Siddam Anjaiah

Characterization of Epoxyeicosatrienoic Acid Binding Site in U937 Membranes Using a Novel Radiolabeled Agonist, 20-125I-14,15-Epoxyeicosa-8(Z)-Enoic Acid

Epoxyeicosatrienoic acids (EETs) are important regulators of vascular tone and homeostasis. Whether they initiate signaling through membrane receptors is unclear. We developed 20-iodo-14,15-epoxyeicosa-8(Z)-enoic acid (20-I-14,15-EE8ZE), a radiolabeled EET agonist, to characterize EET binding to membranes of U937 cells. 20-I-14,15-EE8ZE stimulated cAMP production in U937 cells with similar potency, but it decreased efficacy compared with 11,12-EET. Maximum cAMP production increased 4.2-fold, with an EC50 value of 9 μM. Like 14,15-EET, 20-I-14,15-EE8ZE relaxed bovine coronary arteries, with a similar EC50 value. Both 20-I-14,15-EE8ZE agonist activities were blocked by the EET antagonist 14,15-epoxyeicosa-5(Z)enoic acid (14,15-EE5ZE). Specific 20-125I-14,15-EE8ZE binding to U937 membranes reached equilibrium within 10 min and remained unchanged for 30 min at 4°C. The binding was saturable, reversible, and exhibited KD and Bmax values of 11.8 ± 1.1 nM and 5.8 ± 0.2 pmol/mg protein, respectively. Pretreatment of the membranes with guanosine 5′-O-(3-thio)triphosphate reduced the Bmax in a concentration-related manner. 20-125I-14,15-EE8ZE binding was inhibited by eicosanoids with potency order of 11,12-EET >14,15-EE5ZE ≈ 14,15-EET » 15-hydroxyeicosatetraenoic acid > 14,15-EET-thiirane >14,15-dihydroxyeicosatrienoic acid. This order is in agreement with the efficacy and potency of cAMP production. In summary, 20-125I-14,15-EE8ZE is a radiolabeled EET agonist that is useful to study binding and metabolism. Using this radioligand, we have identified a specific high-affinity and high-abundance EET binding site in U937 cell membranes. This binding site could represent a specific EET receptor, which is probably a G protein-coupled receptor.

20-HETE increases superoxide production and activates NAPDH oxidase in pulmonary artery endothelial cells.

Reactive oxygen species (ROS) signal vital physiological processes including cell growth, angiogenesis, contraction, and relaxation of vascular smooth muscle. Because cytochrome P-450 family 4 (CYP4)/20-hydroxyeicosatetraenoic acid (20-HETE) has been reported to enhance angiogenesis, pulmonary vascular tone, and endothelial nitric oxide synthase function, we explored the potential of this system to stimulate bovine pulmonary artery endothelial cell (BPAEC) ROS production. Our data are the first to demonstrate that 20-HETE increases ROS in BPAECs in a time- and concentration-dependent manner as detected by enhanced fluorescence of oxidation products of dihydroethidium (DHE) and dichlorofluorescein diacetate. An analog of 20-HETE elicits no increase in ROS and blocks 20-HETE-evoked increments in DHE fluorescence, supporting its function as an antagonist. Endothelial cells derived from bovine aortas exhibit enhanced ROS production to 20-HETE quantitatively similar to that of BPAECs. 20-HETE-induced ROS production in BPAECs is blunted by pretreatment with polyethylene-glycolated SOD, apocynin, inhibition of Rac1, and a peptide-based inhibitor of NADPH oxidase subunit p47(phox) association with gp91. These data support 20-HETE-stimulated, NADPH oxidase-derived, and Rac1/2-dependent ROS production in BPAECs. 20-HETE promotes translocation of p47(phox) and tyrosine phosphorylation of p47(phox) in a time-dependent manner as well as increased activated Rac1/2, providing at least three mechanisms through which 20-HETE activates NADPH oxidase. These observations suggest that 20-HETE stimulates ROS production in BPAECs at least in part through activation of NADPH oxidase within minutes of application of the lipid.

Development of Epoxyeicosatrienoic Acid Analogs with in Vivo Anti-Hypertensive Actions

Epoxyeicosatrienoic acids (EETs) contribute importantly to the regulation of vascular tone and blood pressure control. The purpose of this study was to develop stable EET analogs and test their in vivo blood pressure lowering effects in hypertensive rats. Using the pharmacophoric moiety of EETs, ether EET analogs were designed with improved solubility and resistance to auto-oxidation and metabolism by soluble epoxide hydrolase. Ether EET analogs were chosen based on their ability to dilate afferent arterioles and subsequently tested for blood pressure lowering effects in rodent models of hypertension. Initially, 11,12-ether-EET-8-ZE failed to lower blood pressure in angiotensin hypertension or spontaneously hypertensive rats (SHR). Esterification of the carboxylic group of 11,12-ether-EET-8-ZE prevented blood pressure increase in SHR when injected at 2 mg/day for 12 days (MAP Δ change at day 8 of injection was −0.3 ± 2 for treated and 12 ± 1 mmHg for control SHR). Amidation of the carboxylic group with aspartic acid produced another EET analog (NUDSA) with a blood pressure lowering effect when injected at 3 mg/day in SHR for 5 days. Amidation of the carboxylic group with lysine amino acid produced another analog with minimal blood pressure lowering effect. These data suggest that esterification of the carboxylic group of 11,12-ether-EET-8-ZE produced the most effective ether-EET analog in lowering blood pressure in SHR and provide the first evidence to support the use of EET analogs in treatment of cardiovascular diseases.

A 20-hydroxyeicosatetraenoic acid agonist, N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine, opposes the fall in blood pressure and vascular reactivity in endotoxin-treated rats.

Endotoxic shock is a systemic inflammatory response that is associated with an increase in nitric oxide production and a decrease in the formation of 20-hydroxyeicosatetraenoic acid (20-HETE), which may contribute to the fall in blood pressure and vascular reactivity. The present study examined the effects of a synthetic analogue of 20-HETE, N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine (5,14-HEDGE), on the fall in blood pressure and vascular responsiveness to vasoscontrictors and acetylcholine in rats treated with endotoxin. The MAP fell by 31 mmHg, and the heart rate rose by 90 beats/min in male Wistar rats treated with endotoxin (10 mg/kg, intraperitoneally). The fall in MAP was associated with a decrease in the vasoconstrictor response to norepinephrine in isolated aorta and superior mesenteric artery and increased levels of nitrite in the serum, kidney, heart, and vascular tissues. The effects of endotoxin were prevented by 5,14-HEDGE (30 mg/kg, s.c.) given 1 h after injection of endotoxin. Furthermore, a competitive antagonist of vasoconstrictor effects of 20-HETE, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (30 mg/kg, s.c.), prevented the beneficial effects of 5,14-HEDGE on MAP and vascular tone in rats treated with endotoxin. These data are consistent with the view that a fall in the production of 20-HETE contributes to the fall in MAP and vascular reactivity in rats treated with endotoxin, and that 5,14-HEDGE has a beneficial effect.

Mitogenic Activity and Signaling Mechanism of 2-(14,15-Epoxyeicosatrienoyl)Glycerol, a Novel Cytochrome P450 Arachidonate Metabolite

ABSTRACT Arachidonic acid is an essential constituent of cell membranes that is esterified to the sn-2 position of glycerophospholipids and is released from selected phospholipid pools by tightly regulated phospholipase cleavage. Metabolism of the released arachidonic acid by the cytochrome P450 enzyme system (cP450) generates biologically active compounds, including epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids. Here we report that 2-(14,15-epoxyeicosatrienoyl)glycerol (2-14,15-EG), a novel cP450 arachidonate metabolite produced in the kidney, is a potent mitogen for renal proximal tubule cells. This effect is mediated by activation of tumor necrosis factor alpha-converting enzyme (ADAM17), which cleaves membrane-bound transforming growth factor α (proTGF-α) and releases soluble TGF-α as a ligand that binds and activates epidermal growth factor receptor (EGFR). The present studies additionally demonstrate that the structurally related 14,15-EET stimulates release of soluble heparin-binding EGF-like growth factor as an EGFR ligand by activation of ADAM9, another member of the ADAM family. Thus, in addition to the characterization of 2-14,15-EGs mitogenic activity and signaling mechanism, our study provides the first example that two structurally related biologically active lipid mediators can activate different metalloproteinases and release different EGFR ligands in the same cell type to activate EGFR and stimulate cell proliferation.

11(R),12(S),15(S)-trihydroxyeicosa-5(Z),8(Z),13(E)-trienoic acid: an endothelium-derived 15-lipoxygenase metabolite that relaxes rabbit aorta

Previous studies indicate that 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA), an endothelium-derived hyperpolarizing factor in the rabbit aorta, mediates a portion of the relaxation response to acetylcholine by sequential metabolism of arachidonic acid by 15-lipoxygenase, hydroperoxide isomerase, and epoxide hydrolase. To determine the stereochemical configuration of the endothelial 11,12,15-THETA, its activity and chromatographic migration were compared with activity and migration of eight chemically synthesized stereoisomers of 11,12,15(S)-THETA. Of the eight isomers, only 11(R),12(S),15(S)-trihydroxyeicosa-5(Z),8(Z),13(E)-trienoic acid comigrated with the biological 11,12,15-THETA on reverse- and normal-phase HPLC and gas chromatography. The same THETA isomer (10(-7)-10(-4) M) relaxed the rabbit aorta in a concentration-related manner (maximum relaxation = 69 +/- 5%). These relaxations were blocked by apamin (10(-7) M), an inhibitor of small-conductance Ca2+-activated K+ channels. In comparison, 11(S),12(R),15(S),5(Z),8(Z),13(E)-THETA (10(-4) M) relaxed the aorta by 22%. The other six stereoisomers were inactive in this assay. With use of the whole cell patch-clamp technique, it was shown that 10(-4) M 11(R),12(S),15(S),5(Z),8(Z),13(E)-THETA increased outward K+ current in isolated aortic smooth muscle cells by 119 +/- 36% at +60 mV, whereas 10(-4) M 11(R),12(R),15(S),5(Z),8(Z),13(E)-THETA increased outward K+ current by only 20 +/- 2%. The 11(R),12(S),15(S),5(Z),8(Z),13(E)-THETA-stimulated increase in K+ current was blocked by pretreatment with apamin. These studies suggest that 11(R),12(S),15(S)-trihydroxyeicosa-5(Z),8(Z),13(E)-trienoic acid is the active stereoisomer produced by the rabbit aorta. It relaxes smooth muscle by activating K+ channels. The specific structural and stereochemical requirements for K+ channel activation suggest that a specific binding site or receptor of 11,12,15-THETA is involved in these actions.

Identification of 15-hydroxy-11,12-epoxyeicosatrienoic acid as a vasoactive 15-lipoxygenase metabolite in rabbit aorta.

Arachidonic acid (AA) causes endothelium-dependent smooth muscle hyperpolarizations and relaxations that are mediated by a 15-lipoxygenase-I (15-LO-I) metabolite, 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA). We propose that AA is metabolized sequentially by 15-LO-I and hydroperoxide isomerase to an unidentified hydroxyepoxyeicosatrienoic acid (HEETA), which is hydrolyzed by a soluble epoxide hydrolase (sEH) to 11,12,15-THETA. After incubation of aorta with 14C-labeled AA, metabolites were extracted and the HEETAs were resolved by performing HPLC. Mass spectrometric analyses identified 15-Hydroxy-11,12-epoxyeicosatrienoic acid (15-H-11,12-EETA). Incubation of aortic incubates with methanol and acetic acid trapped the acid-sensitive 15-H-11,12-EETA as methoxydihydroxyeicosatrienoic acids (MDHEs) (367 m/z, M-H). Pretreatment of the aortic tissue with the sEH inhibitor 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA; 10(-6) M) increased the formation of 15-H-11,12-EETA, measured as MDHEs. Thus 15-H-11,12-EETA is an acid- and sEH-sensitive precursor of 11,12,15-THETA. Aortic homogenates and endothelial cells contain a 57-kDa protein corresponding to the rabbit sEH. In preconstricted aortic rings, AA (10(-7)-10(-4) M) and acetylcholine (10(-9)-10(-6) M) caused concentration-related relaxations that were enhanced by pretreatment with AUDA. These enhanced relaxations were inhibited by increasing extracellular [K(+)] from 4.8 to 20 mM. AA (3 x 10(-6) M) induced cell membrane hyperpolarization (from -31.0 +/- 1 to -46.8 +/- 2 mV) in aortic strips with an intact endothelium, which was enhanced by AUDA. These results indicate that 15-H-11,12-EETA is produced by the aorta, hydrolyzed by sEH to 11,12,15-THETA, and mediates relaxations by membrane hyperpolarization. 15-H-11,12-EETA represents an endothelium-derived hyperpolarizing factor.

Modulation by Cytochrome P450-4A ω-Hydroxylase Enzymes of Adrenergic Vasoconstriction and Response to Reduced PO2 in Mesenteric Resistance Arteries of Dahl Salt-Sensitive Rats

Please cite this paper as: Raffai, Wang, Roman, Anjaiah, Weinberg, Falck and Lombard (2010). Modulation by Cytochrome P450‐4A ω‐Hydroxylase Enzymes of Adrenergic Vasoconstriction and Response to Reduced PO2 in Mesenteric Resistance Arteries of Dahl Salt‐Sensitive Rats. Microcirculation17(7), 525–535.