Mairead A. Carroll

Vasoactivity of arachidonic acid epoxides

Arachidonic acid (AA) can be metabolized to epoxides and their corresponding diols via the cytochrome P450 epoxygenase pathway. We have compared the vascular activity of four synthetically prepared epoxyeicosatrienoic acids, i.e. 5,6-, 8,9-, 11,12- and 14,15-EET (2-20 microM) on the isolated perfused rat tail artery. The 5,6-EET was equipotent with acetylcholine in dose dependently reducing vascular resistance (ED50 = 3.4 +/- 0.5 microM). The 8,9-, 11,12- and 14,15-EETs of AA did not affect vascular resistance; neither did the 5,6-DHET and delta-lactone, hydrolysis products of 5,6-epoxide. We suggest that the 5,6-epoxide, in contrast to other cytochrome P450-derived products, contributes to the regulation of regional vascular tone.

Epoxyeicosatrienoic Acids Are Released to Mediate Shear Stress–Dependent Hyperpolarization of Arteriolar Smooth Muscle

We hypothesized that shear stress stimulates the release of epoxyeicosatrienoic acids (EETs) from arteriolar endothelium, which directly hyperpolarize smooth muscle. To test this hypothesis, a perfusion system, consisting of two separate, serially connected chambers (A and B), was used. A donor vessel, isolated from gracilis muscle of female NO-deficient mice and rats, was cannulated in chamber A. In chamber B, an endothelium-denuded detector vessel isolated from mesentery of these animals was cannulated. In the presence of indomethacin, 5, 10, and 20 dyne/cm2 shear stress elicited dilation of donor vessels, followed by dilation of detector vessels. Changes in membrane potential of the detector vessel smooth muscle cells in response to the perfusate from 5 and 10 dyne/cm2 shear stress–stimulated donor vessels was also recorded (by ≈−12 to −15 and −20 to −30 mV, respectively). Exposing detector vessels to 30 mmol/L KCl or pretreating them with iberiotoxin abolished their hyperpolarization and dilation to the flow of perfusate. Pretreatment of donor vessels with PPOH, an inhibitor of cytochrome P-450/epoxygenase, eliminated dilator responses in both donor and detector vessels, as well as the hyperpolarization of detector vessels. GC-MS analysis showed increasing release of EETs into the perfusate collected from 1, 5, and 10 dyne/cm2 shear stress–stimulated arterioles, which was abolished by PPOH. Thus, EETs, released from endothelial cells of donor vessels stimulated with shear stress, hyperpolarize smooth muscle of downstream detector vessels, confirming their identity as endothelium-derived hyperpolarizing factors and suggesting that gap junctional communication may not be necessary for shear stress–stimulated EDHF-mediated vasodilation.

Epoxyeicosatrienoic acids mediate adenosine‐induced vasodilation in rat preglomerular microvessels (PGMV) via A2A receptors

Activation of rat adenosine 2A receptors (A2A R) dilates preglomerular microvessels (PGMV), an effect mediated by epoxyeicosatrienoic acids (EETs). Incubation of PGMV with a selective A2A R agonist, 2‐p‐(2‐carboxyethyl) phenethylamino‐5′‐N‐ethylcarboxamidoadenosine (CGS 21680; 100 μM), increased isolated PGMV EET levels to 7.57±1.53 ng mg−1 protein from 1.06±0.22 ng mg−1 protein in controls (P<0.05), without affecting hydroxyeicosatetraenoic acid (HETE) levels (10.8±0.69 vs 11.02±0.74 ng mg−1 protein). CGS 21680‐stimulated EETs was abolished by preincubation with an A2A R antagonist, 4‐(2‐[7‐amino‐2‐(2‐furyl)[1,2,4]triazolo[2,3‐a][1,3,5]triazin‐5‐ylamino]ethyl)phenol (ZM241385) (100 μM). A selective epoxygenase inhibitor, methylsulfonyl‐propargyloxyphenylhexanamide (MS‐PPOH; 12 μM) prevented CGS 21680‐induced increase in EETs, indicating inhibition of de novo synthesis of EETs. In pressurized (80 mmHg) renal arcuate arteries (110–130 μm) preconstricted with phenylephrine (20 nM), superfusion with CGS 21680 (0.01–10 μM) increased the internal diameter (i.d.) concentration‐dependently; vasodilation was independent of nitric oxide and cyclooxygenase activity. CGS 21680 (10 μM) increased i.d. by 32±6 μm; vasodilation was prevented by inhibition of EET synthesis with MS‐PPOH. Addition of 3 nM 5,6‐EET, 8,9‐EET and 11,12‐EET increased i.d. by 53±9, 17±4 and 53±5 μm, respectively, whereas 14,15‐EET was inactive. The responses to 5,6‐EET were, however, significantly inhibited by indomethacin. We conclude that 11,12‐EET is the likely mediator of A2A R‐induced dilation of rat PGMV. Activation of A2A R coupled to de novo EET stimulation may represent an important mechanism in regulating preglomerular microvascular tone.

5,6-epoxyeicosatrienoic acid, a novel arachidonate metabolite. Mechanism of vasoactivity in the rat.

We have reported that 5,6-epoxyeicosatrienoic acid (5,6-EET) was the only cytochrome P-450-dependent arachidonic acid (AA) epoxide to dilate the isolated, perfused caudal artery of the rat. We have investigated the mechanisms by which 5,6-EET dilates the rat-tail artery by studying the effect of deendothelialization and inhibition of AA metabolic pathways (cyclooxygenase, lipoxygenase, and cytochrome P-450 monooxygenase) on the vascular action of the epoxide. Rat isolated caudal arteries were perfused with Krebs-Henseleit solution at 37 degrees C, pH 7.4, and gassed with 95% O2-5% CO2. Arterial tone was elevated with phenylephrine; acetylcholine (0.5 nmol) was used to detect the presence of intact, functional endothelium. Doses of 5,6-EET, from 6.25 to 25.0 nmol, were injected close-arterially. After obtaining control responses, the same doses were randomly retested after deendothelialization or in the presence of inhibitors of AA metabolism. Removal of the endothelium decreased by 70% the vasodilator responses to 5,6-EET. The endothelial dependency was a function of the epoxide interacting with cyclooxygenase of the endothelium, because indomethacin (3 microM) and aspirin (50 microM) prevented the vasodilator response to 5,6-EET while not affecting the response to acetylcholine. SKF-525A (1.1 microM) and metyrapone (150 microM) did not affect the responses to the 5,6-EET, whereas clotrimazole (0.7 microM) and nordihydroguaiaretic acid (2.5 microM) had nonspecific effects, decreasing responses to 5,6-EET and acetylcholine. Because 5,6-EET failed to stimulate detectable release of prostanoids into the effluent from the caudal artery, we conclude that 5,6-EET requires conversion by cyclooxygenase for expression of its vasoactivity.

Soluble epoxide hydrolase inhibitor, AUDA, prevents early salt-sensitive hypertension.

In stroke-prone spontaneously hypertensive rats (SHRSP) end-organ damage is markedly accelerated by high-salt (HS) intake. Since epoxyeicosatrienoic acids (EETs) possess vasodepressor and natriuretic activities, we examined whether a soluble epoxide hydrolase (sEH) inhibitor, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), to inhibit the metabolism of EETs, would protect against pathologic changes in SHRSP. Seven-week-old male SHRSP were treated as follows: normal salt (NS), NS + AUDA, HS and HS + AUDA. Systolic blood pressure (SBP) (205 +/- 4 v 187 +/- 7 mmHg) and proteinuria (3.7 +/- 0.2 v 2.6 +/- 0.2 mg/6 h), but not plasma EETs (11.0 +/- 0.9 v 9.7 +/- 1.1 ng/ml), were significantly increased at 9 weeks of age in HS v NS SHRSP. HS was associated with fibrinoid degeneration and hypertrophy of arterioles in the kidney and perivascular fibrosis and contraction band necrosis in the heart. AUDA ameliorated these early salt-dependent changes in saline-drinking SHRSP and increased plasma levels of EETs but did not affect water and electrolyte excretion. sEH inhibition may provide a therapeutic strategy for treating salt-sensitive hypertension and its sequelae.

Estrogen elicits cytochrome P450--mediated flow-induced dilation of arterioles in NO deficiency: role of PI3K-Akt phosphorylation in genomic regulation.

Abstract— This study investigated the mechanisms responsible for the estrogen-dependent, cytochrome P450 (CYP)-mediated dilator responses to shear stress in arterioles of NO-deficient female rats and mice. Flow-induced dilation (FID) was assessed in isolated arterioles from NG-nitro-l-arginine methyl ester (L-NAME)-treated male and ovariectomized female rats before and after overnight incubation with 17&bgr;-estradiol (17&bgr;-E2, 10−9 mol/L). In control conditions, prostaglandins (PGs) mediated FID, because indomethacin (INDO) abolished the responses. After incubation of the vessels with 17&bgr;-E2, the basal tone of arterioles was significantly reduced and FID was augmented. INDO did not affect the dilation of the vessels incubated with 17&bgr;-E2. Dilations of these vessels, however, were eliminated by PPOH and miconazole, inhibitors of CYP/epoxygenase. Simultaneous incubation of the vessels with 17&bgr;-E2 plus ICI, 182,780, an estrogen receptor antagonist, or wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K) phosphorylation or the transcriptional inhibitor DRB, prevented the reduced arteriolar tone and the enhanced CYP-mediated FID caused by incubation of vessels with17&bgr;-E2. Western blot analysis indicated a significantly increased phospho-Akt level in arterioles incubated with 17&bgr;-E2 compared with those without 17&bgr;-E2. The enhanced phospho-Akt in response to 17&bgr;-E2 was localized, by immunohistochemistry, to arteriolar endothelial cells. Moreover, GC-MS analysis indicated a significantly increased production of epoxyeicosatrienoic acids, vasodilator metabolites of CYP/epoxygenase, in arterioles incubated with 17&bgr;-E2, a response that was prevented by ICI 182780 and wortmannin, respectively. Thus, estrogen, via a receptor-dependent, PI3K/Akt-mediated pathway, transcriptionally upregulates CYP activity, leading to an enhanced arteriolar response to shear stress.

5,6-Epoxyeicosatrienoic Acid Mediates the Enhanced Renal Vasodilation to Arachidonic Acid in the SHR

Abstract—We have shown a cytochrome P450–dependent renal vasodilator effect of arachidonic acid in response to inhibition of cyclooxygenase and elevation of perfusion pressure, which was enhanced in the spontaneously hypertensive rat (SHR) and linked to increased production of and/or responsiveness to epoxyeicosatrienoic acids (EETs). In the SHR, vasodilation elicited by low doses of arachidonic acid was attenuated by the nitric oxide synthase inhibitor Nw-nitro-l-arginine (50 &mgr;mol/L), whereas the responses to high doses were unaffected. Inhibition of epoxygenases with miconazole (0.3 &mgr;mol/L) in the presence of Nw-nitro-l-arginine greatly reduced the renal vasodilator response to all doses of arachidonic acid. Tetraethylammonium (10 mmol/L), a nonselective K+ channel blocker, abolished the nitric oxide–independent renal vasodilator effect of arachidonic acid as well as the vasodilator effect of 5,6-EET, confirming that EET-dependent vasodilation involves activation of K+ channels. Under conditions of elevated perfusion pressure (200 mm Hg) and cyclooxygenase inhibition, 5,6-EET, 8, 9-EET, and 11,12-EET caused renal vasodilatation in both SHR and Wistar-Kyoto rats (WKY), whereas 14,15-EET produced vasoconstriction. 5,6-EET was the most potent renal vasodilator of the EET regioisomers in the SHR by a factor of 4 or more. In the SHR, 5,6-EET- and 11,12-EET–induced renal vasodilatation was >2-fold greater than that registered in WKY. Thus, the augmented vasodilator responses to arachidonic acid in the SHR is through activation of K+ channels, and 5,6-EET is the most likely mediator.

A new class of lipid mediators: cytochrome P450 arachidonate metabolites.

The first reports on the metabolism of arachidonic acid (AA) by cytochrome P450 (CYP) mono-oxygenases appeared in 1981.1 2 The biochemical studies of Capdevila and colleagues have provided the “ground substance” for all future studies. Our initial report in 1984 on the metabolism of AA via CYP pathways by the rabbit medullary thick ascending limb (mTAL) provided evidence that AA metabolism in this segment of the nephron was primarily via the CYP pathway.3 Previously the mTAL was thought to lack the biosynthetic machinery for metabolising AA because of negative immunocytochemical evidence regarding cyclo-oxygenase (COX) capabilities. A subsequent study identified two principal CYP derived AA products generated by the rabbit mTAL, one inhibiting Na+-K+-ATPase and the other relaxing blood vessels.4 Two principal CYP products—20-hydroxyeicosatetraenoic acid (20-HETE) generated by ω/ω-1 hydroxylases and 11,12-epoxyeicosatrienoic acid (11,12-EET) generated by epoxygenases—had been identified in a study of renal zonal CYP derived AA metabolites (fig 1).5 No one then could have anticipated the rise of 20-HETE to its position of pre-eminence among renal eicosanoids, one that functions as a key component in both tubular and vascular mechanisms essential to the regulation of renal haemodynamics and extracellular fluid volume. The first study that pointed to an essential role for 20-HETE in the kidney indicated that it modulated the Na+-K+-2Cl– cotransporter in the mTAL, the target for furosemide (frusemide) and the other “high ceiling” diuretics.6 Our companion study had identified 20-HETE as the principal product of AA metabolism in mTAL.7 Figure 1 Arachidonic acid (AA) metabolism by cytochrome P450 dependent mono-oxygenases to ω- and ω-1-hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatrienoic acids (epoxides, EETs), and dihydroxyeicosatrienoic acids (diols, DHTs). 20-HETE and 5,6-EET can be converted by cyclo-oxygenase to analogues of prostaglandins. 20-HETE is the pre-eminent renal eicosanoid. …

Epoxyeicosatrienoic acid-mediated renal vasodilation to arachidonic acid is enhanced in SHR.

We tested the hypothesis that cyclooxygenase-independent vasodilation produced by arachidonic acid (AA) is mediated by epoxyeicosatrienoic acids (EETs) and is blunted in the spontaneously hypertensive rat (SHR). At normal perfusion pressure (PP; 70 to 90 mm Hg), AA constricted the renal vasculature in both SHR and normotensive Wistar-Kyoto rats, an effect abolished by cyclooxygenase inhibition, and converted to vasodilation when PP was raised to ≈200 mm Hg. Unexpectedly, renal vasodilation elicited by AA was greater in the SHR at high PP; for example, 2.5, 5, and 10 &mgr;g of AA produced PP declines of 54±9, 92±10, and 112±5 mm Hg, respectively, in SHR compared with 26±3, 45±5, and 77±6 mm Hg in Wistar-Kyoto rats (P <0.01). However, the renal vasodilator responses to acetylcholine (0.1 &mgr;g) and sodium nitroprusside (1 &mgr;g) did not differ between strains, indicating that vascular responsiveness to AA was independent of intrinsic changes in vascular smooth muscle. Hyperresponsiveness of the renal vasculature to AA may be unique for the SHR, because it did not occur in Sprague-Dawley rats with angiotensin II–induced hypertension. 5,8,11,14-Eicosatetraynoic acid (ETYA; 4 &mgr;mol/L), an inhibitor of all AA pathways, attenuated the vasodilator responses to AA, as did treatment with stannous chloride, which depletes cytochrome P450 enzymes, suggesting that a cytochrome P450 AA metabolite mediated the renal vasodilation. N-Methylsulfonyl-12,12-dibromododec-11-en-amide (DDMS; 2 &mgr;mol/L), a selective &ohgr;-hydroxylase inhibitor, did not affect AA-induced vasodilation, whereas selective inhibition of epoxygenases with either miconazole (0.3 &mgr;mol/L) or N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH; 12 &mgr;mol/L) did, indicating that one or more EETs were involved in the renal vasodilator action of AA at high PP. This conclusion was supported by the demonstration that AA greatly enhanced the renal efflux of EETs at high PP but not at basal PP.

Increases in plasma trans-EETs and blood pressure reduction in spontaneously hypertensive rats.

Epoxyeicosatrienoic acids (EETs) are vasodilator, natriuretic, and antiinflammatory lipid mediators. Both cis- and trans-EETs are stored in phospholipids and in red blood cells (RBCs) in the circulation; the maximal velocity (V(max)) of trans-EET hydrolysis by soluble epoxide hydrolase (sEH) is threefold that of cis-EETs. Because RBCs of the spontaneously hypertensive rat (SHR) exhibit increased sEH activity, a deficiency of trans-EETs in the SHR was hypothesized to increase blood pressure (BP). This prediction was fulfilled, since sEH inhibition with cis-4-[4-(3-adamantan-1-ylureido)cyclohexyloxy]benzoic acid (AUCB; 2 mg·kg(-1)·day(-1) for 7 days) in the SHR reduced mean BP from 176 ± 8 to 153 ± 5 mmHg (P < 0.05), whereas BP in the control Wistar-Kyoto rat (WKY) was unaffected. Plasma levels of EETs in the SHR were lower than in the age-matched control WKY (16.4 ± 1.6 vs. 26.1 ± 1.8 ng/ml; P < 0.05). The decrease in BP in the SHR treated with AUCB was associated with an increase in plasma EETs, which was mostly accounted for by increasing trans-EET from 4.1 ± 0.2 to 7.9 ± 1.5 ng/ml (P < 0.05). Consistent with the effect of increased plasma trans-EETs and reduced BP in the SHR, the 14,15-trans-EET was more potent (ED(50) 10(-10) M; maximum dilation 59 ± 15 μm) than the cis-isomer (ED(50) 10(-9) M; maximum dilation 30 ± 11 μm) in relaxing rat preconstricted arcuate arteries. The 11,12-EET cis- and trans-isomers were equipotent dilators as were the 8,9-EET isomers. In summary, inhibition of sEH resulted in a twofold increase in plasma trans-EETs and reduced mean BP in the SHR. The greater vasodilator potency of trans- vs. cis-EETs may contribute to the antihypertensive effects of sEH inhibitors.