Increased Expression of Cyclooxygenase-2 Mediates Enhanced Contraction to Endothelin ETA Receptor Stimulation in Endothelial Nitri
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Yingbi Zhou, Srabani Mitra, Saradhadevi
参见附件。
the Davis Heart & Lung Research Institute, The Ohio State University, Columbus.
Abstract
The aim of this study was to determine whether prolonged loss of NO activity, in endothelial NO synthase knockout (eNOS–/–) mice, influences endothelin (ET) ETA receptor-mediated smooth muscle contraction and, if so, to define the underlying mechanism(s). In isolated endothelium-denuded abdominal aortas, contractions to the selective ETA receptor agonist ET-1(1-31) were significantly increased in aortas from eNOS–/– compared with wild-type (WT) mice. In contrast, contractions to the 1-adrenergic agonist phenylephrine or the thromboxane (TX) A2 analog U-46619 were similar between eNOS–/– and WT mice. Immunofluorescent and Western blot analysis demonstrated that the aortic expression of ETA receptors was decreased in eNOS–/– compared with WT mice. Contractions evoked by ET-1(1-31), but not phenylephrine, were reduced by inhibition of cyclooxygenase-2 (COX-2) (indomethacin or celecoxib) or of TXA2/prostaglandin H2 receptors (SQ-29548). After COX inhibition, contractions to ET-1(1-31) were no longer increased and were actually decreased in eNOS–/– compared with WT aortas. Western blot analysis revealed that endothelium-denuded abdominal aortas express COX-2, but not COX-1, and that expression of COX-2 was significantly increased in eNOS–/– compared with WT mice. Contractions to the COX substrate arachidonic acid were also increased in eNOS–/– aortas. Furthermore, ET-1(1-31) but not phenylephrine stimulated production of the TXA2 metabolite TXB2, which was increased in eNOS–/– compared with WT aortas. Therefore, COX-2 plays a crucial and selective role in ETA-mediated smooth muscle contraction. Furthermore, COX-2 expression is increased in eNOS–/– mice, which overcomes a reduced expression of ETA receptors and enables a selective increase in contraction to ETA receptor stimulation.
Key Words: endothelin-1(1-31) arachidonic acid thromboxane A2 prostaglandin H2
Introduction
Endothelins (ETs) are a family of peptides that play important roles in regulating cardiovascular function.1,2 ET-1, which causes potent and long-lasting vasoconstriction, is the major form produced by endothelial cells.3 To date, 2 types of ET receptors have been identified: ETA is selectively activated by ET-1(1-31), whereas ETB is selectively stimulated by sarafotoxin S6c.1,2,4,5 In vascular smooth muscle, ETA is the most abundant receptor with activation causing contraction in all vascular regions, irrespective of species.1,2 Although ETB can cause smooth muscle contraction in certain vascular beds,6 activation of ETB is generally associated with vasodilation through the stimulated release of endothelial nitric oxide (NO).7–9
ETA-mediated contraction of vascular smooth muscle may play an important role in the abnormal vasoreactivity associated with cardiovascular diseases, including coronary atherosclerosis and hypertension.1,2,10–12 Although impaired bioavailability of endothelial NO, which is associated with vascular diseases, causes a general increase in constrictor activity, it can also have some selective effects on ET activity. Decreased NO activity can cause increased production of ETs11,13–17 and enhanced constriction to these peptides by impairing ETB-mediated dilation.7,8,18 The aim of the present study was to determine whether a prolonged decrease in NO activity could directly influence ETA receptor-mediated contraction in smooth muscle and, if so, to define the underlying mechanism(s).
Materials and Methods
Chemicals
Indomethacin, N-nitro-L-arginine methyl ester (L-NAME), phenylephrine, sarafotoxin S6c, BQ123, and U-46619 were obtained from Sigma (St Louis, Mo); the selective ETA receptor agonist ET-1(1-31) from Peptide International (Louisville, Ky); the TxA2/PGH2 receptor antagonist S-Q29548 from ICN Pharmaceuticals (Costa Mesa, Calif); and cyclooxygenase (COX) substrate arachidonic acid from Calbiochem (La Jolla, Calif). The selective COX-2 inhibitor celecoxib was kindly provided by Pharmacia (St Louis, Mo). All other chemicals were of the highest commercially available quality.
Animal and Tissue Preparation
Wild-type (WT) and eNOS–/– mice (8 to 12 weeks) from The Jackson Laboratory (Bar Harbor, Me) were euthanized by CO2 inhalation. This procedure was approved by the Institutional Laboratory Animal Care and Use Committee of The Ohio State University. The abdominal aorta, which contracts strongly to ETA receptor activation,19 was rapidly excised and placed in ice-cold PSS (physiological salt solution; in mmol/L: NaCl 123, KCl 4.7, NaHCO3 15.5, KH2PO4 1.2, MgCl2 1.2, CaCl2 1.25, and D-glucose 11.5). Vessels were dissected free of adventitial fat and connective tissue. For tension measurements, aortic segments were cut into 1-mm vascular rings. For all other assays, aortas were cut open and the endothelial cells removed with a moist cotton swab. In some experiments, aortic segments were separated into adventitial and medial layers. All surgical procedures were performed under a binocular microscope.
Isometric Force Measurement
Isometric force measurement was performed as described previously.19,20 Briefly, the vascular ring was mounted between 2 tungsten wires, each with a diameter of 50 μm, in a 37°C water-circulating tissue bath. One wire was stationary, whereas the other was connected to a force transducer (AE 801, Sensor One, Horten, Norway). Because the endothelium might release vasoactive mediators under basal conditions, most experiments were performed on endothelium-denuded blood vessels. Therefore, unless stated otherwise, the endothelium was denuded from all aortic rings. This was achieved by rotating the rings around the tungsten wires while the passive tension was kept at 100 mg.20 Thereafter, tissues were stimulated with 60 mmol/L K+-PSS (equimolar replacement of NaCl with KCl) every 15 minutes, and the resting tension increased in a stepwise manner. After the equilibration, the resting tension was adjusted to approximately 300 mg, at which the maximal response to K+ was obtained.
Contraction evoked by an agonist was expressed as a percentage of the response to 60 mmol/L K+. Concentration-effect curves to U-46619 or phenylephrine were generated by increasing the concentration of the agonists in half-log increments, once the contraction to the previous concentration had stabilized. Because responses to ET-1(1-31) were transient, contractions to the agonist were assessed by administering only a single concentration of the agonist. In all experiments, only a single curve (U46619, phenylephrine) or a single contraction [ET-1(1-31)] was analyzed on each aortic specimen. In addition, L-NAME (1 mmol/L), which did not affect ETA-mediated contraction in eNOS–/– mice, was added 20 minutes before the application of an agonist in WT mouse specimens. COX inhibitors (indomethacin, 3 μmol/L; celecoxib, 3 μmol/L) or the TxA2/PGH2 receptor antagonist (SQ-29548, 1 μmol/L) were applied 10 minutes before the application of an agonist.
Western Blot
Proteins (20 μg for COX analysis, 40 μg for ETA analysis) isolated from endothelium-denuded aortas were separated by electrophoresis on 10% SDS-PAGE and transferred onto a nitrocellulose membrane. After incubation with a monoclonal anti-COX-1 (Cayman Chemical, Ann Arbor, Mich; dilution: 1:1000), a polyclonal anti-COX-2 antibody (Cayman Chemical; dilution: 1:1000), or a monoclonal anti-ETA receptor antibody (BD Biosciences, Palo Alto, Calif; dilution 1:800), the nitrocellulose membranes were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody. The immunocomplex was detected with ECL plus kit (Amersham, Buckinghamshire, UK), and the band density was analyzed using a personal densitometer (Molecular Dynamics, Sunnyvale, Calif).
Immunofluorescence
Intact mouse abdominal aortic segment was frozen and cut into 5-μm sections. Tissue sections were attached to glass slides and fixed with acetone. After treatment with primary antibody (1:100 dilution, sheep anti-ETA; Alexis, San Diego, Calif),21 tissues sections were incubated with anti-sheep Alexa flour-488 (Molecular Probes, Eugene, Ore). The fluorescent staining was detected using a Zeiss 510 laser scanning microscope equipped with a x63 water immersion objective (NA 1.2). The image was analyzed by using Image J (NIH, Rockville, Md) software. The fluorescent intensity was expressed as the average pixel value, which is the sum of intensities of all the pixels in the area of selection divided by number of pixels.
Measurement of TXA2 Metabolite TXB2
Endothelium-denuded aortic strips (10 mm) were equilibrated in PSS at 37°C for 30 minutes. Thereafter, vessel strips were sequentially incubated in 150 μL of PSS (baseline) and 100 nmol/L ET-1(1-31) or 10 μmol/L PE in 150 μL of PSS for 10 minutes, respectively. The PSS was then removed, snap frozen with liquid nitrogen, and stored at –80°C. TXB2 was measured with an EIA kit (Amersham International), according to the instructions of the manufacturer.
Data Analysis
Data are expressed as means±SEM for n number of experiments, where n equals the number of animals from which blood vessels were studied. Statistical evaluation of the data were performed by Student’s t test for either paired or unpaired observations. When more than 2 means were compared, ANOVA was used: either a 1-way ANOVA with Dunnett’s post hoc test or 2-way ANOVA followed by Bonferroni’s post hoc test (GraphPad 4 Software, San Diego, Calif). Values were considered to be statistically significant when P was <0.05.
Results
Contraction to ETA Receptor Activation Is Increased in eNOS–/– Aorta
The selective ETA receptor agonist ET-1(1-31) (10 to 100 nmol/L) caused contractions of endothelium-denuded abdominal aortas that were significantly increased in blood vessels from eNOS–/– compared with WT mice (Figure 1). In contrast, contractions to the selective 1-adrenergic agonist phenylephrine (0.3 to 10 μmol/L) or to the TxA2 mimic U-46619 (30 to 300 nmol/L) were not significantly different between WT and eNOS–/– aortas (Figure 1C and 1D). The selective ETB receptor agonist S6c (300 nmol/L) did not cause contraction of these abdominal aortas (Figure 1B). Similar to results observed in endothelium-denuded specimens, contractions to ET-1(1-31) (30 nmol/L) in aortas with intact endothelium were also increased in eNOS–/– compared with WT mice (92.9±6.9% and 48.3±6.8% of contraction to K+, respectively, n=5, P<0.01; Figure 2).
Expression of ETA Receptor Is Decreased in eNOS–/– Aorta
The expression of ETA receptor in abdominal aortas of WT and eNOS–/– mice was assessed using immunofluorescent and Western blot analysis. The immunofluorescence of ETA receptor was localized to vascular smooth muscle cells, but not endothelial cells, of WT and eNOS–/– aortas (Figure 3). The intensity of expression was markedly reduced in eNOS–/– cells (average intensity: 9.0±1.2 compared with 92.9±1.7 in WT mice; n=4; P<0.01). Western blot analysis also demonstrated a reduced expression of ETA receptors in endothelium-denuded abdominal aorta from eNOS–/– compared with WT mice (18±4% of expression in WT aortas, n=6, P<0.01; Figure 3).
Role of COX in Contraction to ETA Receptor Activation
Inhibition of COX by indomethacin (3 μmol/L) or of TxA2/PGH2 receptors by SQ-29548 (1 μmol/L) significantly reduced the contraction evoked by 30 nmol/L ET-1(1-31) but did not affect contraction to 2 μmol/L phenylephrine, which was of similar magnitude (Figure 4A). Indomethacin (3 μmol/L) had a greater inhibitory effect against contractions to ET-1(1-31) in eNOS–/– compared with WT aortas. Indeed, after indomethacin, contractions to ET-1 were no longer increased and were actually decreased in eNOS–/– compared with WT aortas (Figure 4B). In addition, SQ-29548 (1 μmol/L) or the selective COX-2 inhibitor celecoxib (3 μmol/L) decreased the contraction evoked by ET-1(1-31) to a similar extent as indomethacin in eNOS–/– mice (Figure 4C and 4D).
COX Expression and Function in WT and eNOS–/– Aorta
COX-1 was not detected in endothelium-denuded abdominal aortas from WT or eNOS–/– mice. In contrast, COX-2 expression could be clearly identified in endothelium-denuded abdominal aortas as well as medial smooth muscle layer but not in the adventitia (Figure 5). In addition, expression of COX-2 was significantly increased in aortas from eNOS–/– compared with WT mice (2.8±0.2 fold, n=4, P<0.01) (Figure 5). To directly assess the role of this increased COX-2 expression, the COX substrate arachidonic acid (10 or 30 μmol/L) was administered to endothelium-denuded abdominal aortas. Arachidonic acid caused contraction that was significantly greater in aortas from eNOS–/– compared with WT mice (Figure 6A). Contractions to arachidonic acid were inhibited by 3 μmol/L indomethacin (Figure 6B).
Generation of the TxA2 metabolite TxB2 was determined in endothelium-denuded abdominal aortas from WT and eNOS–/– mice. ET-1(1-31) (100 nmol/L), but not the 1-adrenergic agonist phenylephrine (10 μmol/L), increased the generation of TxB2 from abdominal aortas of WT and eNOS–/– mice (Figure 7). The ET-1(1-31)–induced production of TxB2 was significantly higher in aortas from eNOS–/– compared with WT mice (434.3±52.4 and 223.9±65.3 pg/mL, respectively, n=5, P<0.05).
Discussion
Endothelin-mediated vasoconstriction contributes to abnormal vasoreactivity of cardiovascular diseases, including hypertension and atherosclerosis.1,2,10–12 This has been attributed to impaired bioavailability of endothelial NO, resulting in increased production of ETs and a generalized increase in vasoconstrictor responsiveness.11,13–17 The present study has identified an additional mechanism whereby the persistent loss of NO can selectively increase vasoconstriction to ET. ETA receptor activation caused contraction of abdominal aortas, which was selectively increased in aortas from eNOS–/– compared with WT mice. This increased contractility occurred despite a decreased expression of ETA receptors in aortic smooth muscle of eNOS–/– mice. However, expression of COX-2 was increased in the aortic smooth muscle of the eNOS–/– aortas, and the stimulated generation of COX-derived contractile mediators dramatically amplified ETA-mediated contraction. These results suggest that COX-2 metabolites may contribute to ET-induced vasoconstriction during the development of cardiovascular disease.
ETA receptors expressed on smooth muscle cells are primarily responsible for vasoconstriction mediated by ETs.1,2 ET-1(1-31), a selective ETA agonist, caused contraction of endothelium-denuded abdominal aortas that was significantly increased in aortas from eNOS–/– compared with WT mice. However, contractions evoked by the 1-adrenergic agonist phenylephrine or the TXA2 analogue U-46619 were similar between aortas of eNOS–/– and WT mice. The ETB receptor agonist S6c (300 nmol/L) did not cause contraction of either blood vessel, which is consistent with the limited role of these receptors in mouse aortic contraction.19 Therefore, the persistent loss of eNOS and NO activity is associated with a selective increase in contractile responsiveness to ETA receptor activation. Furthermore, the enhanced contraction of eNOS–/– aortas to ET-1(1-31) was observed in blood vessels with and without endothelium, indicating that it reflects altered responsiveness of vascular smooth muscle to ETA receptor stimulation.
Western blot and immunofluorescent techniques were used to assess ETA receptor expression. As expected, ETA receptors were detected in smooth muscle, but not in the endothelium, of abdominal aortas from WT or eNOS–/– mice. However, the expression of ETA receptors was surprisingly decreased in aortas from eNOS–/– mice. An explanation for this could be that the loss of NO may facilitate endothelial ET production, resulting in an increase in tissue or circulating ETs, which could then cause downregulation of smooth muscle ETA receptors.14,15,17,22 Indeed, vessels from deoxycorticosterone acetate salt hypertensive rats (which exhibit decreased endothelial NO activity) were reported to have elevated local or circulating ET level and decreased expression of ETA receptors.23–26 However, ETA receptor expression is also decreased in aortas from the spontaneously hypertensive rats, which appear to have normal vascular ET-1 content.22,23,27 Moreover, in eNOS–/– mice, ET immunoreactivity in plasma was reported to be similar to that of control mice.18 Therefore, downregulation of ETA receptors could also be mediated by mechanisms that are independent of increased local or circulating ET levels. Regardless of the underlying mechanism, decreased expression of ETA receptors in eNOS–/– mice would be expected to result in decreased rather than increased contraction to ETA receptor stimulation.
In aortas from WT mice, the COX inhibitor indomethacin or TXA2/PGH2 receptor antagonist SQ-29548 inhibited contractions to ET-1(1-31) but did not affect responses to phenylephrine. These results suggested that contractions to ETA receptor activation are distinct from those induced by 1-adrenergic activation in being mediated by COX-derived metabolites. In a similar manner to ET-11(1-31), the COX substrate arachidonic acid caused indomethacin-sensitive contractions of endothelium-denuded aortas that were significantly increased in blood vessels from eNOS–/– compared with WT mice. Therefore, we considered that increased COX activity might be responsible for the enhanced contraction to ETA receptor stimulation in eNOS–/– mice. This was confirmed by observations that ET-1(1-31) caused increased production of the TXA2 metabolite TXB2 in endothelium-denuded abdominal aorta of eNOS–/– mice compared with WT mice and that indomethacin prevented the increased contraction of eNOS–/– aortas to ET-1(1-31). In fact, after indomethacin, contractions to ETA receptor activation by ET-1(1-31) were decreased in aortas from eNOS–/– compared with WT mice. These results indicate that increased activity of COX in eNOS–/– aortas enables ETA receptor stimulation to overcome the decreased expression of the receptor, which would otherwise result in decreased contractility.
Two isoforms of COX, COX-1 and COX-2, have been identified in cultured vascular smooth muscle cells.28,29 In eNOS–/– mice, the selective COX-2 inhibitor celecoxib or TXA2/PGH2 receptor antagonist SQ-29548 inhibited the contraction induced by ET-1(1-31) to a similar extent as the nonselective COX inhibitor indomethacin. This suggests that enhanced contraction to ET-1(1-31) in eNOS–/– mice is mediated through the COX-2 isoform. Indeed, endothelium-denuded abdominal aortas from WT and eNOS–/– mice were found not to express COX-1, whereas COX-2 was found to be present in the medial layer of both blood vessels, with increased expression in aortas of eNOS–/– compared with WT mice. Therefore, these results identify an important role for COX-2 in ETA-mediated contraction of mouse abdominal aorta. They further demonstrate that increased expression of smooth muscle COX-2 mediates the enhanced contraction to ETA receptor activation in eNOS–/– abdominal aortas. Indeed, the TXA2 analog U-46619 caused similar contractions in aortas from eNOS–/– and WT mice, suggesting that increased COX-dependent contractions to ET(1-31) and arachidonic acid reflected increased mediator production rather than altered sensitivity of TXA2/PGH2 receptors.
Although COX-2 was originally identified as an inducible enzyme, subsequent studies have suggested that COX-2 is also constitutively expressed in some cell types.28–32 The results of the present study suggest that COX-2 is constitutively expressed by smooth muscle of mouse vasculature and can contribute to contractile responses. In addition, COX-2 expression and activity was increased in the smooth muscle of eNOS–/– mice. Similarly, smooth muscle expression of COX-2 is also increased in vascular diseases that are associated with impaired NO bioactivity, including atherosclerosis and diabetes.33–37 Therefore, a persistent loss of NO activity might be a common event that results in increased expression of COX-2. Indeed, transcription of the COX-2 gene is dependent on the activity of factors such as nuclear factor B,38,39 which are sensitive to inhibition by endothelial NO.40–42 On the other hand, the exact mechanism for increased expression of COX-2 in eNOS–/– mice, and how decreased endothelial NO activity contributes to increased expression of COX-2 in these disease states, has not been defined. In patients with essential hypertension and in spontaneously hypertensive rats, ET-1 caused constriction of resistance blood vessels that was highly dependent on COX-derived constrictor mediators.43–45 This COX dependency was not observed in normotensive subjects and was not observed during -adrenergic constriction.43,44 ET-1 or phenylephrine caused similar vasoconstrictor responses in hypertensive and normotensive patients.44 However, after COX inhibition, constriction to ET-1 (but not phenylephrine) was dramatically reduced in hypertensive compared with normotensive individuals.44 Therefore, as occurred in the present study, hypertensive-resistance arteries appear to have a selective reduction in sensitivity to ET-1, which is countered by a selective increase in the contribution of COX to the ET-1 response. Based on the present study, this may reflect decreased expression of ETA receptors countered by increased smooth muscle expression of COX-2. These results emphasize an important pathophysiological role of COX-derived metabolites in the vascular response to ET-1.
The findings of the present study also highlight important differences in the contribution of COX-2–derived metabolites to contractile responses between ETA and other receptors, including 1-adrenoceptor and TXA2/PGH2 receptor. The selective role of COX-2 in contractions to ETA receptor activation likely reflects differences in signaling pathways initiated by these distinct receptor species. These may involve steps that are essential to receptor-mediated PG or TXA2 synthesis, including the activation of phospholipase A2, which leads to the release of arachidonic acid from membrane lipid pool, and the modulation of COX and other downstream enzymes, including TXA2 synthase, which metabolize arachidonic acid to biologically active end products.32,33,37 For example, ETA receptor can activate phospholipase A2 and can also stimulate superoxide production via NADPH oxidase in vascular smooth muscle,26,46 which facilitates formation of lipid peroxides that are required for the activation of COX enzymes.32,33
In summary, smooth muscle contraction to ETA receptor stimulation was selectively increased in aortas from eNOS–/– compared with WT mice. This was associated with a decreased expression of ETA receptors, but increased expression of COX-2, in aortic smooth muscle cells of eNOS–/– compared with WT mice. Indeed, the increased contraction to ETA receptor stimulation was associated with increased production of the TXA2 metabolite TXB2 and was paralleled by increased contraction to the COX substrate arachidonic acid. Furthermore, contractile responses to stimulation of ETA receptors were selectively reduced by inhibition of COX-2 or TXA2/PGH2 receptors. After COX inhibition, contractions to ETA receptor activation were no longer increased and were actually decreased in aortas from eNOS–/– compared with WT aortas. Therefore, these results suggest that increased expression of COX-2 mediates enhanced smooth muscle contraction to ETA receptor activation in eNOS–/– mouse abdominal aorta, overcoming a decreased expression of ETA receptors that would otherwise result in reduced contraction to the mediator.
Acknowledgments
This work was supported by NIH grants HL 67331 and HL 80119 (to N.A.F.).
Footnotes
Presented in part at the 78th Scientific Sessions of the American Heart Association, Dallas, Tex, November 13–16, 2005, and published in abstract form (available at http://circ.ahajournals.org).
N.A.F. is a consultant for Merck & Co Inc.
Both authors contributed equally to this study.
Original received December 5, 2005; revision received March 27, 2006; accepted April 18, 2006.
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the Davis Heart & Lung Research Institute, The Ohio State University, Columbus.
Abstract
The aim of this study was to determine whether prolonged loss of NO activity, in endothelial NO synthase knockout (eNOS–/–) mice, influences endothelin (ET) ETA receptor-mediated smooth muscle contraction and, if so, to define the underlying mechanism(s). In isolated endothelium-denuded abdominal aortas, contractions to the selective ETA receptor agonist ET-1(1-31) were significantly increased in aortas from eNOS–/– compared with wild-type (WT) mice. In contrast, contractions to the 1-adrenergic agonist phenylephrine or the thromboxane (TX) A2 analog U-46619 were similar between eNOS–/– and WT mice. Immunofluorescent and Western blot analysis demonstrated that the aortic expression of ETA receptors was decreased in eNOS–/– compared with WT mice. Contractions evoked by ET-1(1-31), but not phenylephrine, were reduced by inhibition of cyclooxygenase-2 (COX-2) (indomethacin or celecoxib) or of TXA2/prostaglandin H2 receptors (SQ-29548). After COX inhibition, contractions to ET-1(1-31) were no longer increased and were actually decreased in eNOS–/– compared with WT aortas. Western blot analysis revealed that endothelium-denuded abdominal aortas express COX-2, but not COX-1, and that expression of COX-2 was significantly increased in eNOS–/– compared with WT mice. Contractions to the COX substrate arachidonic acid were also increased in eNOS–/– aortas. Furthermore, ET-1(1-31) but not phenylephrine stimulated production of the TXA2 metabolite TXB2, which was increased in eNOS–/– compared with WT aortas. Therefore, COX-2 plays a crucial and selective role in ETA-mediated smooth muscle contraction. Furthermore, COX-2 expression is increased in eNOS–/– mice, which overcomes a reduced expression of ETA receptors and enables a selective increase in contraction to ETA receptor stimulation.
Key Words: endothelin-1(1-31) arachidonic acid thromboxane A2 prostaglandin H2
Introduction
Endothelins (ETs) are a family of peptides that play important roles in regulating cardiovascular function.1,2 ET-1, which causes potent and long-lasting vasoconstriction, is the major form produced by endothelial cells.3 To date, 2 types of ET receptors have been identified: ETA is selectively activated by ET-1(1-31), whereas ETB is selectively stimulated by sarafotoxin S6c.1,2,4,5 In vascular smooth muscle, ETA is the most abundant receptor with activation causing contraction in all vascular regions, irrespective of species.1,2 Although ETB can cause smooth muscle contraction in certain vascular beds,6 activation of ETB is generally associated with vasodilation through the stimulated release of endothelial nitric oxide (NO).7–9
ETA-mediated contraction of vascular smooth muscle may play an important role in the abnormal vasoreactivity associated with cardiovascular diseases, including coronary atherosclerosis and hypertension.1,2,10–12 Although impaired bioavailability of endothelial NO, which is associated with vascular diseases, causes a general increase in constrictor activity, it can also have some selective effects on ET activity. Decreased NO activity can cause increased production of ETs11,13–17 and enhanced constriction to these peptides by impairing ETB-mediated dilation.7,8,18 The aim of the present study was to determine whether a prolonged decrease in NO activity could directly influence ETA receptor-mediated contraction in smooth muscle and, if so, to define the underlying mechanism(s).
Materials and Methods
Chemicals
Indomethacin, N-nitro-L-arginine methyl ester (L-NAME), phenylephrine, sarafotoxin S6c, BQ123, and U-46619 were obtained from Sigma (St Louis, Mo); the selective ETA receptor agonist ET-1(1-31) from Peptide International (Louisville, Ky); the TxA2/PGH2 receptor antagonist S-Q29548 from ICN Pharmaceuticals (Costa Mesa, Calif); and cyclooxygenase (COX) substrate arachidonic acid from Calbiochem (La Jolla, Calif). The selective COX-2 inhibitor celecoxib was kindly provided by Pharmacia (St Louis, Mo). All other chemicals were of the highest commercially available quality.
Animal and Tissue Preparation
Wild-type (WT) and eNOS–/– mice (8 to 12 weeks) from The Jackson Laboratory (Bar Harbor, Me) were euthanized by CO2 inhalation. This procedure was approved by the Institutional Laboratory Animal Care and Use Committee of The Ohio State University. The abdominal aorta, which contracts strongly to ETA receptor activation,19 was rapidly excised and placed in ice-cold PSS (physiological salt solution; in mmol/L: NaCl 123, KCl 4.7, NaHCO3 15.5, KH2PO4 1.2, MgCl2 1.2, CaCl2 1.25, and D-glucose 11.5). Vessels were dissected free of adventitial fat and connective tissue. For tension measurements, aortic segments were cut into 1-mm vascular rings. For all other assays, aortas were cut open and the endothelial cells removed with a moist cotton swab. In some experiments, aortic segments were separated into adventitial and medial layers. All surgical procedures were performed under a binocular microscope.
Isometric Force Measurement
Isometric force measurement was performed as described previously.19,20 Briefly, the vascular ring was mounted between 2 tungsten wires, each with a diameter of 50 μm, in a 37°C water-circulating tissue bath. One wire was stationary, whereas the other was connected to a force transducer (AE 801, Sensor One, Horten, Norway). Because the endothelium might release vasoactive mediators under basal conditions, most experiments were performed on endothelium-denuded blood vessels. Therefore, unless stated otherwise, the endothelium was denuded from all aortic rings. This was achieved by rotating the rings around the tungsten wires while the passive tension was kept at 100 mg.20 Thereafter, tissues were stimulated with 60 mmol/L K+-PSS (equimolar replacement of NaCl with KCl) every 15 minutes, and the resting tension increased in a stepwise manner. After the equilibration, the resting tension was adjusted to approximately 300 mg, at which the maximal response to K+ was obtained.
Contraction evoked by an agonist was expressed as a percentage of the response to 60 mmol/L K+. Concentration-effect curves to U-46619 or phenylephrine were generated by increasing the concentration of the agonists in half-log increments, once the contraction to the previous concentration had stabilized. Because responses to ET-1(1-31) were transient, contractions to the agonist were assessed by administering only a single concentration of the agonist. In all experiments, only a single curve (U46619, phenylephrine) or a single contraction [ET-1(1-31)] was analyzed on each aortic specimen. In addition, L-NAME (1 mmol/L), which did not affect ETA-mediated contraction in eNOS–/– mice, was added 20 minutes before the application of an agonist in WT mouse specimens. COX inhibitors (indomethacin, 3 μmol/L; celecoxib, 3 μmol/L) or the TxA2/PGH2 receptor antagonist (SQ-29548, 1 μmol/L) were applied 10 minutes before the application of an agonist.
Western Blot
Proteins (20 μg for COX analysis, 40 μg for ETA analysis) isolated from endothelium-denuded aortas were separated by electrophoresis on 10% SDS-PAGE and transferred onto a nitrocellulose membrane. After incubation with a monoclonal anti-COX-1 (Cayman Chemical, Ann Arbor, Mich; dilution: 1:1000), a polyclonal anti-COX-2 antibody (Cayman Chemical; dilution: 1:1000), or a monoclonal anti-ETA receptor antibody (BD Biosciences, Palo Alto, Calif; dilution 1:800), the nitrocellulose membranes were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody. The immunocomplex was detected with ECL plus kit (Amersham, Buckinghamshire, UK), and the band density was analyzed using a personal densitometer (Molecular Dynamics, Sunnyvale, Calif).
Immunofluorescence
Intact mouse abdominal aortic segment was frozen and cut into 5-μm sections. Tissue sections were attached to glass slides and fixed with acetone. After treatment with primary antibody (1:100 dilution, sheep anti-ETA; Alexis, San Diego, Calif),21 tissues sections were incubated with anti-sheep Alexa flour-488 (Molecular Probes, Eugene, Ore). The fluorescent staining was detected using a Zeiss 510 laser scanning microscope equipped with a x63 water immersion objective (NA 1.2). The image was analyzed by using Image J (NIH, Rockville, Md) software. The fluorescent intensity was expressed as the average pixel value, which is the sum of intensities of all the pixels in the area of selection divided by number of pixels.
Measurement of TXA2 Metabolite TXB2
Endothelium-denuded aortic strips (10 mm) were equilibrated in PSS at 37°C for 30 minutes. Thereafter, vessel strips were sequentially incubated in 150 μL of PSS (baseline) and 100 nmol/L ET-1(1-31) or 10 μmol/L PE in 150 μL of PSS for 10 minutes, respectively. The PSS was then removed, snap frozen with liquid nitrogen, and stored at –80°C. TXB2 was measured with an EIA kit (Amersham International), according to the instructions of the manufacturer.
Data Analysis
Data are expressed as means±SEM for n number of experiments, where n equals the number of animals from which blood vessels were studied. Statistical evaluation of the data were performed by Student’s t test for either paired or unpaired observations. When more than 2 means were compared, ANOVA was used: either a 1-way ANOVA with Dunnett’s post hoc test or 2-way ANOVA followed by Bonferroni’s post hoc test (GraphPad 4 Software, San Diego, Calif). Values were considered to be statistically significant when P was <0.05.
Results
Contraction to ETA Receptor Activation Is Increased in eNOS–/– Aorta
The selective ETA receptor agonist ET-1(1-31) (10 to 100 nmol/L) caused contractions of endothelium-denuded abdominal aortas that were significantly increased in blood vessels from eNOS–/– compared with WT mice (Figure 1). In contrast, contractions to the selective 1-adrenergic agonist phenylephrine (0.3 to 10 μmol/L) or to the TxA2 mimic U-46619 (30 to 300 nmol/L) were not significantly different between WT and eNOS–/– aortas (Figure 1C and 1D). The selective ETB receptor agonist S6c (300 nmol/L) did not cause contraction of these abdominal aortas (Figure 1B). Similar to results observed in endothelium-denuded specimens, contractions to ET-1(1-31) (30 nmol/L) in aortas with intact endothelium were also increased in eNOS–/– compared with WT mice (92.9±6.9% and 48.3±6.8% of contraction to K+, respectively, n=5, P<0.01; Figure 2).
Expression of ETA Receptor Is Decreased in eNOS–/– Aorta
The expression of ETA receptor in abdominal aortas of WT and eNOS–/– mice was assessed using immunofluorescent and Western blot analysis. The immunofluorescence of ETA receptor was localized to vascular smooth muscle cells, but not endothelial cells, of WT and eNOS–/– aortas (Figure 3). The intensity of expression was markedly reduced in eNOS–/– cells (average intensity: 9.0±1.2 compared with 92.9±1.7 in WT mice; n=4; P<0.01). Western blot analysis also demonstrated a reduced expression of ETA receptors in endothelium-denuded abdominal aorta from eNOS–/– compared with WT mice (18±4% of expression in WT aortas, n=6, P<0.01; Figure 3).
Role of COX in Contraction to ETA Receptor Activation
Inhibition of COX by indomethacin (3 μmol/L) or of TxA2/PGH2 receptors by SQ-29548 (1 μmol/L) significantly reduced the contraction evoked by 30 nmol/L ET-1(1-31) but did not affect contraction to 2 μmol/L phenylephrine, which was of similar magnitude (Figure 4A). Indomethacin (3 μmol/L) had a greater inhibitory effect against contractions to ET-1(1-31) in eNOS–/– compared with WT aortas. Indeed, after indomethacin, contractions to ET-1 were no longer increased and were actually decreased in eNOS–/– compared with WT aortas (Figure 4B). In addition, SQ-29548 (1 μmol/L) or the selective COX-2 inhibitor celecoxib (3 μmol/L) decreased the contraction evoked by ET-1(1-31) to a similar extent as indomethacin in eNOS–/– mice (Figure 4C and 4D).
COX Expression and Function in WT and eNOS–/– Aorta
COX-1 was not detected in endothelium-denuded abdominal aortas from WT or eNOS–/– mice. In contrast, COX-2 expression could be clearly identified in endothelium-denuded abdominal aortas as well as medial smooth muscle layer but not in the adventitia (Figure 5). In addition, expression of COX-2 was significantly increased in aortas from eNOS–/– compared with WT mice (2.8±0.2 fold, n=4, P<0.01) (Figure 5). To directly assess the role of this increased COX-2 expression, the COX substrate arachidonic acid (10 or 30 μmol/L) was administered to endothelium-denuded abdominal aortas. Arachidonic acid caused contraction that was significantly greater in aortas from eNOS–/– compared with WT mice (Figure 6A). Contractions to arachidonic acid were inhibited by 3 μmol/L indomethacin (Figure 6B).
Generation of the TxA2 metabolite TxB2 was determined in endothelium-denuded abdominal aortas from WT and eNOS–/– mice. ET-1(1-31) (100 nmol/L), but not the 1-adrenergic agonist phenylephrine (10 μmol/L), increased the generation of TxB2 from abdominal aortas of WT and eNOS–/– mice (Figure 7). The ET-1(1-31)–induced production of TxB2 was significantly higher in aortas from eNOS–/– compared with WT mice (434.3±52.4 and 223.9±65.3 pg/mL, respectively, n=5, P<0.05).
Discussion
Endothelin-mediated vasoconstriction contributes to abnormal vasoreactivity of cardiovascular diseases, including hypertension and atherosclerosis.1,2,10–12 This has been attributed to impaired bioavailability of endothelial NO, resulting in increased production of ETs and a generalized increase in vasoconstrictor responsiveness.11,13–17 The present study has identified an additional mechanism whereby the persistent loss of NO can selectively increase vasoconstriction to ET. ETA receptor activation caused contraction of abdominal aortas, which was selectively increased in aortas from eNOS–/– compared with WT mice. This increased contractility occurred despite a decreased expression of ETA receptors in aortic smooth muscle of eNOS–/– mice. However, expression of COX-2 was increased in the aortic smooth muscle of the eNOS–/– aortas, and the stimulated generation of COX-derived contractile mediators dramatically amplified ETA-mediated contraction. These results suggest that COX-2 metabolites may contribute to ET-induced vasoconstriction during the development of cardiovascular disease.
ETA receptors expressed on smooth muscle cells are primarily responsible for vasoconstriction mediated by ETs.1,2 ET-1(1-31), a selective ETA agonist, caused contraction of endothelium-denuded abdominal aortas that was significantly increased in aortas from eNOS–/– compared with WT mice. However, contractions evoked by the 1-adrenergic agonist phenylephrine or the TXA2 analogue U-46619 were similar between aortas of eNOS–/– and WT mice. The ETB receptor agonist S6c (300 nmol/L) did not cause contraction of either blood vessel, which is consistent with the limited role of these receptors in mouse aortic contraction.19 Therefore, the persistent loss of eNOS and NO activity is associated with a selective increase in contractile responsiveness to ETA receptor activation. Furthermore, the enhanced contraction of eNOS–/– aortas to ET-1(1-31) was observed in blood vessels with and without endothelium, indicating that it reflects altered responsiveness of vascular smooth muscle to ETA receptor stimulation.
Western blot and immunofluorescent techniques were used to assess ETA receptor expression. As expected, ETA receptors were detected in smooth muscle, but not in the endothelium, of abdominal aortas from WT or eNOS–/– mice. However, the expression of ETA receptors was surprisingly decreased in aortas from eNOS–/– mice. An explanation for this could be that the loss of NO may facilitate endothelial ET production, resulting in an increase in tissue or circulating ETs, which could then cause downregulation of smooth muscle ETA receptors.14,15,17,22 Indeed, vessels from deoxycorticosterone acetate salt hypertensive rats (which exhibit decreased endothelial NO activity) were reported to have elevated local or circulating ET level and decreased expression of ETA receptors.23–26 However, ETA receptor expression is also decreased in aortas from the spontaneously hypertensive rats, which appear to have normal vascular ET-1 content.22,23,27 Moreover, in eNOS–/– mice, ET immunoreactivity in plasma was reported to be similar to that of control mice.18 Therefore, downregulation of ETA receptors could also be mediated by mechanisms that are independent of increased local or circulating ET levels. Regardless of the underlying mechanism, decreased expression of ETA receptors in eNOS–/– mice would be expected to result in decreased rather than increased contraction to ETA receptor stimulation.
In aortas from WT mice, the COX inhibitor indomethacin or TXA2/PGH2 receptor antagonist SQ-29548 inhibited contractions to ET-1(1-31) but did not affect responses to phenylephrine. These results suggested that contractions to ETA receptor activation are distinct from those induced by 1-adrenergic activation in being mediated by COX-derived metabolites. In a similar manner to ET-11(1-31), the COX substrate arachidonic acid caused indomethacin-sensitive contractions of endothelium-denuded aortas that were significantly increased in blood vessels from eNOS–/– compared with WT mice. Therefore, we considered that increased COX activity might be responsible for the enhanced contraction to ETA receptor stimulation in eNOS–/– mice. This was confirmed by observations that ET-1(1-31) caused increased production of the TXA2 metabolite TXB2 in endothelium-denuded abdominal aorta of eNOS–/– mice compared with WT mice and that indomethacin prevented the increased contraction of eNOS–/– aortas to ET-1(1-31). In fact, after indomethacin, contractions to ETA receptor activation by ET-1(1-31) were decreased in aortas from eNOS–/– compared with WT mice. These results indicate that increased activity of COX in eNOS–/– aortas enables ETA receptor stimulation to overcome the decreased expression of the receptor, which would otherwise result in decreased contractility.
Two isoforms of COX, COX-1 and COX-2, have been identified in cultured vascular smooth muscle cells.28,29 In eNOS–/– mice, the selective COX-2 inhibitor celecoxib or TXA2/PGH2 receptor antagonist SQ-29548 inhibited the contraction induced by ET-1(1-31) to a similar extent as the nonselective COX inhibitor indomethacin. This suggests that enhanced contraction to ET-1(1-31) in eNOS–/– mice is mediated through the COX-2 isoform. Indeed, endothelium-denuded abdominal aortas from WT and eNOS–/– mice were found not to express COX-1, whereas COX-2 was found to be present in the medial layer of both blood vessels, with increased expression in aortas of eNOS–/– compared with WT mice. Therefore, these results identify an important role for COX-2 in ETA-mediated contraction of mouse abdominal aorta. They further demonstrate that increased expression of smooth muscle COX-2 mediates the enhanced contraction to ETA receptor activation in eNOS–/– abdominal aortas. Indeed, the TXA2 analog U-46619 caused similar contractions in aortas from eNOS–/– and WT mice, suggesting that increased COX-dependent contractions to ET(1-31) and arachidonic acid reflected increased mediator production rather than altered sensitivity of TXA2/PGH2 receptors.
Although COX-2 was originally identified as an inducible enzyme, subsequent studies have suggested that COX-2 is also constitutively expressed in some cell types.28–32 The results of the present study suggest that COX-2 is constitutively expressed by smooth muscle of mouse vasculature and can contribute to contractile responses. In addition, COX-2 expression and activity was increased in the smooth muscle of eNOS–/– mice. Similarly, smooth muscle expression of COX-2 is also increased in vascular diseases that are associated with impaired NO bioactivity, including atherosclerosis and diabetes.33–37 Therefore, a persistent loss of NO activity might be a common event that results in increased expression of COX-2. Indeed, transcription of the COX-2 gene is dependent on the activity of factors such as nuclear factor B,38,39 which are sensitive to inhibition by endothelial NO.40–42 On the other hand, the exact mechanism for increased expression of COX-2 in eNOS–/– mice, and how decreased endothelial NO activity contributes to increased expression of COX-2 in these disease states, has not been defined. In patients with essential hypertension and in spontaneously hypertensive rats, ET-1 caused constriction of resistance blood vessels that was highly dependent on COX-derived constrictor mediators.43–45 This COX dependency was not observed in normotensive subjects and was not observed during -adrenergic constriction.43,44 ET-1 or phenylephrine caused similar vasoconstrictor responses in hypertensive and normotensive patients.44 However, after COX inhibition, constriction to ET-1 (but not phenylephrine) was dramatically reduced in hypertensive compared with normotensive individuals.44 Therefore, as occurred in the present study, hypertensive-resistance arteries appear to have a selective reduction in sensitivity to ET-1, which is countered by a selective increase in the contribution of COX to the ET-1 response. Based on the present study, this may reflect decreased expression of ETA receptors countered by increased smooth muscle expression of COX-2. These results emphasize an important pathophysiological role of COX-derived metabolites in the vascular response to ET-1.
The findings of the present study also highlight important differences in the contribution of COX-2–derived metabolites to contractile responses between ETA and other receptors, including 1-adrenoceptor and TXA2/PGH2 receptor. The selective role of COX-2 in contractions to ETA receptor activation likely reflects differences in signaling pathways initiated by these distinct receptor species. These may involve steps that are essential to receptor-mediated PG or TXA2 synthesis, including the activation of phospholipase A2, which leads to the release of arachidonic acid from membrane lipid pool, and the modulation of COX and other downstream enzymes, including TXA2 synthase, which metabolize arachidonic acid to biologically active end products.32,33,37 For example, ETA receptor can activate phospholipase A2 and can also stimulate superoxide production via NADPH oxidase in vascular smooth muscle,26,46 which facilitates formation of lipid peroxides that are required for the activation of COX enzymes.32,33
In summary, smooth muscle contraction to ETA receptor stimulation was selectively increased in aortas from eNOS–/– compared with WT mice. This was associated with a decreased expression of ETA receptors, but increased expression of COX-2, in aortic smooth muscle cells of eNOS–/– compared with WT mice. Indeed, the increased contraction to ETA receptor stimulation was associated with increased production of the TXA2 metabolite TXB2 and was paralleled by increased contraction to the COX substrate arachidonic acid. Furthermore, contractile responses to stimulation of ETA receptors were selectively reduced by inhibition of COX-2 or TXA2/PGH2 receptors. After COX inhibition, contractions to ETA receptor activation were no longer increased and were actually decreased in aortas from eNOS–/– compared with WT aortas. Therefore, these results suggest that increased expression of COX-2 mediates enhanced smooth muscle contraction to ETA receptor activation in eNOS–/– mouse abdominal aorta, overcoming a decreased expression of ETA receptors that would otherwise result in reduced contraction to the mediator.
Acknowledgments
This work was supported by NIH grants HL 67331 and HL 80119 (to N.A.F.).
Footnotes
Presented in part at the 78th Scientific Sessions of the American Heart Association, Dallas, Tex, November 13–16, 2005, and published in abstract form (available at http://circ.ahajournals.org).
N.A.F. is a consultant for Merck & Co Inc.
Both authors contributed equally to this study.
Original received December 5, 2005; revision received March 27, 2006; accepted April 18, 2006.
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