Published online before print July 16, 2007, doi: 10.1161/HYPERTENSIONAHA.107.088948
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(Hypertension. 2007;50:525.)
© 2007 American Heart Association, Inc.
Original Articles |
Prostaglandin E2 Induces Vascular Relaxation by E-Prostanoid 4 Receptor-Mediated Activation of Endothelial Nitric Oxide Synthase
From the Department of Physiology and Pharmacology (A-M.H., L.E.R., P.B.L.H., S.S.N., O.S., B.L.J.), University of Southern Denmark, Odense, Denmark; Institute of Clinical Pharmacology (R.M.N.), Johann Wolfgang Goethe-University, Frankfurt, Germany; Department of Pharmacology (P.V.), University of Hong Kong, Hong Kong; and the Department of Pharmacology (S.N.), Kyoto University Faculty of Medicine, Kyoto, Japan.
Correspondence to Boye L. Jensen, Department of Physiology and Pharmacology, University of Southern Denmark, Winslowparken 21, 3, DK-5000 Odense C, Denmark. E-mail: bljensen{at}health.sdu.dk
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Abstract |
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The present experiments were designed to test the hypothesis that prostaglandin (PG) E2 causes vasodilatation through activation of endothelial NO synthase (eNOS). Aortic rings from mice with targeted deletion of eNOS and E-prostanoid (EP) receptors were used for contraction studies. Blood pressure changes in response to PGE2 were measured in conscious mice. Single doses of PGE2 caused concentration-dependent relaxations during contractions to phenylephrine (EC50=5*10–8 mol/L). Relaxation after PGE2 was absent in rings without endothelium and in rings from eNOS–/– mice and was abolished by NG-nitro-L-arginine methyl ester and the soluble guanylate cyclase inhibitor 1H1,2,4-oxadiazolo-[4,3-a]quinoxalin-1-one. In PGE2-relaxed aortic rings, the cGMP content increased significantly. PGE2-induced relaxations were abolished by the EP4 receptor antagonist AE3–208 (10–8 mol/L) and mimicked by an EP4 agonist (AE1–329, 10–7 mol/L) in the presence of endothelium and eNOS only. Relaxations were attenuated significantly in rings from EP4–/– mice but normal in EP2–/–. Inhibitors of the cAMP-protein kinase A pathway attenuated, whereas the inhibitor of protein phosphatase 1C, calyculin (10–8 mol/L), abolished the PGE2-mediated relaxation. In aortic rings, PGE2 dephosphorylated eNOS at Thr495. Chronically catheterized eNOS–/– mice were hypertensive (137±3.6 mm Hg, n=13, versus 101±3.9 mm Hg, n=9) and exhibited a lower sensitivity of blood pressure reduction in response to PGE2 compared with wild-type mice. There was no difference in the blood pressure response to nifedipine. These findings show that PGE2 elicits EP4 receptor-mediated, endothelium-dependent stimulation of eNOS activity by dephosphorylation at Thr495 resulting in guanylyl cyclase–dependent vasorelaxation and accumulation of cGMP in aortic rings.
Key Words: cyclooxygenase • cGMP • hypertension • phosphorylation • thromboxane
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Prostaglandin (PG) E2 is a physiologically important vasodilator in several vascular beds.1 PGE2 can bind to and stimulate a family of specific E-prostanoid (EP) receptors with
4 distinct subtypes, EP1 to EP4. Of those, the EP2 and EP4 receptors are functionally associated with vasodilatation.1 EP2 receptors are less abundant than EP4 in most tissues but are involved in the systemic vasodepressor response to PGE2 infusion, and targeted deletion of EP2 results in salt-sensitive hypertension.2,3 PGE2 signaling through the EP4 receptor contributes to the systemic vasodepressor response to PGE2 in mice,4,5 attenuates ischemia-reperfusion injury in the heart,6 and maintains the patency of the ductus arteriosus in fetal life.7 Experiments with inhibitors of NO synthase have provided data to suggest that NO is an extracellullar mediator for PGE2-mediated vasodilatation in various vascular beds.8,9 Local infusion of cyclooxygenase inhibitors or NO synthase inhibitors in the renal medulla precipitates hypertension, which would be compatible with a serial coupling between the pathways also at this site.10,11 However, it is not known whether there exists a direct interplay between PGE2 and NO formation in the vasculature, as shown previously to be the case for PGD2.12 PGE2 EP2 and EP4 receptors are coupled to adenylyl cyclase.1 The EP4 receptor also activates a phosphatidylinositol 3-kinase and protein kinase B/Akt.13 Both the cAMP/protein kinase A (PKA) and phosphatidylinositol 3-kinase-protein kinase B/Akt pathways may augment endothelial NO synthase (eNOS) activity at the posttranslational level. Thus, reciprocal phosphorylation at eNOS-Ser1177 and dephosphorylation at eNOS-Thr495 (human sequences) control enzyme activity.14–16 Whether the phosphorylation state of eNOS in the vasculature is altered by PGE2 is unknown. The present study was designed to test the hypothesis that vasodilatation mediated by PGE2 is caused by EP4 receptor-mediated phosphorylation/dephosphorylation of eNOS with subsequent increased production of NO. The experiments were performed on rings of isolated aortae from wild-type mice and mice with targeted disruption of eNOS and EP receptors. In addition, PGE2 was infused to conscious unstressed wild-type mice and eNOS–/– mice with chronic indwelling catheters to study, at the systemic level, the dependency of PGE2-mediated blood pressure changes on eNOS. |
Materials and Methods |
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Isometric Force Measurements in Aorta Rings
Please see the online data supplement available at http://hyper.ahajournals.org for details on mouse strains. Two rings were prepared per mouse. Four rings (
3-mm long) per experiment were suspended in a Halpern-Mulvany myograph (model 610 mol/L, Danish Myo Technology A/S) using 200-µm pins for recording of isometric force (PowerLab, ADI Instruments). Rings incubated at 37°C in physiological salt solution equilibrated with 5% CO2 in air at a pH of 7.4. The rings were normalized at resting tension of 23 mN and allowed to equilibrate for 15 minutes. Please see the data supplement for details, drugs, and reagents.
Measurements of eNOS Phosphorylation and cGMP Content
Aortic rings were mounted as described and contracted with phenylephrine (3x10–7 mol/L). PGE2 (10–7 mol/L) or vehicle was added, and preparations that relaxed >50% to PGE2 and their controls were frozen rapidly in liquid nitrogen. Five rings were pooled per condition. The tissue was homogenized and divided in 2 fractions, 1 for cyclic nucleotide measurement (enzyme immunoassay kits 5810213 and 581001, Cayman Chemical) and 1 for Western blotting (anti-phospho-eNOS-Thr495 1:2000, Upstate; anti-phospho-Ser1177 1:200, Cell Signaling; and anti-
actin 1:1000, Abcam). Please see the data supplement for details on Western blotting and PCR.
Blood Pressure Measurement
Catheters were placed in the femoral artery and vein for measurement of arterial blood pressure and intravenous infusions, respectively. The catheters were produced as described by Mattson.17 Mice recovered for 4 days before the experiments were performed. On the day of the experiment, the artery line was connected to a pressure transducer (Föhr Medical Instruments GMBH) connected to an amplifier. The measured arterial blood pressure was converted into a digital signal by an analog to digital converter (National Instruments, USB 6008) and read by a computer using Laboratory View (National Instruments) software. Data were collected at 200 Hz. Systolic, diastolic, and mean blood pressure and heart rate (HR) were determined. For detailed protocols, please see the data supplement.
Data Analysis and Statistics
Constriction was expressed either relative to the maximal constriction of the respective substance set to be 100% or relative to phenylephrine-induced constriction. Relaxation was expressed as percentage of phenylephrine-induced tone. Data are shown as mean±SEM. For comparison of single and group mean values, 1-way and 2-way ANOVA followed by Bonferroni’s multiple comparison test were performed, respectively, using GraphPad Prism software (GraphPad Prism). One sample t test was used for comparing a single mean with a hypothetical value. Values of P<0.05 were considered to indicate statistically significant differences.
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Results |
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Concentration-Response to PGE2 and Roles of Endothelium and NO Synthase
Phenylephrine caused concentration-dependent, sustained contractions of mouse aortic rings with a mean –log EC50 of 7.45±0.19 (n=5; data not shown). Acetylcholine (10–6 mol/L) caused relaxation of preconstricted rings (78.5±3.1%; n=5; data not shown). Phenylephrine at EC80 (3x10–7 mol/L) was used in subsequent experiments to constrict the rings. Addition of PGE2 as a single bolus to constricted rings caused a rapid and transient relaxation that was present over the full concentration range tested (
10–5 mol/L; Figure 1A). At 10–7 mol/L, PGE2 caused a relaxation of 45.5±6.3% (n=6), and this concentration was used in subsequent experiments to identify the receptor(s) and cellular transduction pathway involved. Maximal relaxation (69±7.5%) was observed at 10–6 mol/L of PGE2.
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When PGE2 was added cumulatively with no washing steps between each concentration (10–11 to 10–5 mol/L, 10 minutes at each concentration), PGE2 had no dilatory effect but elicited contraction at high concentrations (in quiescent rings: pEC50 4.89±0.06, n=5; and in preconstricted rings: pEC50 5.89±0.06, n=4; data not shown). As observed previously,18 PGE2-induced contraction was inhibited by thromboxane receptor antagonists (S18886; –log IC50=7.91±0.10, n=5; and SQ29548: –log IC50=7.47±0.02, n=4; data not shown). PGE2-induced contractions were absent in rings from mice with targeted disruption of the thromboxane receptor, although they contracted to phenylephrine (data not shown; n=5). In subsequent experiments, a TP antagonist (S18886) was present (10–7 mol/L), and only bolus applications of prostanoids and analogs were given.
Removal of the endothelium abolished relaxations to acetylcholine (10–6 mol/L, data not shown) and to PGE2 (Figure 1B). Subsequent exposure to the NO donor sodium nitroprusside (SNP) (10–6 mol/L) relaxed the same rings to the level of basal tone (data not shown; n=6). Incubation of endothelium-intact rings with NG-nitro-L-arginine methyl ester (L-NAME; 10–4 mol/L, 30 minutes) eliminated PGE2-induced relaxations (Figure 1B; n=11). In rings prepared from eNOS–/– mice, acetylcholine (data not shown) and PGE2 did not alter vascular tone (Figure 1B; n=6). The same rings displayed complete relaxation after application of SNP (10–6 mol/L; data not shown). Inhibition of soluble guanylate cyclase with 1H1,2,4-oxadiazolo-[4,3-a]quinoxalin-1-one (10–6 mol/L) prevented PGE2-mediated relaxation in a reversible fashion (Figure 1B; n=6). In PGE2-dilated rings (48±3%; n=40), the cGMP content increased significantly compared with control rings (Figure 1C; n=5).
EP Receptors Involved in the Relaxation to PGE2
An EP4 receptor-selective antagonist (AE3–208 10–8 mol/L; 15 minutes) abolished the relaxation to PGE2 (Figure 2A; n=8). In rings from EP4–/– mice, the relaxation to PGE2 was attenuated significantly but not abolished (Figure 2A; n=4). The EP4 receptor-specific agonist AE1–329 (10–7 mol/L) produced significant relaxation in rings from wild-type mice (Figure 2A, right; n=11) but had no effect on vascular tone in rings from eNOS–/– mice (n=4) and in rings from wild-type mice treated with L-NAME (10–4 mol/L; 30 minutes; n=8; Figure 2A, right). In rings prepared from EP2 receptor-deficient mice (EP2–/–), PGE2 (10–7 mol/L) caused relaxations that were indistinguishable from those observed in rings from wild-type mice (Figure 2B; n=4). The EP2 receptor-specific agonist butaprost (10–6 mol/L) marginally affected tone in preconstricted rings (Figure 2B; 4.8±1.4% relaxation; n=7). PCR analysis of RNA from whole aorta showed significant expression of predominantly EP4 receptors (Figure 2C).
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X174DNA/HaeIII fragments.
Signaling Pathways for PGE2-Induced Activation of eNOS
The cAMP-dependent protein kinase A inhibitors Rp-8-Br-cAMPs (10–4 mol/L; 45 minutes) and KT5720 (10–6 mol/L; 45 minutes; n=7) significantly attenuated the PGE2-induced relaxation (Figure 3A; n=6). The adenylate cyclase inhibitor SQ22536 (10–4 mol/L; 45 minutes; n=6) yielded similar results (Figure 3A). Relaxation to acetylcholine was not affected significantly by blockers of the cAMP-PKA pathway (data not shown). In PGE2-relaxed rings (50.3±3%; n=25), cAMP was not significantly elevated compared with control (38.5±7 pmol/mg versus 30.1±9 pmol/mg; n=5; P>0.1). Inhibitors of phosphatidylinositol 3-kinase (LY294002: 10–5 mol/L, n=4; and wortmannin: 10–7 and 10–6 mol/L, n=4 each; data not shown) and mitogen-activated protein kinase (PD98059; 2x10–5 mol/L; 30 minutes; n=3) did not affect the PGE2-induced relaxation significantly (Figure 3B). Incubation of rings with an inhibitor of protein phosphatase 1C, calyculin A,19 (10–8 mol/L; 30 minutes) had no effect on relaxations to acetylcholine (data not shown) but abolished those to PGE2 (Figure 3B; n=11). In phenylephrine-contracted mouse aortic rings, Ser1177 phospho-eNOS was hardly detectable by Western blotting (data not shown), whereas Thr495-phospho-eNOS was readily detectable (Figure 3C). Exposure of preconstricted aortae rings to PGE2 caused significant dephosphorylation at Thr495 (Figure 3C; n=5), whereas Ser1177 phospho-eNOS did not change significantly (data not shown).
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Arterial Blood Pressure Response to PGE2 in Wild-Type and eNOS–/– Mice
In wild-type mice, the resting mean systolic blood pressure was 101±3.9 mm Hg, and HR was 640±23 bpm (n=9). In eNOS–/– mice, arterial pressure was elevated significantly (137±3.6 mm Hg; n=13; Figure 4A and 1
B) and HR was not different from control (625±23 bpm). Injection of a saline bolus had no effect on arterial blood pressure or HR in either wild-type or eNOS–/– (Figure 4B). Two consecutive applications of PGE2 (bolus of 50 µg/kg; 1-hour interval) led to a similar decrease in arterial blood pressure during first and second application in wild-type mice (pressure after PGE2 in mm Hg; 56±2.5 and 57±0.9; n=4; first and second application, respectively) and in eNOS–/– mice (100±9.5 and 99±4.6; n=6; Figure 4B). The maximal response occurred within the first minutes after the bolus injection. Dose-response experiments showed that PGE2 had no significant effect in either strain at 10 µg/kg (data not shown). At 25 µg/kg, PGE2 caused a significant decrease in arterial blood pressure in wild-type mice only (Figure 4C; from 100±7.0 to 83±6.7 mm Hg; n=5). There was no difference in HR in response to 25 µg/kg of PGE2 in wild-type compared with eNOS–/– mice (11.5±3.4% versus 6.9±2.2% increase; P=0.29). A bolus of 50 µg/kg of PGE2 decreased blood pressure significantly in both wild-type and eNOS–/– mice (Figure 4C). The relative blood pressure decrease to 25 µg/kg and 50 µg/kg of PGE2 was significantly larger in wild-type mice compared with eNOS–/– mice (Figure 4C). There was no significant difference in the blood pressure response to nifedipine, which lowered arterial blood pressure by 37±4% and 46±6% in eNOS–/– and wild-type mice (Figure 4C). There was no significant difference in the time to recovery of blood pressure: in wild-type mice, 3.2±1.0 minutes and eNOS–/– mice, 3.4±1.3 minutes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study demonstrates an endothelium- and eNOS-dependent, EP4 receptor-mediated relaxation of an isolated vessel preparation by PGE2 that is associated with cGMP accumulation and dephosphorylation of eNOS at Thr495 (Figure 5). The novel finding may provide an explanation for the rapid effects of cyclooxygenase inhibitors or prostanoids on NO-dependent physiological responses in certain vascular beds.8–11 The use of an integrated vascular preparation showed that, after prolonged exposure to PGE2, the EP4 receptor-mediated relaxation waned, and at high concentrations of PGE2, cross-reactivity with thromboxane receptors was apparent, as reported also in rat aorta.18 It is well established that EP4 receptors exhibit ligand-mediated desensitization primarily because of internalization.20,21 When vessels had not been exposed to PGE2 before, the vessels responded by relaxation at the full range of concentrations.
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Removal of the endothelium, L-NAME treatment, or deletion of eNOS abolished the ability of PGE2 to cause relaxation, so we infer that smooth-muscle EP4 or EP2 receptors are of minor significance for relaxation after PGE2 in the mouse aorta. The initial sequence of events must depend on EP4 receptor-mediated activation of eNOS, followed by NO-mediated stimulation of soluble guanylate cyclase with accumulation of cGMP in the smooth muscle (Figure 5). The data indicate that PGE2 activates eNOS through formation of cAMP by the endothelium; vasorelaxation was inhibited by 3 separate blockers of PKA and adenylate cyclase; calcium-coupled PGE2 EP1 receptors were not detected in the aorta, and the response depended on phosphoprotein phosphatase 1C (PP1C), which is activated predominantly by the cAMP pathway19 (Figure 5). cAMP did not increase significantly in PGE2-relaxed vascular rings. This finding does not exclude that PGE2 induces formation of cAMP restricted to the limited pool of endothelial cells in the preparation. The notion of cAMP-PKA–dependent activation of eNOS by PP1C-mediated dephosphorylation at Thr495 is consistent with data obtained in cultured endothelial cells.14,19 PGE2 phosphorylates eNOS at Ser1177 in cultured human endothelial cells at passage 3 to 6.22 This discrepancy with the present study is likely because of the very different experimental conditions. It may also reflect that NO-mediated responses display segmental heterogeneity; they predominate in conduit vessels like the aorta, and eNOS is also more strongly expressed in this segment compared with smaller vessels.23,24
The in vivo experiments demonstrated a fall in arterial blood pressure in response to PGE2, both in eNOS–/– and wild-type mice. The response to PGE2 was less pronounced in mice with targeted disruption of eNOS than in wild-type animals. This differential sensitivity is compatible with a significant contribution of eNOS to the systemic vasodilator response to PGE2. However, the effect of PGE2 on blood pressure also involves mechanisms clearly not dependent on eNOS. Both peripheral vascular and central nervous mechanisms may be involved.25 In the mouse, EP2 and EP4 receptors contribute to the acute depressor effect of PGE2 on arterial blood pressure.4,5 Thus, EP2 receptors could be responsible for the residual systemic vasodepressor action of PGE2 in eNOS–/– mice, and EP4 receptors associated with vascular smooth muscle in the arteriolar segments could also participate.
Vasoconstriction because of cross-activation of thromboxane receptors by prostanoids and isoprostanes has been observed in various preparations.18,26 No significant expression of EP receptors associated with constriction (EP1 and EP3) was observed in mouse aorta in the present and a previous study.27 Moreover, the dissociation constant values for PGE2 at EP1 and EP3 receptors are significantly lower than the concentrations of PGE2 required to mediate constriction in the present study.28 These observations permit the conclusion that PGE2 is a low-affinity agonist at the thromboxane receptor in the mouse aorta, as observed previously in the rat aorta.18
Perspectives
The present data provide evidence for a novel pathway between PGE2 and eNOS activity initiated by endothelial EP4 receptor occupation and mediated by PP1C-dependent dephosphorylation of eNOS. This pathway is relevant not only for physiological regulation of vascular tone and blood flow but may also contribute to other vascular processes influenced by eNOS activity, such as platelet aggregation and smooth muscle cell proliferation.
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Acknowledgments |
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We thank Lis Teusch and Mette Fredenslund for excellent technical assistance and Prof Ulf Simonsen, Aarhus University, for assistance with Western blotting protocols. We thank Prof Peter Bie and Ole Madsen for assistance with blood pressure measurements.
Sources of Funding
The present work was supported by grants from the Danish Research Council for Health and Disease, Danish Heart Foundation, NOVO Nordisk Foundation, AJ Andersen and Hustrus Foundation, Else Poulsens Legacy, Danish Kidney Association Research Fund, and Helen and Ejnar Bjørnows Foundation.
Disclosures
None.
Received February 14, 2007; first decision March 12, 2007; accepted June 21, 2007.
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