Relative contribution of cyclooxygenases, epoxyeicosatrienoic acids, and pH to the cerebral blood flow response to vibrissal stimulation

The increase in cerebral blood flow (CBF) during neuronal activation can be only partially attenuated by individual inhibitors of epoxyeicosatrienoic acids (EETs), cyclooxgenase-2, group I metabotropic glutamate receptors (mGluR), neuronal nitric oxide synthase (nNOS), N-methyl-D-aspartate receptors, or adenosine receptors. Some studies that used a high concentration (500 μM) of the cyclooxygenase-1 inhibitor SC-560 have implicated cyclooxygenase-1 in gliovascular coupling in certain model systems in the mouse. Here, we found that increasing the concentration of SC-560 from 25 μM to 500 μM over whisker barrel cortex in anesthetized rats attenuated the CBF response to whisker stimulation. However, exogenous prostaglandin E(2) restored the response in the presence of 500 μM SC-560 but not in the presence of a cyclooxygenase-2 inhibitor, thereby suggesting a limited permissive role for cyclooxygenase-1. Furthermore, inhibition of the CBF response to whisker stimulation by an EET antagonist persisted in the presence of SC-560 or a cyclooxygenase-2 inhibitor, thereby indicating that the EET-dependent component of vasodilation did not require cyclooxygenase-1 or -2 activity. With combined inhibition of cyclooxygenase-1 and -2, mGluR, nNOS, EETs, N-methyl-D-aspartate receptors, and adenosine 2B receptors, the CBF response was reduced by 60%. We postulated that the inability to completely block the CBF response was due to tissue acidosis resulting from impaired clearance of metabolically produced CO2. We tested this idea by increasing the concentration of superfused bicarbonate from 25 to 60 mM and found a markedly reduced CBF response to hypercapnia. However, increasing bicarbonate had no effect on the initial or steady-state CBF response to whisker stimulation with or without combined inhibition. We conclude that the residual response after inhibition of several known vasodilatory mechanisms is not due to acidosis arising from impaired CO2 clearance when the CBF response is reduced. An unidentified mechanism apparently is responsible for the rapid, residual cortical vasodilation during vibrissal stimulation.     

Interaction of nitric oxide, 20-HETE, and EETs during functional hyperemia in whisker barrel cortex

Nitric oxide (NO) modulates vasodilation in cerebral cortex during sensory activation. NO is known to inhibit the synthesis of 20-HETE, which has been implicated in arteriolar constriction during astrocyte activation in brain slices. We tested the hypothesis that the attenuated cerebral blood flow (CBF) response to whisker stimulation seen after NO synthase (NOS) inhibition requires 20-HETE synthesis and that the ability of an epoxyeicosatrienoic acids (EETs) antagonist to reduce the CBF response is blunted after NOS inhibition but restored with simultaneous blockade of 20-HETE synthesis. In anesthetized rats, the increase in CBF during whisker stimulation was attenuated after the blockade of neuronal NOS with 7-nitroindazole. Subsequent administration of the 20-HETE synthesis inhibitor N-hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine (HET0016) restored the CBF response to control levels. After the administration of 7-nitroindazole, the inhibitory effect of an EETs antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) on the CBF response was lost, whereas the simultaneous administration of 7-nitroindazole and HET0016 restored the inhibitory effect of 14,15-EEZE. The administration of HET0016 alone had only a small effect on the evoked CBF response in rats. Furthermore, in neuronal NOS(+/+) and NOS(-/-) mice, HET0016 administration did not increase the CBF response to whisker stimulation. In neuronal NOS(+/+) mice, HET0016 also blocked the reduction in the response seen with acute NOS inhibition. These results indicate that 20-HETE synthesis normally does not substantially restrict functional hyperemia. Increased NO production during functional activation may act dynamically to suppress 20-HETE synthesis or downstream signaling and permit EETs-dependent vasodilation. With the chronic loss of neuronal NOS in mice, other mechanisms apparently suppress 20-HETE synthesis or signaling.     

Role of cytochrome P-450 metabolites in the regulation of renal function and blood pressure in 2-kidney 1-clip hypertensive rats

Alterations in renal function contribute to Goldblatt two-kidney, one-clip (2K1C) hypertension. A previous study indicated that bioavailability of cytochrome P-450 metabolites epoxyeicosatrienoic acids (EETs) is decreased while that of 20-hydroxyeicosatetraenoic acids (20-HETE) is increased in this model. We utilized the inhibitor of soluble epoxide hydrolase cis-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (c-AUCB) and HET-0016, the inhibitor of 20-HETE production, to study the role of EETs and 20-HETE in the regulation of renal function. Chronic c-AUCB treatment significantly decreased systolic blood pressure (SBP) (133 ± 1 vs. 163 ± 3 mmHg) and increased sodium excretion (1.23 ± 0.10 vs. 0.59 ± 0.03 mmol/day) in 2K1C rats. HET-0016 did not affect SBP and sodium excretion. In acute experiments, renal blood flow (RBF) was decreased in 2K1C rats (5.0 ± 0.2 vs. 6.9 ± 0.2 ml·min(-1)·g(-1)). c-AUCB normalized RBF in 2K1C rats (6.5 ± 0.6 ml·min(-1)·g(-1)). HET-0016 also increased RBF in 2K1C rats (5.8 ± 0.2 ml·min(-1)·g(-1)). Although RBF and glomerular filtration rate (GFR) remained stable in normotensive rats during renal arterial pressure (RAP) reductions, both were significantly reduced at 100 mmHg RAP in 2K1C rats. c-AUCB did not improve autoregulation but increased RBF at all RAPs and shifted the pressure-natriuresis curve to the left. HET-0016-treated 2K1C rats exhibited impaired autoregulation of RBF and GFR. Our data indicate that c-AUCB displays antihypertensive properties in 2K1C hypertension that are mediated by an improvement of RBF and pressure natriuresis. While HET-0016 enhanced RBF, its anti-natriuretic effect likely prevented it from producing a blood pressure-lowering effect in the 2K1C model.     

Suppression of cortical functional hyperemia to vibrissal stimulation in the rat by epoxygenase inhibitors

Application of glutamate to glial cell cultures stimulates the formation and release of epoxyeicosatrienoic acids (EETs) from arachidonic acid by cytochome P-450 epoxygenases. Epoxygenase inhibitors reduce the cerebral vasodilator response to glutamate and N-methyl-D-aspartate. We tested the hypothesis that epoxygenase inhibitors reduce the somatosensory cortical blood flow response to whisker activation. In chloralose-anesthetized rats, percent changes in cortical perfusion over whisker barrel cortex were measured by laser-Doppler flowmetry during whisker stimulation. Two pharmacologically distinct inhibitors were superfused subdurally: 1) N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH), an epoxygenase substrate inhibitor; and 2) miconazole, a reversible cytochrome P-450 inhibitor acting on the heme moiety. Superfusion with 5 micromol/l MS-PPOH decreased the hyperemic response to whisker stimulation by 28% (from 25 +/- 9 to 18 +/- 7%, means +/- SD, n = 8). With 20 micromol/l MS-PPOH superfusion, the response was decreased by 69% (from 28 +/- 9% to 9 +/- 4%, n = 8). Superfusion with 20 micromol/l miconazole decreased the flow response by 67% (from 31 +/- 6% to 10 +/- 3%, n = 8). Subsequent superfusion with vehicle restored the response to 26 +/- 11%. Indomethacin did not prevent MS-PPOH inhibition of the flow response, suggesting that EET-related vasodilation was not dependent solely on cyclooxygenase metabolism of 5,6-EET. Neither MS-PPOH nor miconazole changed baseline flow, reduced the blood flow response to an adenosine A(2) agonist, or decreased somatosensory evoked potentials. The marked reduction of the cortical flow response to whisker stimulation with two different types of epoxygenase inhibitors indicates that EETs play an important role in the physiological coupling of blood flow to neural activation.     

Modulation of V1-receptor-mediated renal vasoconstriction by epoxyeicosatrienoic acids

This study examined the effects of renal arterial infusion of a selective cytochrome P-450 epoxygenase inhibitor, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH; 2 mg/kg plus 1.5 mg.kg(-1).h(-1)), on renal hemodynamic responses to infusions of [Phe(2),Ile(3),Orn(8)]vasopressin and ANG II into the renal artery of anesthetized rabbits. MS-PPOH did not affect basal renal blood flow (RBF) or cortical or medullary blood flow measured by laser-Doppler flowmetry (CLDF/MLDF). In vehicle-treated rabbits, [Phe(2),Ile(3),Orn(8)]vasopressin (30 ng.kg(-1).min(-1)) reduced MLDF by 62 +/- 7% but CLDF and RBF were unaltered. In MS-PPOH-treated rabbits, RBF and CLDF fell by 51 +/- 8 and 59 +/- 13%, respectively, when [Phe(2),Ile(3),Orn(8)]vasopressin was infused. MS-PPOH had no significant effects on the MLDF response to [Phe(2),Ile(3),Orn(8)]vasopressin (43 +/- 9% reduction). ANG II (20 ng.kg(-1).min(-1)) reduced RBF by 45 +/- 10% and CLDF by 41 +/- 14%, but MLDF was not significantly altered. MS-PPOH did not affect blood flow responses to ANG II. Formation of epoxyeicosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids (DiHETEs) was 49% lower in homogenates prepared from the renal cortex of MS-PPOH-treated rabbits than from vehicle-treated rabbits. MS-PPOH had no effect on the renal formation of 20-hydroxyeicosatetraenoic acid (20-HETE). Incubation of renal cortical homogenates from untreated rabbits with [Phe(2),Ile(3),Orn(8)]vasopressin (0.2-20 ng/ml) did not affect formation of EETs, DiHETEs, or 20-HETE. These results do not support a role for de novo EET synthesis in modulating renal hemodynamic responses to ANG II. However, EETs appear to selectively oppose V(1)-receptor-mediated vasoconstriction in the renal cortex but not in the medullary circulation and contribute to the relative insensitivity of cortical blood flow to V(1)-receptor activation [corrected].     

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.     

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.     

Metabotropic Glutamate Receptor Agonists Potentiate Cyclic AMP Formation Induced by Forskolin or β-Adrenergic Receptor Activation in Cerebral Cortical Astrocytes in Culture

Abstract: The metabotropic glutamate receptor (mGluR) agonist 1-aminocyclopentane-1 S ,3 R -dicarboxylic acid (ACPD) potentiated the accumulation of cyclic AMP induced by either β-adrenergic receptor stimulation (isoproterenol) or direct activation of adenylyl cyclase (AC) with forskolin in rat cerebral cortical astrocytes grown in a defined medium. In contrast, ACPD inhibits the cyclic AMP response in astrocytes cultured in a serum-containing medium. Pharmacological characterization indicated that a group I mGluR, of which only mGluR5 is detectable in these cells, is involved in the potentiation of cyclic AMP accumulation. Potentiation was elicited by mGluR I agonists [e.g., ( R,S )-3,5-dihydroxyphenylglycine (DHPG)], but not by mGluR II or III agonists; it was pertussis toxin resistant and abolished by procedures suppressing mGluR5 function (phorbol ester pretreatment or DHPG-induced receptor down-regulation). Nevertheless, it appears that products generated through the mGluR5 transduction pathway, such as elevated [Ca²⁺]_i or activated protein kinase C (PKC), are not involved in the potentiation as it was not influenced by either the intracellular calcium chelator BAPTA-AM or the PKC inhibitor Ro 31-8220. An inhibitor of phospholipase C, U-73122, markedly attenuated mGluR5-activated phosphoinositide hydrolysis but did not significantly affect the DHPG potentiation of the cyclic AMP response. A mechanism is proposed in which the potentiating effect on AC could be mediated by free βγ complex that is liberated after the agonist-bound mGluR5 interacts with its coupled G protein.     

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.     

Role of cytochrome P450-dependent arachidonic acid metabolites in liver physiology and pathophysiology

Arachidonic acid (AA) can undergo monooxygenation or epoxidation by enzymes in the cytochrome P450 (CYP) family in the brain, kidney, lung, vasculature, and the liver. CYP-AA metabolites, 19- and 20-hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatrienoic acids (EETs) and diHETEs have different biological properties based on sites of production and can be stored in tissue lipids and released in response to hormonal stimuli. 20-HETE is a vasoconstrictor, causing blockade of Ca(++)-activated K(+) (KCa) channels. Inhibition of the formation of nitric oxide (NO) by 20-HETE mediates most of the cGMP-independent component of the vasodilator response to NO. 20-HETE elicits a potent dilator response in human and rabbit pulmonary vascular and bronchiole rings that is dependent on an intact endothelium and COX. 20-HETE is also a vascular oxygen sensor, inhibits Na(+)/K(+)-ATPase activity, is an endogenous inhibitor of the Na(+)-K(+)-2Cl(-)cotransporter, mediates the mitogenic actions of vasoactive agents and growth factors in many tissues and plays a significant role in angiogenesis. EETs, produced by the vascular endothelium, are potent dilators. EETs hyperpolarize VSM cells by activating KCa channels. Several investigators have proposed that one or more EETs may serve as endothelial-derived hyperpolarizing factors (EDHF). EETs constrict human and rabbit bronchioles, are potent mediators of insulin and glucagon release in isolated rat pancreatic islets, and have anti-inflammatory activity. Compared with other organs, the liver has the highest total CYP content and contains the highest levels of individual CYP enzymes involved in the metabolism of fatty acids. In humans, 50-75% of CYP-dependent AA metabolites formed by liver microsomes are omega/omega-OH-AA, mainly w-OH-AA, i.e. 20HETE, and 13-28% are EETs. Very little information is available on the role of 19- and 20-HETE and EETs in liver function. EETs are involved in vasopressin-induced glycogenolysis, probably via the activation of phosphorylase. In the portal vein, inhibition of EETs exerts profound effects on a variety of K-channel activities in smooth muscles of this vessel. 20-HETE is a weak, COX-dependent, vasoconstrictor of the portal circulation. EETs, particularly 11,12-EET, cause vasoconstriction of the porto-sinusoidal circulation. Increased synthesis of EETs in portal vessels and/or sinusoids or increased levels in blood from the meseneric circulation may participate in the pathophysiology of portal hypertension of cirrhosis. CYP-dependent AA metabolites are involved in the pathophysiology of portal hypertension, not only by increasing resistance in the porto-sinusoidal circulation, but also by increasing portal inflow through mesenteric vasodilatation. In patients with cirrhosis, urinary 20-HETE is several-fold higher than PGs and TxB2, whereas in normal subjects, 20-HETE and PGs are excreted at similar rates. Thus, 20-HETE is probably produced in increased amounts in the preglomerular microcirculation accounting for the functional decrease of flow and increase in sodium reabsorption. In conclusion, CYP-AA metabolites represent a group of compounds that participate in the regulation of liver metabolic activity and hemodynamics. They appear to be deeply involved in abnormalities related to liver diseases, particularly cirrhosis, and play a key role in the pathophysiology of portal hypertension and renal failure.     

Soluble epoxide hydrolase contamination of specific catalase preparations inhibits epoxyeicosatrienoic acid vasodilation of rat renal arterioles

Cytochrome P-450 metabolites of arachidonic acid, the epoxyeicosatrienoic acids (EETs) and hydrogen peroxide (H(2)O(2)), are important signaling molecules in the kidney. In renal arteries, EETs cause vasodilation whereas H(2)O(2) causes vasoconstriction. To determine the physiological contribution of H(2)O(2), catalase is used to inactivate H(2)O(2). However, the consequence of catalase action on EET vascular activity has not been determined. In rat renal afferent arterioles, 14,15-EET caused concentration-related dilations that were inhibited by Sigma bovine liver (SBL) catalase (1,000 U/ml) but not Calbiochem bovine liver (CBL) catalase (1,000 U/ml). SBL catalase inhibition was reversed by the soluble epoxide hydrolase (sEH) inhibitor tAUCB (1 μM). In 14,15-EET incubations, SBL catalase caused a concentration-related increase in a polar metabolite. Using mass spectrometry, the metabolite was identified as 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), the inactive sEH metabolite. 14,15-EET hydrolysis was not altered by the catalase inhibitor 3-amino-1,2,4-triazole (3-ATZ; 10-50 mM), but was abolished by the sEH inhibitor BIRD-0826 (1-10 μM). SBL catalase EET hydrolysis showed a regioisomer preference with greatest hydrolysis of 14,15-EET followed by 11,12-, 8,9- and 5,6-EET (V(max) = 0.54 ± 0.07, 0.23 ± 0.06, 0.18 ± 0.01 and 0.08 ± 0.02 ng DHET·U catalase(-1)·min(-1), respectively). Of five different catalase preparations assayed, EET hydrolysis was observed with two Sigma liver catalases. These preparations had low specific catalase activity and positive sEH expression. Mass spectrometric analysis of the SBL catalase identified peptide fragments matching bovine sEH. Collectively, these data indicate that catalase does not affect EET-mediated dilation of renal arterioles. However, some commercial catalase preparations are contaminated with sEH, and these contaminated preparations diminish the biological activity of H(2)O(2) and EETs.     

Soluble epoxide hydrolase inhibition and gene deletion are protective against myocardial ischemia-reperfusion injury in vivo

Soluble epoxide hydrolase (sEH) metabolizes epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids. EETs are formed from arachidonic acid during myocardial ischemia and play a protective role against ischemic cell death. Deletion of sEH has been shown to be protective against myocardial ischemia in the isolated heart preparation. We tested the hypothesis that sEH inactivation by targeted gene deletion or pharmacological inhibition reduces infarct size (I) after regional myocardial ischemia-reperfusion injury in vivo. Male C57BL\6J wild-type or sEH knockout mice were subjected to 40 min of left coronary artery (LCA) occlusion and 2 h of reperfusion. Wild-type mice were injected intraperitoneally with 12-(3-adamantan-1-yl-ureido)-dodecanoic acid butyl ester (AUDA-BE), a sEH inhibitor, 30 min before LCA occlusion or during ischemia 10 min before reperfusion. 14,15-EET, the main substrate for sEH, was administered intravenously 15 min before LCA occlusion or during ischemia 5 min before reperfusion. The EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (EEZE) was given intravenously 15 min before reperfusion. Area at risk (AAR) and I were assessed using fluorescent microspheres and triphenyltetrazolium chloride, and I was expressed as I/AAR. I was significantly reduced in animals treated with AUDA-BE or 14,15-EET, independent of the time of administration. The cardioprotective effect of AUDA-BE was abolished by the EET antagonist 14,15-EEZE. Immunohistochemistry revealed abundant sEH protein expression in left ventricular tissue. Strategies to increase 14,15-EET, including sEH inactivation, may represent a novel therapeutic approach for cardioprotection against myocardial ischemia-reperfusion injury.     

Astrocyte inositol triphosphate receptor type 2 and cytosolic phospholipase a(2) alpha regulate arteriole responses in mouse neocortical brain slices

Functional hyperemia of the cerebral vascular system matches regional blood flow to the metabolic demands of the brain. One current model of neurovascular control holds that glutamate released by neurons activates group I metabotropic glutamate receptors (mGluRs) on astrocytes, resulting in the production of diffusible messengers that act to regulate smooth muscle cells surrounding cerebral arterioles. The acute mouse brain slice is an experimental system in which changes in arteriole diameter can precisely measured with light microscopy. Stimulation of the brain slice triggers specific cellular responses that can be correlated to changes in arteriole diameter. Here we used inositol trisphosphate receptor type 2 (IP(3)R2) and cytosolic phospholipase A(2) alpha (cPLA(2)α) deficient mice to determine if astrocyte mGluR activation coupled to IP(3)R2-mediated Ca(2+) release and subsequent cPLA(2)α activation is required for arteriole regulation. We measured changes in astrocyte cytosolic free Ca(2+) and arteriole diameters in response to mGluR agonist or electrical field stimulation in acute neocortical mouse brain slices maintained in 95% or 20% O(2). Astrocyte Ca(2+) and arteriole responses to mGluR activation were absent in IP(3)R2(-) (/-) slices. Astrocyte Ca(2+) responses to mGluR activation were unchanged by deletion of cPLA(2)α but arteriole responses to either mGluR agonist or electrical stimulation were ablated. The valence of changes in arteriole diameter (dilation/constriction) was dependent upon both stimulus and O(2) concentration. Neuron-derived NO and activation of the group I mGluRs are required for responses to electrical stimulation. These findings indicate that an mGluR/IP(3)R2/cPLA(2)α signaling cascade in astrocytes is required to transduce neuronal glutamate release into arteriole responses.     

Contribution of adenosine A2A and A2B receptors and heme oxygenase to AMPA-induced dilation of pial arterioles in rats

Nitric oxide (NO) has been implicated in mediation of cerebral vasodilation during neuronal activation and, specifically, in pharmacological activation of N-methyl-d-aspartate (NMDA) and kainate receptors. Possible mediators of cerebral vasodilation to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) have not been well studied in mature brain, although heme oxygenase (HO) activity has been implicated in newborn pigs. In anesthetized rats, 5 min of topical superfusion of 30 and 100 microM AMPA on the cortical surface through a closed cranial window resulted in increases in pial arteriolar diameter. The dilatory response to AMPA was not inhibited by superfusion of an NO synthase inhibitor, a cyclooxygenase-2 inhibitor, or a cytochrome P-450 epoxygenase inhibitor, all of which have been shown to inhibit the cortical blood flow response to sensory activation. However, the 48 +/- 13% dilation to 100 microM AMPA was attenuated 56-71% by superfusion of the adenosine A(2A) receptor antagonist ZM-241385, the A(2B) receptor antagonist alloxazine, and the HO inhibitor chromium mesoporphyrin. Combination of the latter three inhibitors did not attenuate the dilator response more than the individual inhibitors, whereas an AMPA receptor antagonist fully blocked the vasodilation to AMPA. These results indicate that cortical pial arteriolar dilation to AMPA does not require activation of NO synthase, cyclooxygenase-2, or cytochrome P-450 epoxygenase but does depend on activation of adenosine A(2A) and A(2B) receptors. In addition, CO derived from HO appears to play a role in the vascular response to AMPA receptor activation in mature brain by a mechanism that is not additive with that of adenosine receptor activation.     

Role of 20-hydroxyeicosatetraenoic acid in the renal and vasoconstrictor actions of angiotensin II

The present study examined the effects of ANG II on the renal synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE) and its contribution to the renal vasoconstrictor and the acute and chronic pressor effects of ANG II in rats. ANG II (10(-11) to 10(-7) mol/l) reduced the diameter of renal interlobular arteries treated with inhibitors of nitric oxide synthase and cyclooxygenase, lipoxygenase, and epoxygenase by 81 +/- 8%. Subsequent blockade of the synthesis of 20-HETE with 17-octadecynoic acid (1 micromol/l) increased the ED(50) for ANG II-induced constriction by a factor of 15 and diminished the maximal response by 61%. Graded intravenous infusion of ANG II (5-200 ng/min) dose dependently increased mean arterial pressure (MAP) in thiobutylbarbitol-anesthetized rats by 35 mmHg. Acute blockade of the formation of 20-HETE with dibromododecenyl methylsulfimide (DDMS; 10 mg/kg) attenuated the pressor response to ANG II by 40%. An intravenous infusion of ANG II (50 ng. kg(-1). min(-1)) in rats for 5 days increased the formation of 20-HETE and epoxyeicosatrienoic acids (EETs) in renal cortical microsomes by 60 and 400%, respectively, and increased MAP by 78 mmHg. Chronic blockade of the synthesis of 20-HETE with intravenous infusion of DDMS (1 mg. kg(-1). h(-1)) or EETs and 20-HETE with 1-aminobenzotriazole (ABT; 2.2 mg. kg(-1). h(-1)) attenuated the ANG II-induced rise in MAP by 40%. Control urinary excretion of 20-HETE averaged 350 +/- 23 ng/day and increased to 1,020 +/- 105 ng/day in rats infused with ANG II (50 ng. kg(-1). min(-1)) for 5 days. In contrast, urinary excretion of 20-HETE only rose to 400 +/- 40 and 600 +/- 25 ng/day in rats chronically treated with ANG II and ABT or DDMS respectively. These results suggest that acute and chronic elevations in circulating ANG II levels increase the formation of 20-HETE in the kidney and peripheral vasculature and that 20-HETE contributes to the acute and chronic pressor effects of ANG II.     

Crosstalk between EET and HO-1 downregulates Bach1 and adipogenic marker expression in mesenchymal stem cell derived adipocytes

Epoxygenase activity and synthesis of epoxyeicosatrienoic acids (EETs) have emerged as important modulators of obesity and diabetes. We examined the effect of the EET-agonist 12-(3-hexylureido)dodec-8(2) enoic acid on mesenchymal stem cell (MSC) derived adipocytes proliferation and differentiation. MSCs expressed substantial levels of EETs and inhibition of soluble epoxide hydrolase (sEH) increased the level of EETs and decreased adipogenesis. EET agonist treatment increased HO-1 expression by inhibiting a negative regulator of HO-1 expression, Bach-1. EET treatment also increased βcatenin and pACC levels while decreasing PPARγ C/EBPα and fatty acid synthase levels. These changes were manifested by a decrease in the number of large inflammatory adipocytes, TNFα, IFNγ and IL-1α, but an increase in small adipocytes and in adiponectin levels. In summary, EET agonist treatment inhibits adipogenesis and decreases the levels of inflammatory cytokines suggesting the potential action of EETs as intracellular lipid signaling modulators of adipogenesis and adiponectin.     

Hemoglobin, NO, and 20-HETE interactions in mediating cerebral vasoconstriction following SAH

Recent studies have indicated that 20-hydroxyeicosatetraenoic acid (20-HETE) contributes to the fall in cerebral blood flow (CBF) after subarachnoid hemorrhage (SAH), but the factors that stimulate the production of 20-HETE are unknown. This study examines the role of vasoactive factors released by clotting blood vs. the scavenging of nitric oxide (NO) by hemoglobin (Hb) in the fall in CBF after SAH. Intracisternal (icv) injection of blood produced a greater and more prolonged (120 vs. 30 min) decrease in CBF than that produced by a 4% solution of Hb. Pretreating rats with N(omega)-nitro-l-arginine methyl ester (l-NAME; 10 mg/kg iv) to block the synthesis of NO had no effect on the fall in CBF produced by an icv injection of blood. l-NAME enhanced rather than attenuated the fall in CBF produced by an icv injection of Hb. Blockade of the synthesis of 20-HETE with TS-011 (0.1 mg/kg iv) prevented the sustained fall in CBF produced by an icv injection of blood and the transient vasoconstrictor response to Hb. Hb (0.1%) reduced the diameter of the basilar artery (BA) of rats in vitro by 10 +/- 2%. This response was reversed by TS-011 (100 nM). Pretreatment of vessels with l-NAME (300 muM) reduced the diameter of BA and blocked the subsequent vasoconstrictor response to the addition of Hb to the bath. TS-011 returned the diameter of vessels exposed to l-NAME and Hb to that of control. These results suggest that the fall in CBF after SAH is largely due to the release of vasoactive factors by clotting blood rather than the scavenging of NO by Hb and that 20-HETE contributes the vasoconstrictor response of cerebral vessels to both Hb and blood.     

Role of astrocytes in cerebrovascular regulation.

Astrocytes send processes to synapses and blood vessels, communicate with other astrocytes through gap junctions and by release of ATP, and thus are an integral component of the neurovascular unit. Electrical field stimulations in brain slices demonstrate an increase in intracellular calcium in astrocyte cell bodies transmitted to perivascular end-feet, followed by a decrease in vascular smooth muscle calcium oscillations and arteriolar dilation. The increase in astrocyte calcium after neuronal activation is mediated, in part, by activation of metabotropic glutamate receptors. Calcium signaling in vitro can also be influenced by adenosine acting on A2B receptors and by epoxyeicosatrienoic acids (EETs) shown to be synthesized in astrocytes. Prostaglandins, EETs, arachidonic acid, and potassium ions are candidate mediators of communication between astrocyte end-feet and vascular smooth muscle. In vivo evidence supports a role for cyclooxygenase-2 metabolites, EETs, adenosine, and neuronally derived nitric oxide in the coupling of increased blood flow to increased neuronal activity. Combined inhibition of the EETs, nitric oxide, and adenosine pathways indicates that signaling is not by parallel, independent pathways. Indirect pharmacological results are consistent with astrocytes acting as intermediaries in neurovascular signaling within the neurovascular unit. For specific stimuli, astrocytes are also capable of transmitting signals to pial arterioles on the brain surface for ensuring adequate inflow pressure to parenchymal feeding arterioles. Therefore, evidence from brain slices and indirect evidence in vivo with pharmacological approaches suggest that astrocytes play a pivotal role in regulating the fundamental physiological response coupling dynamic changes in cerebral blood flow to neuronal synaptic activity. Future work using in vivo imaging and genetic manipulation will be required to provide more direct evidence for a role of astrocytes in neurovascular coupling.     

Fatty acid-binding proteins inhibit hydration of epoxyeicosatrienoic acids by soluble epoxide hydrolase

Epoxyeicosatrienoic acids (EETs) are potent regulators of vascular homeostasis and are bound by cytosolic fatty acid-binding proteins (FABPs) with K(d) values of approximately 0.4 microM. To determine whether FABP binding modulates EET metabolism, we examined the effect of FABPs on the soluble epoxide hydrolase (sEH)-mediated conversion of EETs to dihydroxyeicosatrienoic acids (DHETs). Kinetic analysis of sEH conversion of racemic [(3)H]11,12-EET yielded K(m) = 0.45 +/- 0.08 microM and V(max) = 9.2 +/- 1.4 micromol min(-1) mg(-)(1). Rat heart FABP (H-FABP) and rat liver FABP were potent inhibitors of 11,12-EET and 14,15-EET conversion to DHET. The resultant inhibition curves were best described by a substrate depletion model, with K(d) = 0.17 +/- 0.01 microM for H-FABP binding to 11,12-EET, suggesting that FABP acts by reducing EET availability to sEH. The EET depletion by FABP was antagonized by the co-addition of arachidonic acid, oleic acid, linoleic acid, or 20-hydroxyeicosatetraenoic acid, presumably due to competitive displacement of FABP-bound EET. Collectively, these findings imply that FABP might potentiate the actions of EETs by limiting their conversion to DHET. However, the effectiveness of this process may depend on metabolic conditions that regulate the levels of competing FABP ligands.     

Reno-protective mechanisms of epoxyeicosatrienoic acids in cardiovascular disease

Cardiovascular disease (CVD) is the leading cause of mortality worldwide, and it is well known that end-stage renal disease (ESRD) is a profound consequence of the progression of CVD. Present treatments only slow CVD progression to ESRD, and it is imperative that new therapeutic strategies are developed to prevent the incidence of ESRD. Because epoxyeicosatrienoic acids (EETs) have been shown to elicit reno-protective effects in hypertensive animal models, the current review will focus on addressing the reno-protective mechanisms of EETs in CVD. The cytochrome P-450 epoxygenase catalyzes the oxidation of arachidonic acid to EETs. EETs have been identified as endothelium-derived hyperpolarizing factors (EDHFs) with vasodilatory, anti-inflammatory, antihypertensive, and antiplatelet aggregation properties. EETs also have profound effects on vascular migration and proliferation and promote angiogenesis. The progression of CVD has been linked to decreased EETs levels, leading to the concept that EETs should be therapeutically targeted to prevent end-organ damage associated with CVD. However, EETs are quickly degraded by the enzyme soluble epoxide hydrolase (sEH) to their less active diols, dihydroxyeicosatrienoic acids (DHETs). As such, one way to increase EETs level is to inhibit their degradation to DHETs by using sEH inhibitors. Inhibition of sEH has been shown to effectively reduce blood pressure and organ damage in experimental models of CVD. Another approach to target EETs is to develop EET analogs with improved solubility and resistance to auto-oxidation and metabolism by sEH. For example, stable ether EET analogs dilate afferent arterioles and lower blood pressure in hypertensive rodent animal models. EET agonists also improve insulin signaling and vascular function in animal models of metabolic syndrome.