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.

Endothelium-dependent blunting of myogenic responsiveness after chronic hypoxia

2002 ◽  
Vol 283 (6) ◽  
pp. H2202-H2209 ◽  
Author(s):  
Scott Earley ◽  
Benjimen R. Walker
Keyword(s):  
Membrane Potential ◽  
Vessel Wall ◽  
Chronic Hypoxia ◽  
Barometric Pressure ◽  
No Synthase ◽  
Resting Membrane Potential ◽  
Resistance Arteries ◽  
Inner Diameter ◽  
Normoxic Control ◽  
Increased Pressure

Blunted agonist-induced vasoconstriction after chronic hypoxia is associated with endothelium-dependent vascular smooth muscle (VSM) cell hyperpolarization and decreased vessel-wall Ca2+concentration ([Ca2+]). We hypothesized that myogenic vasoconstriction and pressure-induced Ca2+ influx would also be attenuated in vessels from chronically hypoxic (CH) rats. Mesenteric resistance arteries isolated from CH [barometric pressure (BP), 380 Torr for 48 h] or normoxic control (BP, 630 Torr) rats were cannulated and pressurized. VSM cell resting membrane potential was recorded at intraluminal pressures of 40–120 Torr under normoxic conditions. VSM cells in vessels from CH rats were hyperpolarized compared with control rats at all pressures. Inner diameter was maintained for vessels from control rats, whereas vessels from CH rats developed less tone as pressure was increased. Pressure-induced increases in vessel-wall [Ca2+] were also attenuated for arteries from CH rats. Endothelium removal restored myogenic constriction to vessels from CH rats and normalized VSM cell resting membrane potential and pressure-induced Ca2+ responses to control levels. Myogenic constriction and pressure-induced vessel-wall [Ca2+] increases remained blunted in the presence of nitric oxide (NO) synthase inhibition for arteries from CH rats. We conclude that blunted myogenic reactivity after chronic hypoxia results from a non-NO, endothelium-dependent VSM cell hyperpolarizing influence.

scholarly journals Cytochrome P-450 metabolites of 2-arachidonoylglycerol play a role in Ca2+-induced relaxation of rat mesenteric arteries

2008 ◽  
Vol 294 (5) ◽  
pp. H2363-H2370 ◽  
Author(s):  
Emmanuel M. Awumey ◽  
Sylvie K. Hill ◽  
Debra I. Diz ◽  
Richard D. Bukoski
Keyword(s):  
Antihypertensive Therapy ◽  
Sensory Nerve ◽  
Mesenteric Arteries ◽  
Phospholipase A ◽  
Epoxyeicosatrienoic Acids ◽  
Resistance Arteries ◽  
Isolated Arteries ◽  
Endogenous Cannabinoid ◽  
Cytochrome P 450 ◽  
Endogenous Cannabinoid System

The perivascular sensory nerve (PvN) Ca2+-sensing receptor (CaR) is implicated in Ca2+-induced relaxation of isolated, phenylephrine (PE)-contracted mesenteric arteries, which involves the vascular endogenous cannabinoid system. We determined the effect of inhibition of diacylglycerol (DAG) lipase (DAGL), phospholipase A2 (PLA2), and cytochrome P-450 (CYP) on Ca2+-induced relaxation of PE-contracted rat mesenteric arteries. Our findings indicate that Ca2+-induced vasorelaxation is not dependent on the endothelium. The DAGL inhibitor RHC 802675 (1 μM) and the CYP and PLA2 inhibitors quinacrine (5 μM) (EC50: RHC 802675 2.8 ± 0.4 mM vs. control 1.4 ± 0.3 mM; quinacrine 4.8 ± 0.4 mM vs. control 2.0 ± 0.3 mM; n = 5) and arachidonyltrifluoromethyl ketone (AACOCF3, 1 μM) reduced Ca2+-induced relaxation of mesenteric arteries. Synthetic 2-arachidonoylglycerol (2-AG) and glycerated epoxyeicosatrienoic acids (GEETs) induced concentration-dependent relaxation of isolated arteries. 2-AG relaxations were blocked by iberiotoxin (IBTX) (EC50: control 0.96 ± 0.14 nM, IBTX 1.3 ± 0.5 μM) and miconazole (48 ± 3%), and 11,12-GEET responses were blocked by IBTX (EC50: control 55 ± 9 nM, IBTX 690 ± 96 nM) and SR-141716A. The data suggest that activation of the CaR in the PvN network by Ca2+ leads to synthesis and/or release of metabolites of the CYP epoxygenase pathway and metabolism of DAG to 2-AG and subsequently to GEETs. The findings indicate a role for 2-AG and its metabolites in Ca2+-induced relaxation of resistance arteries; therefore this receptor may be a potential target for the development of new vasodilator compounds for antihypertensive therapy.

Gender-dependent modulation of α1-adrenergic responses in rat mesenteric arteries

2003 ◽  
Vol 284 (5) ◽  
pp. H1737-H1743 ◽  
Author(s):  
Alyson P. McKee ◽  
Dee A. Van Riper ◽  
Cathy A. Davison ◽  
Harold A. Singer
Keyword(s):  
Smooth Muscle ◽  
Endothelial Damage ◽  
Selective Inhibitor ◽  
Mesenteric Arteries ◽  
Resistance Arteries ◽  
Adrenergic Agonist ◽  
Contractile Responses ◽  
Cyclooxygenase 1 ◽  
Acid Metabolites ◽  
Selective Increase

The purpose of this study was to test the hypothesis that pathways modulating vasoconstriction in rat mesenteric resistance arteries are gender dependent. Net contractile responses to phenylephrine were significantly increased by endothelium disruption in arteries from males but not females. This gender-dependent effect was stimulus specific, because disruption of endothelium increased reactivity to serotonin comparably in arteries from both genders. Ovariectomy unmasked an increase in net α1-adrenergic contractile responsiveness after endothelium disruption, suggesting α1-adrenergic-stimulated production of endothelial vasodilators is suppressed in control females by gonadal sex steroids. Production of modulatory endothelium-derived vasodilators in males is balanced by production of vasoconstricting arachidonic acid metabolites. This was revealed by decreased α1-adrenergic contractile responses in arteries from males after pretreatment with indomethacin or the cyclooxygenase-1 selective inhibitor SC-560. The indomethacin-induced effect persisted after endothelium disruption, indicating smooth muscle as the source of cyclooxygenase-1-derived vasoconstrictors and was attenuated after orchiectomy. This study indicates gender differences in the expression of two pathways modulating α1-adrenergic sensitivity in mesenteric arteries: an endothelium-dependent vasodilator pathway and a balancing smooth muscle cyclooxygenase-1-dependent vasoconstrictor pathway. One consequence of these differences is that endothelial damage produces a selective increase in α1-adrenergic agonist reactivity in arteries from males.

Decreased epoxygenase and increased epoxide hydrolase expression in the mesenteric artery of obese Zucker rats

2005 ◽  
Vol 288 (1) ◽  
pp. R188-R196 ◽  
Author(s):  
Xueying Zhao ◽  
Aparajita Dey ◽  
Olga P. Romanko ◽  
David W. Stepp ◽  
Mong-Heng Wang ◽  
...  
Keyword(s):  
Mesenteric Artery ◽  
Epoxide Hydrolase ◽  
Soluble Epoxide Hydrolase ◽  
Mesenteric Arteries ◽  
Zucker Rats ◽  
Epoxyeicosatrienoic Acids ◽  
Obese Zucker Rat ◽  
Protein Expressions ◽  
Obese Zucker Rats ◽  
Cytochrome P 450

Previous studies suggest that epoxyeicosatrienoic acids (EETs) are vasodilators of the mesenteric artery; however, the production and regulation of EETs in the mesenteric artery remain unclear. The present study was designed 1) to determine which epoxygenase isoform may contribute to formation of EETs in mesenteric arteries and 2) to determine the regulation of mesenteric artery cytochrome P-450 (CYP) enzymes in obese Zucker rats. Microvessels were incubated with arachidonic acid, and CYP enzyme activity was determined. Mesenteric arteries demonstrate detectable epoxygenase and hydroxylase activities. Next, protein and mRNA expressions were determined in microvessels. Although renal microvessels express CYP2C23 mRNA and protein, mesenteric arteries lacked CYP2C23 expression. CYP2C11 and CYP2J mRNA and protein were expressed in mesenteric arteries and renal microvessels. In addition, mesenteric artery protein expression was evaluated in lean and obese Zucker rats. Compared with lean Zucker rats, mesenteric arterial CYP2C11 and CYP2J proteins were decreased by 38 and 43%, respectively, in obese Zucker rats. In contrast, soluble epoxide hydrolase mRNA and protein expressions were significantly increased in obese Zucker rat mesenteric arteries. In addition, nitric oxide-independent dilation evoked by acetylcholine was significantly attenuated in mesenteric arteries of obese Zucker rats. These data suggest that the main epoxygenase isoforms expressed in mesenteric arteries are different from those expressed in renal microvessels and that decreased epoxygenases and increased soluble epoxide hydrolase are associated with impaired mesenteric artery dilator function in obese Zucker rats.

Role of CO in attenuated vasoconstrictor reactivity of mesenteric resistance arteries after chronic hypoxia

2002 ◽  
Vol 282 (1) ◽  
pp. H30-H37 ◽  
Author(s):  
Rayna J. Gonzales ◽  
Benjimen R. Walker
Keyword(s):  
Chronic Hypoxia ◽  
Protoporphyrin Ix ◽  
Aortic Ring ◽  
Mesenteric Arteries ◽  
Zinc Protoporphyrin ◽  
Sprague Dawley ◽  
Resistance Arteries ◽  
Sprague Dawley Rats ◽  
Oxide Synthase

Chronic hypoxia (CH) is associated with a persistent reduction in systemic vasoconstrictor reactivity. Experiments on aortic ring segments isolated from CH rats suggest that enhanced vascular expression of heme oxygenase (HO) and resultant production of the vasodilator carbon monoxide (CO) may underlie this attenuated vasoreactivity after hypoxia. Similar to the aorta, small arteries from CH rats exhibit blunted reactivity; however, the regulatory role of CO in the resistance vasculature has not been established. Therefore, we examined the significance of HO activity on responsiveness to phenylephrine (PE) in the mesenteric circulation of control and CH rats. To document that the mesenteric bed demonstrates reduced reactivity after CH, we determined the vasoconstrictor responses of conscious, chronically instrumented male Sprague-Dawley rats to PE under control conditions and then immediately after exposure to 48 h CH (0.5 atm). All rats showed reduced mesenteric vasoconstriction to PE after CH. To examine the role of CO in reduced reactivity, small mesenteric arteries (100–200 μm intraluminal diameter) from control and 48-h CH rats were isolated and mounted on glass cannulas, pressurized to 60 mmHg and superfused with increasing concentrations of PE under normoxic conditions. Similar to the intact circulation, vessels from CH rats exhibited reduced vasoconstrictor sensitivity to PE compared with controls that persisted in the presence of nitric oxide synthase inhibition. The HO inhibitor, zinc protoporphyrin IX (5 μM) enhanced reactivity only in CH vessels. Additionally, a range of concentrations of the HO substrate heme-l-lysinate caused vasodilation in CH vessels but not in controls. Thus we conclude that CO contributes a significant vasodilator influence in resistance vessels after CH that may account for diminished vasoconstrictor responsiveness under these conditions.

scholarly journals Roles of CYP2C29 and RXRγ in vascular EET synthesis of female mice

2010 ◽  
Vol 298 (4) ◽  
pp. R862-R869 ◽  
Author(s):  
Dong Sun ◽  
Yang-Ming Yang ◽  
Houli Jiang ◽  
Hongyan Wu ◽  
Caroline Ojaimi ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Shear Stress ◽  
Mesenteric Arteries ◽  
Epoxyeicosatrienoic Acids ◽  
Rt Pcr ◽  
Related Transcription Factor ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide ◽  
Cytochrome P 450 ◽  
Stress Dose

We aimed to identify which cytochrome P-450 (CYP) family/subfamily, as well as related transcription factor(s), is responsible for the estrogen-dependent synthesis of epoxyeicosatrienoic acids (EETs) to initiate shear stress-induced vasodilation. Microarray analysis indicated a significant upregulation of CYP2C29 and retinoid X receptor γ (RXRγ) in isolated mesenteric arteries/arterioles of female endothelial nitric oxide synthase-knockout mice, a result that was validated by real-time RT-PCR. The cannulated vessels were then perfused with 2 and 10 dyn/cm2 shear stress, followed by collection of the perfusate to determine EET concentrations and isoforms. Shear stress dose-dependently stimulated the release of EETs into the perfusate, associated with an EET-mediated vasodilation, in which predominantly 14,15-EET and 11,12-EET contributed to the responses (∼87.4% of total EETs). Transfection of vessels with CYP2C29 siRNA eliminated the release of EETs into the perfusate, which was evidenced by an abolished vasodilation, and confirmed by RT-PCR and Western blot analyses. Knockdown of RXRγ in these vessels significantly inhibited the production of EETs, parallel to a reduced vasodilation. RXRγ siRNA not only silenced the vascular RXRγ expression, but synchronously downregulated CYP2C29 expression, leading to a reduced EET synthesis. In conclusion, our data provide the first evidence for a specific signaling cascade, by which estrogen potentially activates the CYP2C29 gene in the absence of nitric oxide, to synthesize EETs in response to shear stress, via an RXRγ-related regulatory mechanism.

Increased nitric oxide production following chronic hypoxia contributes to attenuated systemic vasoconstriction

2003 ◽  
Vol 284 (5) ◽  
pp. H1655-H1661 ◽  
Author(s):  
Scott Earley ◽  
Benjimen R. Walker
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cell ◽  
Chronic Hypoxia ◽  
Barometric Pressure ◽  
Mesenteric Arteries ◽  
No Production ◽  
Western Blots ◽  
Fura 2 ◽  
Enhanced Production ◽  
Nos Isoforms

Attenuated vasoconstrictor reactivity following chronic hypoxia (CH) is associated with endothelium-dependent vascular smooth muscle (VSM) cell hyperpolarization and diminished intracellular [Ca2+]. We tested the hypothesis that increased production of nitric oxide (NO) after CH contributes to blunted vasoconstrictor responsiveness. We found that basal NO production of mesenteric arteries from CH rats (barometric pressure = 380 Torr; 48 h) was greater than that of controls (barometric pressure = 630 Torr). In addition, studies employing pressurized mesenteric arteries (100–200 μM ID) abluminally loaded with the Ca2+ indicator fura 2-AM demonstrated that although NO synthase (NOS) inhibition normalized agonist-induced vasoconstrictor responses between groups, VSM cell [Ca2+] in vessels from CH rats remained diminished compared with controls. To determine whether elevated NO production following CH results from increased NOS protein levels, we performed Western blots for NOS isoforms by using mesenteric arteries from control and CH rats. Endothelial NOS levels did not differ between groups, and other NOS isoforms were not detected in these samples. Selective endothelial loading of fura 2-AM was employed to test the hypothesis that elevated endothelial cell [Ca2+] following CH accounts for enhanced NOS activity. These experiments demonstrated greater endothelial cell [Ca2+] in mesenteric arteries isolated from CH rats compared with controls. We conclude that enhanced production of NO resulting from elevated endothelial cell [Ca2+] contributes to attenuated reactivity following CH by decreasing VSM cell Ca2+sensitivity.

Cerebral adaptations to chronic anemia in a model of erythropoietin-deficient mice exposed to hypoxia

2009 ◽  
Vol 296 (3) ◽  
pp. R801-R811 ◽  
Author(s):  
Raja El Hasnaoui-Saadani ◽  
Aurélien Pichon ◽  
Dominique Marchant ◽  
Paul Olivier ◽  
Thierry Launay ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Chronic Hypoxia ◽  
Acute Hypoxia ◽  
Hypoxia Inducible Factor ◽  
Barometric Pressure ◽  
Capillary Density ◽  
Wild Type ◽  
Chronic Anemia ◽  
Protein Levels ◽  
Deficient Mice

Anemia and hypoxia in rats result in an increase in factors potentially involved in cerebral angiogenesis. Therefore, the aim of this study was to assess the effect of chronic anemia and/or chronic hypoxia on cerebral cellular responses and angiogenesis in wild-type and anemic transgenic mice. These studies were done in erythropoietin-deficient mice (Epo-TAgh) in normoxia and following acute (one day) and chronic (14 days, barometric pressure = 420 mmHg) hypoxia. In normoxia, Epo-TAgh mice showed an increase in transcript and protein levels of hypoxia-inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF), erythropoietin receptors (EpoR), phospho-STAT-5/STAT-5 ratio, and neuronal neuronal nitric oxide synthase (nNOS) along with a higher cerebral capillary density. In wild-type (WT) mice, acute hypoxia increased all of the studied factors, while in chronic hypoxia, HIF-1α, EpoR, phospho-STAT-5/STAT-5 ratio, nNOS, and inducible NOS remained elevated, with an increase in capillary density. Surprisingly, in Epo-TAgh mice, chronic hypoxia did not further increase any factor except the nitric oxide metabolites, while HIF-1α, EpoR, and phospho-STAT-5/STAT-5 ratio were reduced. Normoxic Epo-TAgh mice developed cerebral angiogenesis through the HIF-1α/VEGF pathway. In acute hypoxia, WT mice up-regulated all of the studied factors, including cerebral NO. Polycythemia and angiogenesis occurred with acclimatization to chronic hypoxia only in WT mice. In Epo-TAgh, the decrease in HIF-1α, VEGF proteins, and phospho-STAT-5 ratio in chronic hypoxia suggest that neuroprotective and angiogenesis pathways are altered.

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.

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.

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