Cytochrome P450 Epoxygenase Research Articles

Beneficial effects of soluble epoxide hydrolase inhibitors in myocardial infarction model: Insight gained using metabolomic approaches

Myocardial infarction (MI) leading to myocardial cell loss represents one of the common causes leading to cardiac failure. We have previously demonstrated the beneficial effects of several potent soluble epoxide hydrolase (sEH) inhibitors in cardiac hypertrophy. sEH catalizes the conversion of epoxyeicosatrienoic acids (EETs) to form the corresponding dihydroxyeicosatrienoic acids (DHETs). EETs are products of cytochrome P450 epoxygenases that have vasodilatory properties. Additionally, EETs inhibit the activation of nuclear factor (NF)-κB-mediated gene transcription. Motivated by the potential to uncover a new class of therapeutic agents for cardiovascular diseases which can be effectively used in clinical setting, we directly tested the biological effects of sEH inhibitors (sEHIs) on the progression of cardiac remodeling using a clinically relevant murine model of MI. We demonstrated that sEHIs were highly effective in the prevention of progressive cardiac remodeling post MI. Using metabolomic profiling of the inflammatory lipid mediators, we documented a significant decrease in EETs/DHETs ratio in MI model predicting a heightened inflammatory state. Treatment with sEHIs resulted in a change in the pattern of lipid mediators from one of inflammation towards resolution. Moreover, the oxylipin profiling showed a striking parallel to the changes in inflammatory cytokines in this model. Our study provides evidence for a possible new therapeutic strategy to improve cardiac function post MI.

14,15-Epoxyeicosa-5,8,11-trienoic Acid (14,15-EET) Surrogates Containing Epoxide Bioisosteres: Influence upon Vascular Relaxation and Soluble Epoxide Hydrolase Inhibition

All-cis-14,15-epoxyeicosa-5,8,11-trienoic acid (14,15-EET) is a labile, vasodilatory eicosanoid generated from arachidonic acid by cytochrome P450 epoxygenases. A series of robust, partially saturated analogues containing epoxide bioisosteres were synthesized and evaluated for relaxation of precontracted bovine coronary artery rings and for in vitro inhibition of soluble epoxide hydrolase (sEH). Depending upon the bioisostere and its position along the carbon chain, varying levels of vascular relaxation and/or sEH inhibition were observed. For example, oxamide 16 and N-iPr-amide 20 were comparable (ED(50) 1.7 microM) to 14,15-EET as vasorelaxants but were approximately 10-35 times less potent as sEH inhibitors (IC(50) 59 and 19 microM, respectively); unsubstituted urea 12 showed useful activity in both assays (ED(50) 3.5 microM, IC(50) 16 nM). These data reveal differential structural parameters for the two pharmacophores that could assist the development of potent and specific in vivo drug candidates.

Differential action of bradykinin on intrarenal regional perfusion in the rat: waning effect in the cortex and major impact in the medulla

The renal kallikrein-kinin system is involved in the control of the intrarenal circulation and arterial pressure but bradykinin (Bk) effects on perfusion of individual kidney zones have not been examined in detail. Effects of Bk infused into renal artery, renal cortex or medulla on perfusion of whole kidney (RBF, renal artery probe) and of the cortex, outer- and inner medulla (CBF, OMBF, IMBF: laser-Doppler fluxes), were examined in anaesthetized rats. Renal artery infusion of Bk, 0.3-0.6 mg kg(-1) h(-1), induced no sustained increase in RBF or CBF. OMBF and IMBF increased initially 6 or 16%, respectively; only the IMBF increase (+10%) was sustained. Pre-treatment with L-NAME, 2.4 mg kg(-1) I.V., prevented the sustained but not initial transient elevation of medullary perfusion. Intracortical Bk infusion, 0.75-1.5 mg kg(-1) h(-1), did not alter RBF or CBF but caused a sustained 33% increase in IMBF. Intramedullary Bk, 0.3 mg kg(-1) h(-1), did not alter RBF or CBF but caused sustained increases in OMBF (+10%) and IMBF (+23%). These responses were not altered by pre-treatment with 1-aminobenzotriazole, 10 mg kg(-1)i.v., a cytochrome P-450 (CYP450) inhibitor, but were prevented or significantly attenuated by L-NAME or intramedullary clotrimazole, 3.9 mg kg(-1) h(-1), an inhibitor of CYP450 epoxygenase and of calcium-dependent K(+) channels (K(Ca)). Thus, cortical vasodilatation induced by Bk is only transient so that the agent is unlikely to control perfusion of the cortex. Bk selectively increases perfusion of the medulla, especially of its inner layer, via activation of the NO system and of K(Ca) channels.

Characterization of transgenic mice with neuron-specific expression of soluble epoxide hydrolase

Soluble epoxide hydrolase (sEH) is the major enzyme responsible for the metabolism and inactivation of epoxyeicosatrienoic acids (EETs). EETs are produced by the cytochrome P450 (CYP) epoxygenase pathway of arachidonic acid (AA) metabolism and tend to be anti-hypertensive, anti-inflammatory and protective against ischemic injury. Since the metabolism of EETs by sEH reduces or eliminates their bioactivity, inhibition of sEH has become a therapeutic strategy for hypertension and inflammation. sEH is found in nearly all tissues so the systemic application of inhibitors is likely to affect more than blood pressure and inflammation. In the central nervous system, EETs are thought to play a role in the regulation of local blood flow, protection from ischemic injury, inhibition of inflammation, the release of peptide hormones and modulation of fever. However, little is known about region- and cell-specific expression of sEH in the brain. In the mouse brain, expression of sEH was found widely in cortical and hippocampal astrocytes and also in a few specific neuron types in the cortex, cerebellum, and medulla. To assess the functional significance of neuronal sEH, we generated a transgenic mouse model, which over-expresses sEH specifically in neurons. Transgenic mice showed increased neuron labeling in cortex and hippocampus with little change in labeling of other brain regions. Despite a 3-fold increase in sEH activity in the brain, there was no change in arterial pressure. This data provides new information required for studying the central roles of the cytochrome P450 epoxygenase pathway.

Tanshinone II A elicited vasodilation in rat coronary arteriole: Roles of nitric oxide and potassium channels

Salvia miltiorrhiza has been widely used in the treatment of various cardiovascular diseases due to its ability to improve coronary microcirculation and increase coronary blood flow. Tanshinone II A, the major active lipophilic ingredient responsible for the beneficial actions of Salvia miltiorrhiza, was shown to induce vasodilation in coronary arteries. But its effects on coronary arterioles remain unknown. The purpose of this study was to investigate the effects of tanshinone II A on isolated rat coronary arteriole and the underlying mechanisms. Coronary arterioles were carefully dissected, cannulated and pressurized. Tanshinone II A-elicited vascular inner diameter change was recorded by a computerized diameter tracking system. To investigate the mechanisms governing the vasodilative effects of tanshinone II A, the roles of endothelium, endothelium-derived vasoactive factors and potassium channels were assessed respectively. Endothelium denudation, inhibition of nitric oxide synthase (NOS), inhibition of the cytochrome P450 epoxygenase, and blockade of the large conductance calcium(Ca 2+)-activated potassium channels (BKca) significantly decreased the vasodilation elicited by Tanshinone II A. The results indicated that tanshinone II A induces an endothelium-dependent vasodilation in coronary arterioles; nitric oxide (NO) and cytochrome P450 metabolites contribute to the vasodilation; activation of BKca channels plays an important role in the vasodilation.

Evidence for role of epoxyeicosatrienoic acids in mediating ischemic preconditioning and postconditioning in dog

Cytochrome P-450 (CYP) epoxygenases and their arachidonic acid (AA) metabolites, the epoxyeicosatrienoic acids (EETs), have been shown to produce marked reductions in infarct size (IS) in canine myocardium either given before an ischemic insult or at reperfusion similar to that produced in ischemic preconditioning (IPC) and postconditioning (POC) protocols. However, no studies have addressed the possibility that EETs serve a beneficial role in IPC or POC. We tested the hypothesis that EETs may play a role in these two phenomena by preconditioning dog hearts with one 5-min period of total coronary occlusion followed by 10 min of reperfusion before 60 min of occlusion and 3 h of reperfusion or by postconditioning with three 30-s periods of reperfusion interspersed with three 30-s periods of occlusion. To test for a role of EETs in IPC and POC, the selective EET antagonists 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) or its derivative, 14,15-epoxyeicosa-5(Z)-enoic acid 2-[2-(3-hydroxy-propoxy)-ethoxy]-ethyl ester (14,15-EEZE-PEG), were administered 10 min before IPC, 5 min after IPC, or 5 min before POC. In a separate series, the selective EET synthesis inhibitor N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH) was administered 10 min before IPC. Infarct size was determined by tetrazolium staining and coronary collateral blood flow at 30 min of occlusion and reperfusion flow at 3 h by radioactive microspheres. Both IPC and POC produced nearly equivalent reductions in IS expressed as a percentage of the area at risk (AAR) [Control 21.2 +/- 1.2%, IPC 8.3 +/- 2.2%, POC 10.1 +/- 1.8% (P < 0.001)]. 14,15-EEZE, 14,15-EEZE-PEG, and MS-PPOH markedly attenuated the cardioprotective effects of IPC and POC (14,15-EEZE and 14,15-EEZE-PEG) at doses that had no effect on IS/AAR when given alone. These results suggest a unique role for endogenous EETs in both IPC and POC.

Flow Dilation in Rat Small Mesenteric Arteries is Mediated by Hydrogen Peroxide Generated from CYP Epoxygenases and Xanthine Oxidase

We have previously demonstrated that hydrogen peroxide (H2O2) mediates flow-induced vascular dilation in rat small mesenteric arteries. In the present study, the source of this flow-induced H2O2 was explored. The arteries were pressurized to 50 mm Hg and preconstricted with phenylephrine. Intraluminal flow reversed the effect of phenylephrine, resulting in vascular dilation. Cytochrome P450 (CYP) inhibitors N-methylsulfonyl-6-(2-proparglyoxyphenyl) hexanoic acid (MS-PPOH, 10 �M) and miconazole (30 �M) reduced the magnitude of peak flow dilation by ~20%-30%, and reduced the duration of dilatory response by ~70-80%. Nevertheless, sulphaphenazole (10 �M), a selective inhibitor of CYP 2C9, had no effect neither on the peak flow dilation nor the duration of dilatory response. Oxypurinol (100 �M), an inhibitor of xanthine oxidase, attenuated the duration of dilatory response by ~60% but exerted no effect on the magnitude of peak flow dilation. Cyclosporin A (2 �M), an inhibitor for mitochondrial permeability transition pore, MitoQ (300 nM), a mitochondria-targeted antioxidant, and apocynin (1 mM), a NADPH oxidase inhibitor, had no effect neither on the magnitude of peak flow dilation nor the duration of dilatory response. To further confirm the role of CYP in flow-induced H2O2 production, a fluorescent probe CM-H2DCFDA was used to monitor the production of H2O2 in the primary endothelial cells isolated from rat small mesenteric arteries. The results showed that flow-induced H2O2 production was markedly reduced in MS-PPOH and miconazole pretreated endothelial cells. Taken together, our results suggest that, during flow dilation, H2O2 is generated from CYP epoxygenases and xanthine oxidase.

Arachidonic acid cytochrome P450 epoxygenase pathway

Cytochrome P450 (CYP) epoxygenases convert arachidonic acid to four epoxyeicosatrienoic acid (EET) regioisomers, 5,6-, 8,9-, 11,12-, and 14,15-EET, that function as autacrine and paracrine mediators. EETs produce vascular relaxation by activating smooth muscle large-conductance Ca2+-activated K+ channels (BKCa). In addition, they have anti-inflammatory effects on blood vessels and in the kidney, promote angiogenesis, and protect ischemic myocardium and brain. CYP epoxygenases also convert eicosapentaenoic acid to vasoactive epoxy-derivatives, and endocannabinoids containing 11,12- and 14,15-EET are formed. Many EET actions appear to be initiated by EET binding to a membrane receptor that activates ion channels and intracellular signal transduction pathways. However, EETs also are taken up by cells, are incorporated into phospholipids, and bind to cytosolic proteins and nuclear receptors, suggesting that some functions may occur through direct interaction of the EET with intracellular effector systems. Soluble epoxide hydrolase (sEH) converts EETs to dihydroxyeicosatrienoic acids (DHETs). Because this attenuates many of the functional effects of EETs, sEH inhibition is being evaluated as a mechanism for increasing and prolonging the beneficial actions of EETs.

Angiotensin II directly regulates intestinal epithelial NHE3 in Caco2BBE cells

BackgroundAngiotensin II (AII) effects on intestinal Na+ transport may be multifactorial. To determine if AII might have a direct effect on intestinal epithelial Na+ transport, we investigated its actions on Na+ transport in human intestinal epithelial Caco2BBE cells.ResultsAII increased apical (brush border) sodium-hydrogen exchanger (NHE)-3, but not NHE2, activity within one hour. Similarly, only apical membrane NHE3 abundance increased at 1–2 hours without any change in total NHE3 protein abundance. From 4–48 hours, AII stimulated progressively larger increases in apical NHE3 activity and surface abundance, which was associated with increases in NHE3 protein expression. At 4–24 hours, NHE3 mRNA increases over baseline expression, suggesting increased gene transcription. This was supported by AII induced increases in rat NHE3 gene promoter-reporter activity. AII induction of NHE3 was blocked by the AII type I receptor antagonist losartan. Acute changes in AII-induced increases in NHE3 exocytosis were blocked by a phospholipase C inhibitor, an arachidonic acid cytochrome P450 epoxygenase inhibitor, as well as phosphatidylinositol 3 kinase (PI3K) inhibitors and Akt inhibitor, partially blocked by a metalloproteinase inhibitor and an EGF (epidermal growth factor) receptor kinase inhibitor, but not affected by an inhibitor of MEK-1 (MAPKK-1, mitogen activated protein kinase kinase-1).ConclusionWe conclude that angiotensin II has a direct role in regulating intestinal fluid and electrolyte absorption which may contribute to its overall effects in regulation systemic volume and blood pressure. AII activates several key signaling pathways that induce acute and chronic changes in NHE3 membrane trafficking and gene transcription.

Epoxyeicosanoids as mediators of neurogenic vasodilation in cerebral vessels

Epoxyeicosatrienoic acids (EETs) are potent vasodilators produced from arachidonic acid by cytochrome P-450 (CYP) epoxygenases and metabolized to vicinal diols by soluble epoxide hydrolase (sEH). In the brain, EETs are produced by astrocytes and the vascular endothelium and are involved in the control of cerebral blood flow (CBF). Recent evidence, however, suggests that epoxygenases and sEH are present in perivascular vasodilator nerve fibers innervating the cerebral surface vasculature. In the present study, we tested the hypothesis that EETs are nerve-derived relaxing factors in the cerebral circulation. We first traced these fibers by retrograde labeling in the rat to trigeminal ganglia (TG) and sphenopalatine ganglia (SPG). We then examined the expression of CYP epoxygenases and sEH in these ganglia. RT-PCR and Western blot analysis identified CYP2J3 and CYP2J4 epoxygenase isoforms and sEH in both TG and SPG, and immunofluorescence double labeling identified CYP2J and sEH immunoreactivity in neuronal cell bodies of both ganglia. To evaluate the functional role of EETs in neurogenic vasodilation, we elicited cortical hyperemia by electrically stimulating efferent cerebral perivascular nerve fibers and by chemically stimulating oral trigeminal fibers with capsaicin. Cortical blood flow responses were monitored by laser-Doppler flowmetry. Local administration to the cortical surface of the putative EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (30 mumol/l) attenuated CBF responses to electrical and chemical stimulation. These results suggest that EETs are produced by perivascular nerves and play a role in neurogenic vasodilation of the cerebral vasculature. The findings have important implications to such clinical conditions as migraine, vasospasm after subarachnoid hemorrhage, and stroke.

Selective Inhibitors of CYP2J2 Related to Terfenadine Exhibit Strong Activity against Human Cancers in Vitro and in Vivo

The cytochrome P450 epoxygenase, CYP2J2, converts arachidonic acid to four regioisomeric epoxyeicosatrienoic acids (EETs). We found recently that this enzyme is dramatically up-regulated in a variety of established human carcinoma cell lines and in human cancerous tissue and promotes the neoplastic phenotype. In the present study, we tested the hypothesis that specific inhibitors of CYP2J2 related to the drug terfenadine are effective antitumor agents. Four of these inhibitors (compounds 4, 5, 11, and 26) were tested for effectiveness in vitro and in vivo. In Tca-8113 cells, the CYP2J2 inhibitors decreased EET production by approximately 60%, whereas they had no effect on CYP2J2 mRNA or protein expression. Compound 26 inhibited the proliferation of human tumor cells, reduced their ability to adhere, invade, and migrate, and attenuated activation of epithelial growth factor receptor signal and kinases and phosphatidylinositol 3 kinase/Akt pathways. Inhibition of CYP2J2 also significantly potentiated human tumor cell apoptosis and caused a corresponding increase in caspase-3 activity and change in expression of apoptosis-related proteins Bax and Bcl-2. In murine xenograft models using MDA-MB-435 cells, treatment with compound 26 significantly repressed tumor growth, decreased lung metastasis, and was associated with increased expression of the anticancer genes CD82 and nm23, without causing toxicity. These data suggest that CYP2J2 inhibitors hold significant promise for use in treatment of neoplastic diseases.

Inhibition of the soluble epoxide hydrolase attenuates monocrotaline-induced pulmonary hypertension in rats

The soluble epoxide hydrolase (sEH) metabolizes epoxyeicosatrienoic acids (EETs) to their less active dihydroxy derivatives. Because EETs have antiinflammatory properties, we determined whether or not inhibition of sEH attenuates disease development in the monocrotaline model of pulmonary hypertension in rats. sEH inhibition was achieved using 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (25 mg/l) and cis- 4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (5 mg/l) administered via drinking water starting 3 days prior to monocrotaline injection (60 mg/kg). Monocrotaline induced the development of progressive pulmonary hypertension. sEH inhibition increased the plasma ratio of EETs to DHETs and attenuated the monocrotaline-induced increase in pulmonary artery medial wall thickness as well as the degree of vascular muscularization. Moreover, right ventricular pressure was significantly lower in the group treated with sEH inhibitors. Pulmonary sEH protein expression and sEH activity, as well as pulmonary cytochrome P450 epoxygenase activity were all impaired in monocrotaline-treated rats as compared with control animals. sEH inhibitors, however, increased the plasma ratio of EETs to dihydroxy epoxyeicosatrienoic acids. Monocrotaline induced the proliferation of pulmonary endothelial and vascular smooth muscle cells in vivo as determined by 5-Bromo-2'-deoxy-Uridine incorporation, and this effect was significantly blunted in animals treated with sEH inhibitors. Proliferation of cultured pulmonary smooth muscle cell, however, was not affected by EETs or sEH inhibitors suggesting that the in-vivo effects are a consequence of a direct EET-mediated protection against the inflammation induced by monocrotaline. sEH inhibition reduces pulmonary vascular remodeling and the development of pulmonary hypertension in the monocrotaline model of primary pulmonary hypertension in rats.

Effects of Lipids on ENaC Activity in Cultured Mouse Cortical Collecting Duct Cells

Direct effects on epithelial Na+ channels (ENaC) activity by lipids, e.g., arachidonic acid (AA), eicosatetraynoic acid (ETYA), linoleic acid (LA), stearic acid (SA), hydroxyeicosatetraenoic acid (HETE), 11,12-epoxyeicosatrienoic acid (EET), (PGF2), and (PGE2), in cultured mouse cortical collecting duct (M1) cells were clarified by using single-channel recordings in this study. In a cell-attached recording, a bath application of 10 microM AA significantly reduced the ENaC open probability (NPo), whereas 10 microM ETYA or 5 microM LA only induced a slight inhibition. The inside-out recording as a standard protocol was thereafter performed to examine effects of these lipids on ENaC activity. Within 10 min after the formation of the inside-out configuration, the NPo of ENaC in cultured mouse cortical collecting duct (M1) cells remained relatively constant. Application of ETYA or LA or SA exhibited a similar inhibition on the channel NPo when applied to the extracellular side, suggesting that fatty acids could exert a nonspecific inhibition on ENaC activity. 11,12-EET, a metabolite of AA via the cytochrome P450 epoxygenase pathway, significantly inhibited the ENaC NPo, whereas 20-HETE, a metabolite of AA via the hydroxylase pathway, only caused a small inhibition of the ENaC NPo, to a similar degree as that seen with ETYA and LA. However, both PGE2 and PGF2alpha significantly enhanced the ENaC NPo. These results suggest that fatty acids exert a nonspecific effect on ENaC activity due to the interaction between the channel proximity and the lipid. The opposite effects of 11,12-EET and prostaglandin (PG) implicate different mechanisms in regulation of ENaC activity by activation of epoxygenase and cyclooxygenase.

Tyrosine Phosphorylation Modulates the Activity of TRPV4 in Response to Defined Stimuli

Src family tyrosine kinases (SFKs) regulate the function of several transient receptor potential (TRP) family members, yet their role in the regulation of the vanilloid subfamily member 4 protein (TRPV4) remains controversial. TRPV4 is a calcium-permeable channel activated by numerous physical and chemical stimuli. Here we show that SFKs mediate tyrosine phosphorylation of TRPV4 in different cell lines. Using mass spectrometric analysis, we identified two novel phosphorylation sites in the cytosolic N- and C-terminal tails of TRPV4. Substitution of either tyrosine with phenylalanine led to a substantial reduction in the overall tyrosine phosphorylation level of TRPV4, suggesting that these two tyrosines constitute major phosphorylation sites. Both mutants efficiently localized to the plasma membrane, indicating that neither tyrosine is required for trafficking of TRPV4 in the secretory pathway. Analysis of the channel function demonstrated a crucial role of the N-terminal tyrosine residue in the activation of TRPV4 by heat, mechanical (shear) stress, hypotonic cell swelling, and phorbol 12-myristate 13-acetate, but not in the activation by synthetic ligand 4alpha-phorbol 12,13-didecanoate. Furthermore, the response of TRPV4 to phorbol 12-myristate 13-acetate was SFK-dependent. Because the SFK-mediated phosphorylation of the N-terminal tyrosine occurred before TRPV4 activation, tyrosine phosphorylation appears to sensitize rather than activate this channel. Reactive oxygen species, known to mediate inflammatory pain, strongly up-regulated TRPV4 phosphorylation in the presence of SFKs. Our findings indicate that tyrosine phosphorylation of TRPV4 represents an important modulatory mechanism, which may underlie the recently described function of TRPV4 in inflammatory hyperalgesia.

High-salt diet enhances mouse aortic relaxation through adenosine A2Areceptor via CYP epoxygenases

We hypothesize that A(2A) adenosine receptors (A(2A) AR) promote aortic relaxation in mice through cytochrome P450 (CYP)-epoxygenases and help to avoid salt sensitivity. Aortas from male mice maintained on a high-salt (HS; 7% NaCl) or normal-salt (NS; 0.45% NaCl) diet for 4-5 wks were used. Concentration-response curves (10(-11)-10(-5) M) for 5'-N-ethylcarboxamidoadenosine (NECA; a nonselective adenosine analog) and CGS 21680 (A(2A) AR agonist) were obtained with different antagonists including ZM 241385 (A(2A) AR antagonist; 10(-6) M), SCH 58261 (A(2A) AR antagonist; 10(-6) M), N(omega)-nitro-l-arginine methyl ester (l-NAME; endothelial nitric oxide synthase inhibitor; 10(-4) M) and inhibitors including methylsulfonyl-propargyloxyphenylhexanamide (MS-PPOH; CYP epoxygenases inhibitor; 10(-5)M), 14,15-epoxyeicosa-5(z)-enoic acid (14,15-EEZE; EET antagonist; 10(-5)M), dibromo-dodecenyl-methylsulfimide (DDMS; CYP4A inhibitor; 10(-5)M), and HET0016 (20-HETE inhibitor; 10(-5)M). At 10(-7) M of NECA, significant relaxation in HS (+22.58 +/- 3.12%) was observed compared with contraction in NS (-10.62 +/- 6.27%, P < 0.05). ZM 241385 changed the NECA response to contraction (P < 0.05) in HS. At 10(-7) M of CGS 21680, significant relaxation in HS (+32.04 +/- 3.08%) was observed compared with NS (+10.45 +/- 1.34%, P < 0.05). SCH 58261, l-NAME, MS-PPOH, and 14,15-EEZE changed the CGS 21680-induced relaxation to contraction (P < 0.05) in HS. Interestingly, DDMS and HET0016 changed CGS 21680 response to relaxation (P < 0.05) in NS; however, there was no significant difference found between DDMS, HET0016-treated HS and NS vs. nontreated HS group (P > 0.05). CYP2C29 protein was 55% and 74% upregulated in HS vs. NS (P < 0.05) mice aorta and kidney, respectively. CYP4A protein was 30.30% and 35.70% upregulated in NS vs. HS (P < 0.05) mice aorta and kidneys, respectively. A(1) AR was downregulated, whereas A(2A) AR was upregulated in HS compared with NS. These data suggest that HS may activate CYP2C29 via A(2A) AR, causing relaxation, whereas NS may contribute to the upregulation of CYP4A causing contraction.

Relaxing effects of 17(18)-EpETE on arterial and airway smooth muscles in human lung

Human cytochrome P-450 epoxygenase enzymes metabolize eicosapentaenoic acid (EPA), an omega-3-polyunsaturated fatty acid (PUFA), and leads to the production of 17(18)-epoxyeicosatetraenoic acid, or 17(18)-EpETE. The aim of the present study was to delineate the mode of action of 17(18)-EpETE on human pulmonary artery (HPA) and distal bronchi. Isometric tension measurements demonstrated that 17(18)-EpETE induced concentration-dependent relaxing effects in pulmonary artery and airway smooth muscles. Iberiotoxin (IbTx) and glyburide (Glyb), known BK(Ca) and K(ATP) channel inhibitors, respectively, reversed the relaxation induced by 17(18)-EpETE on both tissues types. Microelectrode measurements showed that exogenous addition of 17(18)-EpETE hyperpolarized the membrane potential of HPA and bronchial smooth muscle cells. These induced electrophysiological effects were reversed by the addition of 10 nM IbTx and 10 muM Glyb. Complementary experiments performed on human bronchi, using the planar lipid bilayer reconstitution technique, demonstrated that 17(18)-EpETE activated reconstituted BK(Ca) channels at low free Ca(2+) concentration. Moreover, in bronchi, the relaxing responses induced by 17(18)-EpETE were also related to reduced Ca(2+) sensitivity of the myofilaments, since free Ca(2+) concentration-response curves, performed on beta-escin-permeabilized cultured explants, were shifted toward higher Ca(2+). Together, these results provide new insight into the mode of action of 17(18)-EpETE in lung tissues and highlight this eicosanoid as a potent modulator of tone on both HPA and distal bronchi in vitro, which may be of clinical relevance in the pathophysiology of pulmonary hypertension and airway diseases.

Epoxyeicosatrienoic acid prevents postischemic electrocardiogram abnormalities in an isolated heart model

Cytochrome P450 epoxygenases metabolize arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs) which are in turn converted to dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). The main objective of this study was to investigate the protective effects of EETs following ischemic injury using an ex vivo electrocardiogram (EKG) model. Hearts from C57Bl/6, transgenic mice with cardiomyocyte-specific overexpression of CYP2J2 (Tr) and wildtype (WT) littermates were excised and perfused with constant pressure in a Langendorff apparatus. Electrodes were placed superficially at the right atrium and left ventricle to assess EKG waveforms. In ischemic reperfusion experiments hearts were subjected to 20 min of global no-flow ischemia followed by 20 min of reperfusion (R20). The EKG from C57Bl/6 hearts perfused with 1 μM 14,15-EET showed less QT prolongation (QTc) and ST elevation (STE) (QTc = 41 ± 3, STE = 2.3 ± 0.3; R20: QTc = 42 ± 2 ms, STE = 1.2 ± 0.2mv) than control hearts (QTc = 36 ± 2, STE = 2.3 ± 0.2; R20: QTc = 53 ± 3 ms; STE = 3.6 ± 0.4mv). Similar results of reduced QT prolongation and ST elevation were observed in EKG recording from CYP2J2 Tr mice (QTc = 35 ± 1, STE = 1.9 ± 0.1; R20: QTc = 38 ± 4 ms, STE = 1.3 ± 0.2mv) compared to WT hearts. The putative epoxygenase inhibitor MS-PPOH (50 μM) and EET antagonist 14,15-EEZE (10 μM) both abolished the cardioprotective response, implicating EETs in this process. In addition, separate exposure to the K ATP channel blockers glibenclamide (1 μM) and HMR1098 (10 μM), or the PKA protein inhibitor H89 (50 nM) during reperfusion abolished the improved repolarization in both the models. Consistent with a role of PKA, CYP2J2 Tr mice had an enhanced activation of the PKAα regulatory II subunit in plasma membrane following IR injury. The present data demonstrate that EETs can enhance the recovery of ventricular repolarization following ischemia, potentially by facilitating activation of K + channels and PKA-dependent signaling.

Role of CYP epoxygenases in A2AAR-mediated relaxation using A2AAR-null and wild-type mice

We hypothesized that A2A adenosine receptor (A2A AR) activation causes vasorelaxation through cytochrome P-450 (CYP) epoxygenases and endothelium-derived hyperpolarizing factors, whereas lack of A2A AR activation promotes vasoconstriction through Cyp4a in the mouse aorta. Adenosine 5'-N-ethylcarboxamide (NECA; 10(-6) M), an adenosine analog, caused relaxation in wild-type A2A AR (A2A AR+/+; +33.99 +/- 4.70%, P < 0.05) versus contraction in A2A AR knockout (A2A AR(-/-); -27.52 +/- 4.11%) mouse aortae. An A2A AR-specific antagonist (SCH-58261; 1 microM) changed the NECA (10(-6) M) relaxation response to contraction (-35.82 +/- 4.69%, P < 0.05) in A2A AR+/+ aortae, whereas no effect was noted in A2A AR(-/-) aortae. Significant contraction was seen in the absence of the endothelium in A2A AR+/+ (-2.58 +/- 2.25%) aortae compared with endothelium-intact aortae. An endothelial nitric oxide synthase inhibitor (N-nitro-L-arginine methyl ester; 100 microM) and a cyclooxygenase inhibitor (indomethacin; 10 microM) failed to block NECA-induced relaxation in A2A AR+/+ aortae. A selective inhibitor of CYP epoxygenases (methylsulfonyl-propargyloxyphenylhexanamide; 10 microM) changed NECA-mediated relaxation (-22.74 +/- 5.11% at 10(-6) M) and CGS-21680-mediated relaxation (-18.54 +/- 6.06% at 10(-6) M) to contraction in A2A AR+/+ aortae, whereas no response was noted in A2A AR(-/-) aortae. Furthermore, an epoxyeicosatrienoic acid (EET) antagonist [14,15-epoxyeicosa-5(Z)-enoic acid; 10 microM] was able to block NECA-induced relaxation in A2A AR+/+ aortae, whereas omega-hydroxylase inhibitors (10 microM dibromo-dodecenyl-methylsulfimide and 10 microM HET-0016) changed contraction into relaxation in A2A AR(-/-) aorta. Cyp2c29 protein was upregulated in A2A AR+/+ aortae, whereas Cyp4a was upregulated in A2A AR(-/-) aortae. Higher levels of dihydroxyeicosatrienoic acids (DHETs; 14,15-DHET, 11,12-DHET, and 8,9-DHET, P < 0.05) were found in A2A AR+/+ versus A2A AR(-/-) aortae. EET levels were not significantly different between A2A AR+/+ and A2A AR(-/-) aortae. It is concluded that CYP epoxygenases play an important role in A2A AR-mediated relaxation, and the deletion of the A2A AR leads to contraction through Cyp4a.

Epoxyeicosatrienoic acids are part of the VEGF-activated signaling cascade leading to angiogenesis

Cytochrome P-450 (CYP) epoxygenases metabolize arachidonic acid to epoxyeicosatrienoic acid (EET) regioisomers, which activate several signaling pathways to promote endothelial cell proliferation, migration, and angiogenesis. Since vascular endothelial growth factor (VEGF) plays a key role in angiogenesis, we assessed a possible role of EETs in the VEGF-activated signal transduction cascade. Stimulation with VEGF increased CYP2C promoter activity in endothelial cells and enhanced CYP2C8 mRNA and protein expression resulting in increased intracellular EET levels. VEGF-induced endothelial cell tube formation was inhibited by the EET antagonist 14,15-epoxyeicosa-5(Z)-enoicacid (14,15-EEZE), which did not affect the VEGF-induced phosphorylation of its receptor or basic fibroblast growth factor (bFGF)-stimulated tube formation. Moreover, VEGF-stimulated endothelial cell sprouting in a modified spheroid assay was reduced by CYP2C antisense oligonucleotides. Mechanistically, VEGF stimulated the phosphorylation of the AMP-activated protein kinase (AMPK), which has also been linked to CYP induction, and the overexpression of a constitutively active AMPK mutant increased CYP2C expression. On the other hand, a dominant-negative AMPK mutant prevented the VEGF-induced increase in CYP2C RNA and protein expression in human endothelial cells. In vivo (Matrigel plug assay) in mice, endothelial cells were recruited into VEGF-impregnated plugs; an effect that was sensitive to 14,15-EEZE and the inclusion of small interfering RNA directed against the AMPK. The EET antagonist did not affect responses observed in plugs containing bFGF. Taken together, our data indicate that CYP2C-derived EETs participate as second messengers in the angiogenic response initiated by VEGF and that preventing the increase in CYP expression curtails the angiogenic response to VEGF.

Inhibitory effects of epoxyeicosatrienoic acids on volume-activated chloride channels in rat mesenteric arterial smooth muscle

Epoxyeicosatrienoic acids (EETs) are synthesized from arachidonic acid by cytochrome P450 epoxygenases in endothelial cells. It has previously been shown that EETs activate K + channels, which are important for the hyperpolarization and dilation of blood vessels. However, the effects of EETs on other ion channels have been less well studied. We investigated the effects of EETs on volume-activated Cl − channels (VACCs) in rat mesenteric arterial smooth muscle cells. Whole-cell patch clamp recording demonstrated that hypotonic solution and guanosine 5′-[γ-thio]triphosphate (GTPγS) induced a 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB)- and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS)-sensitive VACC current in the primary cultured rat mesenteric arterial smooth muscle cells. The VACC current was inhibited by EETs and the order of potency was 8,9-EET > 5,6-EET > 11,12-EET > 14,15-EET. The inhibitory effects of EETs could be reversed by 14,15 epoxyeicosa-5( Z)-enoic acid (14,15-EEZE, an EET analog), Rp-cGMP and KT-5823 (protein kinase G inhibitors). Interestingly, the inhibitory effects of EETs on VACCs were not influenced by Rp-cAMP (a protein kinase A antagonist) but it could be abolished by NF-449 (a Gs protein inhibitor), indicating the involvement of cAMP but not protein kinase A. In conclusion, our results demonstrate that EETs inhibit VACCs in rat mesenteric arterial smooth muscle cells through a cGMP-dependent pathway, which is probably due to the cross-activation by cAMP. This mechanism may be involved in the regulation of cell volume and membrane potential.