‐CYP2J2‐Tr mice, which was associated with increased energy expenditure and decreased lipogenic genes expression. Furthermore, these antiaging phenotypes of Tie2‐CYP2J2‐Tr mice were also associated with increased muscle blood flow, enhanced active‐phase locomotor activity, and improved mitochondrial dysfunction in skeletal muscle. Collectively, our findings indicated that endothelium‐specific CYP2J2 overexpression alleviated age‐related insulin resistance and metabolic dysfunction, which highlighted CYP epoxygenase‐EET system as a potential target for combating aging‐related metabolic disorders.