Metabolism of adrenic acid to vasodilatory 1α,1β-dihomo-epoxyeicosatrienoic acids by bovine coronary arteries

2007 ◽  
Vol 292 (5) ◽  
pp. H2265-H2274 ◽  
Author(s):  
Xiu-Yu Yi ◽  
Kathryn M. Gauthier ◽  
Lijie Cui ◽  
Kasem Nithipatikom ◽  
John R. Falck ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Smooth Muscle ◽  
Coronary Arteries ◽  
Carbon Chain ◽  
Chain Elongation ◽  
Epoxyeicosatrienoic Acids ◽  
Spectrometric Analysis ◽  
Adrenic Acid ◽  
Acid Metabolites ◽  
Cytochrome P 450

Adrenic acid (docosatetraenoic acid), an abundant fatty acid in the vasculature, is produced by a two-carbon chain elongation of arachidonic acid. Despite its abundance and similarity to arachidonic acid, little is known about its role in the regulation of vascular tone. Gas chromatography/mass spectrometric analysis of bovine coronary artery and endothelial cell lysates revealed arachidonic acid concentrations of 2.06 ± 0.01 and 6.18 ± 0.60 μg/mg protein and adrenic acid concentrations of 0.29 ± 0.01 and 1.56 ± 0.16 μg/mg protein, respectively. In bovine coronary arterial rings preconstricted with the thromboxane mimetic U-46619, adrenic acid (10−9–10−5 M) induced concentration-related relaxations (maximal relaxation = 83 ± 4%) that were similar to arachidonic acid relaxations. Adrenic acid relaxations were blocked by endothelium removal and the K+ channel inhibitor, iberiotoxin (100 nM), and inhibited by the cyclooxygenase inhibitor, indomethacin (10 μM, maximal relaxation = 53 ± 4%), and the cytochrome P-450 inhibitor, miconazole (10 μM, maximal relaxation = 52 ± 5%). Reverse-phase HPLC and liquid chromatography/mass spectrometry isolated and identified numerous adrenic acid metabolites from coronary arteries including dihomo (DH)-epoxyeicosatrienoic acids (EETs) and DH-prostaglandins. DH-EET [16,17-, 13,14-, 10,11-, and 7,8- (10−9–10−5 M)] induced similar concentration-related relaxations (maximal relaxations averaged 83 ± 3%). Adrenic acid (10−6 M) and DH-16,17-EET (10−6 M) hyperpolarized coronary arterial smooth muscle. DH-16,17-EET (10−8–10−6 M) activated iberiotoxin-sensitive, whole cell K+ currents of isolated smooth muscle cells. Thus, in bovine coronary arteries, adrenic acid causes endothelium-dependent relaxations that are mediated by cyclooxygenase and cytochrome P-450 metabolites. The adrenic acid metabolite, DH-16,17-EET, activates smooth muscle K+ channels to cause hyperpolarization and relaxation. Our results suggest a role of adrenic acid metabolites, specifically, DH-EETs as endothelium-derived hyperpolarizing factors in the coronary circulation.

scholarly journals EDHF function in the ductus arteriosus: evidence against involvement of epoxyeicosatrienoic acids and 12S-hydroxyeicosatetraenoic acid

2009 ◽  
Vol 297 (6) ◽  
pp. H2161-H2168 ◽  
Author(s):  
Barbara Baragatti ◽  
Michal Laniado Schwartzman ◽  
Debora Angeloni ◽  
Francesca Scebba ◽  
Enrica Ciofini ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Carbon Monoxide ◽  
Arachidonic Acid ◽  
Ductus Arteriosus ◽  
Epoxyeicosatrienoic Acids ◽  
Spectrometric Analysis ◽  
Hydroxyeicosatetraenoic Acid ◽  
Acid Product ◽  
Acid Metabolites ◽  
Cytochrome P 450

We have previously shown (Ref. 2 ) that endothelium-derived hyperpolarizing factor (EDHF) becomes functional in the fetal ductus arteriosus on removal of nitric oxide and carbon monoxide. From this, it was proposed that EDHF originates from a cytochrome P-450 (CYP450)-catalyzed reaction being inhibited by the two agents. Here, we have examined in the mouse ductus whether EDHF can be identified as an arachidonic acid product of a CYP450 epoxygenase and allied pathways. We did not detect transcripts of the mouse CYP2C subfamily in vessel, while CYP2J subfamily transcripts were expressed with CYP2J6 and CYP2J9. These CYP2J hemoproteins were also detected in the ductus by immunofluorescence microscopy, being colocalized with the endoplasmic reticulum in both endothelial and muscle cells. Distinct CYP450 transcripts were also detected and were responsible for ω-hydroxylation (CYP4A31) and 12R-hydroxylation (CYP4B1). Mass spectrometric analysis showed formation of epoxyeicosatrienoic acids (EETs) in the intact ductus, with 11,12- and 14,15-EETs being more prominent than 5,6- and 8,9-EETs. However, their yield did not increase with nitric oxide/carbon monoxide suppression, nor did it abate with endothelium removal. No evidence was obtained for formation of 12R-hydroxyeicosatrienoic acid and ω-hydroxylation products. 2S-hydroxyeicosatetraenoic acid was instead detected, and, contrary to data implicating this compound as an alternative EDHF, its suppression with baicalein did not modify the EDHF-mediated relaxation to bradykinin. We conclude that none of the more common CYP450-linked arachidonic acid metabolites appears to qualify as EDHF in mouse ductus. We speculate that some novel eicosanoid or a totally unrelated compound requiring CYP450 for its synthesis accounts for EDHF in this vessel.

Role of PGI2 and epoxyeicosatrienoic acids in relaxation of bovine coronary arteries to arachidonic acid

1993 ◽  
Vol 264 (2) ◽  
pp. H327-H335 ◽  
Author(s):  
M. Rosolowsky ◽  
W. B. Campbell
Keyword(s):  
Arachidonic Acid ◽  
Coronary Arteries ◽  
Vascular Tone ◽  
Epoxyeicosatrienoic Acids ◽  
Lipoxygenase Inhibitor ◽  
Physiological Processes ◽  
Coronary Vascular Tone ◽  
Cytochrome P 450 ◽  
Endothelium Dependent Relaxation

Metabolites of arachidonic acid regulate several physiological processes, including vascular tone. The purpose of this study was to determine which metabolites of arachidonic acid are produced by bovine coronary arteries and which may regulate coronary vascular tone. Arachidonic acid induced a concentration-related, endothelium-dependent relaxation [one-half maximum effective concentration (EC50) of 2 x 10(-7) M and a maximal relaxation of 91 +/- 2% at 10(-5) M] of bovine coronary arteries that were contracted with U-46619, a thromboxane mimetic. The concentration of 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha), a metabolite of prostaglandin I2 (PGI2), increased from 82 +/- 6 to 328 +/- 24 pg/ml with arachidonic acid (10(-5) M). Treatment with the cyclooxygenase inhibitor indomethacin attenuated arachidonic acid-induced relaxations by approximately 50% and blocked the synthesis of 6-keto-PGF1 alpha. PGI2 caused a concentration-related relaxation (EC50 of 10(-8) M and a maximal relaxation of 125 +/- 11% at 10(-7) M). BW755C, a cyclooxygenase and lipoxygenase inhibitor, inhibited arachidonic acid-induced relaxation to the same extent as indomethacin. When vessels were treated with both indomethacin and BW755C, the inhibition of relaxation was the same as either inhibitor alone. SKF 525a, a cytochrome P-450 inhibitor, reduced arachidonic acid-induced relaxation by approximately 50%. When SKF 525a was given in combination with indomethacin, the relaxation by arachidonic acid was almost completely inhibited. SKF 525a inhibited the synthesis of epoxyeicosatrienoic acids (EETs).(ABSTRACT TRUNCATED AT 250 WORDS)

Pulmonary cytochrome P-450 2J4 is reduced in a rat model of acute Pseudomonas pneumonia

2003 ◽  
Vol 285 (5) ◽  
pp. L1099-L1105 ◽  
Author(s):  
Asma Yaghi ◽  
J. Alyce Bradbury ◽  
Darryl C. Zeldin ◽  
Sanjay Mehta ◽  
John R. Bend ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Smooth Muscle ◽  
Vascular Tone ◽  
Potential Role ◽  
Pulmonary Arteries ◽  
Epoxyeicosatrienoic Acids ◽  
Rat Lung ◽  
Metabolic Products ◽  
And Control ◽  
Cytochrome P 450

We previously reported that the levels of epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) are depressed in microsomes prepared from lungs of rats with acute Pseudomonas pneumonia. We also showed a potential role for cytochrome P-450 (CYP) metabolites of arachidonic acid (AA) in contractile responses of both normal pulmonary arteries and pulmonary arteries from rats with pneumonia. The CYP2J subfamily enzymes (endogenous source of EETs and HETEs) are constitutively expressed in human and rat lungs where they are localized in vascular smooth muscle and endothelium. The purpose of this study was to determine if CYP2J proteins are modified in pneumonia. Pseudomonas organisms were injected via a tracheostomy in the lungs of rats. Later (44 h), lungs were frozen, and microsomes were prepared from pneumonia and control rat lung homogenates. Lung microsomal proteins were then immunoblotted with anti-CYP2B1/2B2, anti-CYP4A, anti-CYP2J9pep2 (which reacts with rat CYP2J3), anti-CYP2J6pep1 (which reacts with rat CYP2J4), anti-CYP2J2pep4, or anti-CYP2J2pep3 (both of which react with all known CYP2J isozymes). Western blotting revealed a prominent 55-kDa band with anti-CYP2J2pep3, anti-CYP2J2pep4, and anti-CYP2J6pep1 (but not anti-CYP2J9pep2) that was reduced in pneumonia compared with control lung microsomes. The CYP2B bands (51-52 kDa) were less prominent and not different between pneumonia and control lungs. CYP4A proteins (20-HETE sources) were not detected in rat lung microsomes. Therefore, rat lung contains a protein with immunological characteristics similar to CYP2J4, and this CYP is reduced after pneumonia. We speculate that CYP2J (but not CYP2B) enzymes and their AA metabolic products (EETs) are involved in the modulation of pulmonary vascular tone in pneumonia in rats.

scholarly journals 11,12,20-Trihydroxy-eicosa-8(Z)-enoic acid: a selective inhibitor of 11,12-EET-induced relaxations of bovine coronary and rat mesenteric arteries

2012 ◽  
Vol 302 (8) ◽  
pp. H1574-H1583 ◽  
Author(s):  
Ishfaq A. Bukhari ◽  
Abdul Jabbar Shah ◽  
Kathryn M. Gauthier ◽  
Katherine A. Walsh ◽  
Sreenivasulu Reddy Koduru ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Parent Compound ◽  
Inhibitory Effect ◽  
Mesenteric Arteries ◽  
Enoic Acid ◽  
Epoxyeicosatrienoic Acids ◽  
Spectrometric Analysis ◽  
Antagonist Activity ◽  
Cytochrome P 450 ◽  
Antagonist Effect

Arachidonic acid is metabolized to four regioisomeric epoxyeicosatrienoic acids (EETs) by cytochrome P-450. 5,6-, 8,9-, 11,12-, and 14,15-EET are equipotent in relaxing bovine coronary arteries (BCAs). Vasorelaxant effects of EETs are nonselectively antagonized by 14,15-epoxyeicosa-5( Z)-enoic acid. The 11,12-EET analogs, 20-hydroxy-11,12-epoxyeicosa-8( Z)-enoic acid (20-H-11,12-EE8ZE) and 11,12,20-trihydroxyeicosa-8( Z)-enoic acid (11,12,20-THE8ZE) were synthesized and tested for antagonist activity against EET-induced relaxations in BCAs. In U-46619-preconstricted arterial rings, 5,6-, 8,9-, 11,12-, and 14,15-EET caused concentration-dependent relaxations with maximal relaxations ranging from 80 to 96%. Preincubation of arteries with 20-H-11,12-EE8ZE (10−5 M) inhibited relaxations to 14,15- and 11,12-EET, but not 5,6- and 8,9-EET; however, greatest inhibitory effect was against 11,12-EET (maximal relaxation = 80.6 ± 4.6 vs. 26.7 ± 7.4% without and with 20-H-11,12-EE8ZE, respectively). Preincubation with the soluble epoxide hydrolase inhibitor (tAUCB, 10−6 M) significantly enhanced the antagonist effect of 20-H-11,12-EE8ZE against 14,15-EET-induced relaxations (maximal relaxation = 86.6 ± 4.4 vs. 27.8 ± 3.3%, without and with 20-H-11,12-EE8ZE and tAUCB) without any change in its effect against 11,12-EET-induced relaxations. In contrast to the parent compound, the metabolite, 11,12,20-THE8ZE (10−5 M), significantly inhibited relaxations to 11,12-EET and was without effect on other EET regioisomers. Mass spectrometric analysis revealed conversion of 20-H-11,12-EE8ZE to 11,12,20-THE8ZE by incubation with BCA. The conversion was blocked by tAUCB. 14,15-Dihydroxy-eicosa-5 Z-enoic acid (a 14,15-EET antagonist), but not 11,12,20-THE8ZE (an 11,12-EET antagonist), inhibited BCA relaxations to arachidonic acid and flow-induced dilation in rat mesenteric arteries. These results indicate that 11,12,20-THE8ZE is a selective antagonist of 11,12-EET relaxations and a useful pharmacological tool to elucidate the function of 11,12-EET in the cardiovascular system.

Regulation of potassium channels in coronary smooth muscle by adenoviral expression of cytochrome P-450 epoxygenase

2006 ◽  
Vol 290 (1) ◽  
pp. H64-H71 ◽  
Author(s):  
William B. Campbell ◽  
Blythe B. Holmes ◽  
John R. Falck ◽  
Jorge H. Capdevila ◽  
Kathryn M. Gauthier
Keyword(s):  
Arachidonic Acid ◽  
Smooth Muscle ◽  
Epoxyeicosatrienoic Acids ◽  
Channel Activity ◽  
Channel Activation ◽  
Membrane Hyperpolarization ◽  
Coronary Smooth Muscle ◽  
Adenoviral Transduction ◽  
Cytochrome P 450 ◽  
M 14

Epoxyeicosatrienoic acids (EETs) are endothelium-derived cytochrome P-450 (CYP) metabolites of arachidonic acid that relax vascular smooth muscle by large-conductance calcium-activated potassium (BKCa) channel activation and membrane hyperpolarization. We hypothesized that if smooth muscle cells (SMCs) had the capacity to synthesize EETs, endogenous EET production would increase BKCa channel activity. Bovine coronary SMCs were transduced with adenovirus coding the CYP Bacillus megaterium -3 (F87V) (CYP BM-3) epoxygenase that metabolizes arachidonic acid exclusively to 14( S),15( R)-EET. Adenovirus containing the cytomegalovirus promoter- Escherichia coli β-galactosidase was used as a control. With the use of an anti-CYP BM-3 (F87V) antibody, a 124-kDa immunoreactive protein was detected only in CYP BM-3-transduced cells. Protein expression increased with increasing amounts of virus. When CYP BM-3-transduced cells were incubated with [14C]arachidonic acid, HPLC analysis detected 14,15-dihydroxyeicosatrienoic acid (14,15-DHET) and 14,15-EET. The identity of 14,15-EET and 14,15-DHET was confirmed by mass spectrometry. In CYP BM-3-transduced cells, methacholine (10−5 M) increased 14,15-EET release twofold and BKCa channel activity fourfold in cell-attached patches. Methacholine-induced increases in BKCa channel activity were blocked by the CYP inhibitor 17-octadecynoic acid (10−5 M). 14( S),15( R)-EET was more potent than 14( R),15( S)-EET in relaxing bovine coronary arteries and activating BKCa channels. Thus CYP BM-3 adenoviral transduction confers SMCs with epoxygenase activity. These cells acquire the capacity to respond to the vasodilator agonist by synthesizing 14( S),15( R)-EET from endogenous arachidonic acid to activate BKCa channels. These studies indicate that 14( S),15( R)-EET is a sufficient endogenous activator of BKCa channels in coronary SMCs.

scholarly journals H2O2 opens BKCa channels via the PLA2-arachidonic acid signaling cascade in coronary artery smooth muscle

2000 ◽  
Vol 279 (2) ◽  
pp. H475-H483 ◽  
Author(s):  
Robert S. Barlow ◽  
Abdalla M. El-Mowafy ◽  
Richard E. White
Keyword(s):  
Arachidonic Acid ◽  
Smooth Muscle ◽  
Coronary Arteries ◽  
Signaling Cascades ◽  
Signaling Cascade ◽  
Bkca Channels ◽  
Coronary Smooth Muscle ◽  
Kinase C ◽  
Protein Kinase C Activity ◽  
Cytochrome P 450

H2O2 is a reactive oxygen species that contracts or relaxes vascular smooth muscle, but the molecular basis of these effects remains obscure. We previously demonstrated that H2O2 opens the large-conductance, calcium- and voltage-activated (BKCa) potassium channel of coronary myocytes (2) and now report physiological and biochemical evidence that the effect of H2O2 on coronary smooth muscle involves the phospholipase A2 (PLA2)/arachidonic acid (AA) signaling cascades. H2O2 stimulation of BKCa channel activity was inhibited by arachidonyl trifluoromethyl ketone, an inhibitor of cytosolic PLA2. Furthermore, H2O2 stimulated release of [3H]AA from coronary myocytes, and exogenous AA mimicked the effect of H2O2 on BKCachannels. Inhibitors of protein kinase C activity attenuated the effect of H2O2 on BKCa channels, [3H]AA release, or intact coronary arteries. In addition, the effect of H2O2 or AA on BKCachannels was inhibited by blockers of lipoxygenase metabolism. In contrast, inhibitors of cyclooxygenase or cytochrome P-450 had no effect. We propose that H2O2 relaxes coronary arteries by stimulating BKCa channels via the PLA2/AA signaling cascade and that lipoxygenase metabolites mediate this response.

EETs relax airway smooth muscle via an EpDHF effect: BKCa channel activation and hyperpolarization

2001 ◽  
Vol 280 (5) ◽  
pp. L965-L973 ◽  
Author(s):  
Catherine Benoit ◽  
Barbara Renaudon ◽  
Dany Salvail ◽  
Eric Rousseau
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Molecular Mechanisms ◽  
Muscle Tone ◽  
Muscle Relaxation ◽  
Smooth Muscle Relaxation ◽  
Epoxyeicosatrienoic Acids ◽  
Bkca Channel ◽  
Channel Activity ◽  
Cytochrome P 450

Epoxyeicosatrienoic acids (EETs) are produced from arachidonic acid via the cytochrome P-450 epoxygenase pathway. EETs are able to modulate smooth muscle tone by increasing K+ conductance, hence generating hyperpolarization of the tissues. However, the molecular mechanisms by which EETs induce smooth muscle relaxation are not fully understood. In the present study, the effects of EETs on airway smooth muscle (ASM) were investigated using three electrophysiological techniques. 8,9-EET and 14,15-EET induced concentration-dependent relaxations of the ASM precontracted with a muscarinc agonist (carbamylcholine chloride), and these relaxations were partly inhibited by 10 nM iberiotoxin (IbTX), a specific large-conductance Ca2+-activated K+ (BKCa) channel blocker. Moreover, 3 μM 8,9- or 14,15-EET induced hyperpolarizations of −12 ± 3.5 and −16 ± 3 mV, with EC50 values of 0.13 and 0.14 μM, respectively, which were either reversed or blocked on addition of 10 nM IbTX. These results indicate that BKCa channels are involved in hyperpolarization and participate in the relaxation of ASM. In addition, complementary experiments demonstrated that 8,9- and 14,15-EET activate reconstituted BKCa channels at low free Ca2+ concentrations without affecting their unitary conductance. These increases in channel activity were IbTX sensitive and correlated well with the IbTX-sensitive hyperpolarization and relaxation of ASM. Together these results support the view that, in ASM, the EETs act through an epithelium-derived hyperpolarizing factorlike effect.

Cytochrome P-450 epoxygenase products contribute to attenuated vasoconstriction after chronic hypoxia

2003 ◽  
Vol 285 (1) ◽  
pp. H127-H136 ◽  
Author(s):  
Scott Earley ◽  
Andrzej Pastuszyn ◽  
Benjimen R. Walker
Keyword(s):  
Chronic Hypoxia ◽  
Barometric Pressure ◽  
Mesenteric Arteries ◽  
Resting Membrane Potential ◽  
Epoxyeicosatrienoic Acids ◽  
Resistance Arteries ◽  
Protein Levels ◽  
Acid Metabolites ◽  
Channel Dependent ◽  
Cytochrome P 450

The systemic vasculature exhibits attenuated vasoconstriction following chronic hypoxia (CH) that is associated with endothelium-dependent vascular smooth muscle (VSM) cell hyperpolarization. We hypothesized that increased production of arachidonic acid metabolites such as the cyclooxygenase product prostacyclin or cytochrome P-450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) contributes to VSM cell hyperpolarization following CH. VSM cell resting membrane potential ( Em) was measured in superior mesenteric artery strips isolated from rats with control barometric pressure (Pb, ≅630 Torr) and CH (Pb, 380 Torr for 48 h). VSM cell Em was normalized between groups following administration of the CYP inhibitors 17-octadecynoic acid and SKF-525A. VSM cell hyperpolarization after CH was not altered by cyclooxygenase inhibition, whereas the selective CYP2C9 inhibitor sulfaphenazole normalized VSM cell Em between groups. Iberiotoxin also normalized VSM cell Em, which suggests that large-conductance, Ca2+-activated K+ (BKCa) channel activity is increased after CH. Sulfaphenazole administration restored phenylephrine-induced and myogenic vasoconstriction and Ca2+ responses of mesenteric resistance arteries isolated from CH rats to control levels. Western blot experiments demonstrated that CYP2C9 protein levels were greater in mesenteric arteries from CH rats. In addition, 11,12-EET levels were elevated in endothelial cells from CH rats compared with controls. We conclude that enhanced CYP2C9 expression and 11,12-EET production following CH contributes to BKCa channel-dependent VSM cell hyperpolarization and attenuated vasoreactivity.

Fluorescent HPLC assay for 20-HETE and other P-450 metabolites of arachidonic acid

2000 ◽  
Vol 279 (2) ◽  
pp. H863-H871 ◽  
Author(s):  
Kristopher G. Maier ◽  
Lisa Henderson ◽  
Jayashree Narayanan ◽  
Magdalena Alonso-Galicia ◽  
John R. Falck ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Interstitial Fluid ◽  
Internal Standard ◽  
Dienoic Acid ◽  
Renal Tissue ◽  
Gas Chromatography Mass Spectrometry ◽  
Epoxyeicosatrienoic Acids ◽  
Hplc Assay ◽  
Standard Curve ◽  
Cytochrome P 450

This study describes a fluorescent HPLC assay for measuring 20-hydroxyeicosatetraenoic acid (20-HETE) and other cytochrome P-450 metabolites of arachidonic acid in urine, tissue, and interstitial fluid. An internal standard, 20-hydroxyeicosa-6( Z),15( Z)-dienoic acid, was added to samples, and the lipids were extracted and labeled with 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate. P-450 metabolites were separated on a C18 reverse-phase HPLC column. Coelution and gas chromatography-mass spectrometry studies confirmed the identity of the 20-HETE peak. The 20-HETE peak can be separated from those for dihydroxyeicosatrienoic acids, other HETEs, and epoxyeicosatrienoic acids. Known amounts of 20-HETE were used to generate a standard curve (range 1–10 ng, r 2 = 0.98). Recovery of 20-HETE from urine averaged 95%, and the intra-assay variation was <5%. Levels of 20-HETE were measured in 100 μl of urine and renal interstitial fluid or 0.1 mg of renal tissue. The assay was evaluated by studying the effects of 1-aminobenzotriazole (ABT) on the excretion of 20-HETE in rats. ABT reduced excretion of 20-HETE by >65% and inhibited the formation of 20-HETE by renal microsomes. The availability of this assay should facilitate work in this field.

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