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Cardiovascular Research Advance Access first published online on December 18, 2007
This version [Corrected Proof] published online on March 1, 2008
Cardiovascular Research, doi:10.1093/cvr/cvm112
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2007. For permissions please email: journals.permissions@oxfordjournals.org

The role of prostaglandin E and thromboxane-prostanoid receptors in the response to prostaglandin E2 in the aorta of Wistar Kyoto rats and spontaneously hypertensive rats

Eva H.C. Tang1, Boye L. Jensen2, Ole Skott2, George P.H. Leung1, Michel Feletou3, Ricky Y.K. Man1 and Paul M. Vanhoutte1,*

1 Department of Pharmacology, The University of Hong Kong, 2/F, Laboratory Block, Faculty of Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong
2 Department of Physiology and Pharmacology, University of Southern Denmark, Odense, Denmark
3 Department of Angiology, Institut de Recherches Servier, Suresnes, France

* Corresponding author. Tel: +852 28199250; fax: +852 28170859. E-mail address: vanhoutte.hku{at}hku.hk

Received 30 July 2007; revised 2 December 2007; accepted 17 December 2007

Time for primary review: 24 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 Supplementary material
 Funding
 References
 
Aims: The present study examined the hypothesis that prostaglandin E2 (PGE2) through activation of prostaglandin E (EP) receptor contributes to endothelium-dependent contractions.

Methods and results: Western blotting revealed that the protein expression of EP1 receptor was significantly down-regulated in the aorta of the spontaneously hypertensive rat (SHR), but there was no significant difference in the expression of EP2, EP4, and total EP3 receptors between preparations of Wistar Kyoto rats (WKY) and SHR. Isometric tension studies showed that low concentrations of PGE2 caused endothelium-dependent relaxations in WKY but not in aortas of the SHR. High concentrations of PGE2 evoked contractions predominately through the activation of thromboxane-prostanoid (TP) receptors in the WKY, but involves the dual activation EP and TP receptors in the SHR. SQ29,548, BAYu3405 and Terutroban (TP receptor antagonists), and AH6809 (non-selective EP receptor antagonist) abolished, while SC19220 (preferential EP1 receptor antagonist) did not inhibit endothelium-dependent contractions. Both SC19220 and AH6809 significantly inhibited contractions to U46619 [GenBank] (TP receptor agonist).

Conclusion: The present study demonstrates that the contraction caused by PGE2 in the SHR aorta is dependent on the activation of EP1 and TP receptors, but that endothelium-dependent contractions do not require the former. Thus, PGE2 is unlikely to be an endothelium-derived contracting factor in this artery. The ability of AH6809 to inhibit endothelium-dependent contractions can be attributed to its partial antagonism at TP receptors. Nevertheless, the impairment of PGE2-mediated relaxation may contribute to endothelial dysfunction in the aorta of the SHR.

KEYWORDS Endothelial factors; Endothelial function; Vasoconstriction/dilation; Endothelium-derived contracting factor


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 Supplementary material
 Funding
 References
 
Endothelial cells release a mixture of endothelium-derived relaxing (EDRF) and endothelium-derived contracting (EDCF) factors to help maintain the local vascular tone. Exposure of the endothelium to chronic hypertension leads to the enhanced release of EDCF and reduces the production of, or the sensitivity to EDRF causing endothelial dysfunction.1–4 The production of EDCF is prominent in arteries of mature spontaneously hypertensive rats (SHR), but not in those of age-matched normotensive Wistar Kyoto rats (WKY).1,3,4 In the SHR aorta, acetylcholine activates endothelial muscarinic receptors, resulting in an abnormal increase in intracellular calcium concentration that in turn stimulates phospholipase A2 to metabolize fatty acids into arachidonic acid.5 The endothelial cyclooxygenase transforms arachidonic acid to form vasoconstrictor prostanoids.6 They include endoperoxides and prostacyclin which diffuse to, and activate thromboxane-prostanoid (TP) receptors of the underlying vascular smooth muscle, thus causing endothelium-dependent contractions.3,6–8 However, the SHR aorta with endothelium also releases prostaglandin E2 in response to releasers of EDCF.6,9,10 It is unknown whether or not this endothelium-derived prostaglandin E2 contributes to endothelium-dependent contractions in the SHR aorta, as is the case in arteries of diabetic animals.11

There are four known subtypes of EP receptors, denoted as EP1 to EP4, which are encoded by different genes and are G-protein-coupled seven transmembrane receptors.12 Activation of EP receptors has a wide range of effects in inflammation, renal function, the central nervous system, and the gastrointestinal tract.13 EP1 receptor activation causes an increase in cytosolic calcium concentration through coupling to phospholipase C.12,13 EP2 and EP4 receptors are coupled to the activation of adenylyl cyclase and their stimulation increases the concentration of cyclic adenosine monophosphate (cAMP).12,13 EP3 is the most complex EP receptor, with alternate splicing potentially generating a variety of subtypes which are coupled to at least three second messenger systems, usually leading to the inhibition of adenylyl cyclase and/or an increase in cytosolic calcium.12,13

Pharmacological studies indicate the presence of EP receptors in the vasculature.11,14–18 Expression of EP receptors is confirmed in cultured smooth muscle cells of the porcine cerebral artery,19 the human aorta,15 and the human umbilical artery.20 The genetic expression of EP3 and EP4 receptors is augmented in the vascular smooth muscle of the SHR.21 Activation of EP1 and EP3 results predominately in vasoconstriction, whereas the vasodilator effect of prostaglandin E2 is mediated presumably by EP2 and EP4.12,13 EP4 receptor-mediated endothelium-dependent stimulation of endothelial nitric oxide synthase has been demonstrated in the aorta of the mouse.18

The present study examined the hypothesis that prostaglandin E2 through the activation of EP receptors contributes to endothelium-dependent contractions. Whether or not the different EP receptor subtypes are expressed in the rat aorta was evaluated. Furthermore, the inhibitory effect of several EP and TP receptor antagonists was studied in order to define the relative involvement of these prostanoid receptor subtypes in endothelium-dependent contractions.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusions
 Supplementary material
 Funding
 References
 
2.1 Tissue preparation
WKY and SHR (36-weeks old) were maintained under a 12 h light/12 h dark cycle at 21 ± 1°C and were fed with standard laboratory chow and water ad libitum. They were anesthetized with pentobarbital sodium (70 mg/mL/kg; Ganes Chemicals Inc., Pennsville, NJ, USA). Their thoracic aorta was quickly excised and placed in Krebs-Ringer bicarbonate buffer of the following composition (mmol/L): NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, and glucose 11.1 (control solution). The aortas were cleaned of adherent connective tissue and were either cut into rings (approximately 3 mm in length) for the measurement of isometric tension, or immediately frozen in liquid nitrogen and stored at –80°C until protein extraction. The present investigations conform with the Guide for the Care and Use of laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996).

2.2 Immunohistochemistry
Polyclonal antibodies for EP1, EP2, EP3, and EP4 receptors were from Cayman Chemicals (Ann Arbor, MI, USA). For EP1 and EP3 detection, antigen retrieval was carried out by pepsin treatment (0.4% in 0.1 mol/L HCl, 37°C for 30 min). For EP2 detection, antigen retrieval was carried out by pressure cooking in citrate buffer (Dako, Glostrup, Denmark) for 5 min. The sections were blocked with 5% dry milk in 0.05% Tween-Tris-buffered saline (TTBS), and then incubated with diluted primary antibodies (1:500 EP1, 1:1000 EP2, 1:50 EP3) overnight at 4°C. Next, the sections were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (1:2000, Dako) in TTBS for 45 min at room temperature. Signals were visualized by incubation with 0.01% diaminobenzidine (Dako) and 0.02% H2O2. Sections were inspected with an epifluorescence microscope (Olympus BX51, Ballerup, Denmark). Either kidney or brain sections were used for positive control staining.22

2.3 Protein extraction and immunoblotting
Whole aortas were cut into small pieces and then homogenized in lysis buffer (20 mmol/L Tris–HCl, 1% Triton X-100, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerophosphate, 1 mmol/L sodium orthovanadate) supplemented with a cocktail of protease inhibitors [phenylmethylsulfonyl fluoride (100 mmol/L), trypsin inhibitor (10 µg/mL), leupeptin (1 mg/mL), and pepstatin A (2 µg/mL)]. The mixture was centrifuged at 5000 rpm at 4°C for 3 min, and the supernatant was kept at –80°C until use. The protein concentration was determined spectrophotometrically using the Bradford protein assay reagent (Bio-rad, Hercules, CA, USA) using serial dilution of bovine serum albumin as a standard. For gel electrophoresis, 20 µg of tissue homogenate protein were used. The samples were mixed with 1x sample buffer (NuPAGE® LDS Sample Buffer 4x, Invitrogen, Carlsbad, CA, USA) and 1x reducing agent (10x Reducing Agent, Invitrogen), and diluted with ultrapure water to obtain 20 µL. The samples were boiled for 5 min at 95°C and subsequently separated by SDS–PAGE (10%) at 200 V, 500 mA for 50 min. The proteins were transferred electrophoretically onto nitrocellulose membranes. The blotting was performed at 1000 V, 300 mA for 50 min. Subsequently, the membranes were blocked in Tris-buffered saline (TBS) with 5% dry milk at room temperature for 1 h, washed in TTBS, and then incubated with primary antibody (1:500 EP1, 1:2000 EP2, 1:2000 EP3, 1:250 EP4, 1:100 TP receptors, Cayman Chemicals) in TBS for 2 h at room temperature for EP1, EP2, and EP3 receptors or overnight incubation at 4°C for EP4 and TP receptors. Then, the membranes were incubated with HRP-conjugated anti-rabbit antibody (1:6000 in TBS, room temperature, 1 h, Amersham Biosciences, Piscataway, NJ, USA). Bound secondary antibody was detected by chemiluminiscence (Amersham Biosciences) and exposed to X-ray film. If an antibody detected more than one band, the experiment was repeated by incubation of the primary antibody with its respective blocking peptide (Cayman Chemicals) in a 1:2 antibody: blocking peptide w/w ratio according to manufacturer’s instructions, to exclude the possibility of non-specific binding. To reprobe β-actin, membranes were stripped using TTBS (pH 2) for 5 min at room temperature. After washing, they were incubated with TBS with 5% dry milk (for an hour) and subsequently with the monoclonal β-actin antibody (Sigma, St Louis, MO, USA). The optical densities of the protein bands were determined with the computerized program MultiAnalysis (Bio-rad). Densitometric analysis was normalized with the immunoreactive β-actin band. Changes in protein level were expressed in relative terms of those observed in preparations of control rats run on identical gels.

2.4 Isometric tension recordings
Aortic rings from SHR or WKY were equilibrated in organ chambers as described.23 To study endothelium-dependent contractions, quiescent rings were incubated with N{omega}-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor; 100 µmol/L; Sigma) for 40 min in order to optimize the production of EDCF23,24 and then exposed to progressively increasing concentrations of acetylcholine (10 nmol/L to 100 µmol/L, Sigma). To study concentration responses to prostaglandin E2 (Cayman Chemicals), sulprostone (a preferential EP1 and EP3 receptor agonist; Cayman Chemicals) and U46619 [GenBank] (a synthetic TP receptor agonist,25 Biomol, PA, USA), quiescent rings or preparations contracted with phenylephrine (1 µmol/L, Sigma) were exposed to progressively increasing concentrations of the agonists. For quiescent preparations, all changes in tension were expressed as percentage of the reference contraction to 60 mmol/L KCl. For previously contracted rings, all changes in tension were expressed as percentage of the response to phenylephrine. In some preparations, the endothelium was removed mechanically by inserting the tip of a syringe needle into the rings and rolling them back and forth in a sylgard-based container filled with control solution. In other experiments, the rings were treated with indomethacin (cyclooxygenase inhibitor, 10 µmol/L; Sigma),5,8 Terutroban (also known as S18886 or Triplion®; TP receptor antagonist, 0.1 µmol/L; a kind gift of the Institut de Recherches Servier, Suresnes, France),8,26 AH6809 (non-selective EP receptor antagonist, 30 µmol/L, Cayman Chemicals),11,27 SC19220 (selective EP1 receptor antagonist, 100 µmol/L, Cayman Chemicals),11,28 SQ29,548 (selective TP receptor antagonist, 10 µmol/L, Cayman Chemicals),29 or BAYu3405 (selective TP receptor antagonist, 1 µmol/L, Cayman Chemicals)30 for 40 min before concentration–response curves were recorded. The concentrations used of the antagonists were selected from earlier work or from the literature demonstrating their efficacy.

2.5 Statistical analysis
The results are presented as means±SEM with n being the number of individual observations. The area under each dose-response curves were computed and statistical significances between the area under curves of different datasets were analysed using Student’s t-test for comparison of two groups or one-way ANOVA followed by the Bonferroni post-test for multiple comparisons. Western blots data were analysed using one sample t-test that compares the means of the SHR data against the hypothetical WKY mean of 100%. All statistical analysis and area under the curve calculation were performed using Prism version 3a (GraphPad Software, San Diego, CA, USA). Values of P less than 0.05 were considered to indicate statistically significant differences.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusions
 Supplementary material
 Funding
 References
 
3.1 Potassium chloride and phenylephrine-mediated contractions
The contractions evoked by 60 mmol/L KCl in WKY and SHR aortic rings with endothelium were 1.457 ± 0.043 and 1.460 ± 0.041 g, respectively (n = 30). The contractions evoked by 60 mmol/L KCl in WKY and SHR aortic rings without endothelium were 1.181 ± 0.006 and 1.221 ± 0.032 g, respectively (n = 30). The contractions evoked by phenylephrine (1 µmol/L) in WKY and SHR aortic rings with endothelium were 1.443 ± 0.068 and 1.449 ± 0.089 g, respectively (n = 10). The contractions obtained with KCl (60 mmol/L) (in ring with or without endothelium) or phenylephrine (1 µmol/L) (in rings with endothelium) of WKY and SHR were not statistically significantly different.

3.2 Immunochemistry
EP1 was abundantly and evenly expressed throughout the multiple vascular smooth muscle layers and on the monolayer of endothelial cells. The abundance of EP1 was higher in WKY compared to the SHR (Figure Figure 1A). EP2 receptors were detected in aortas of both WKY and SHR, scattered throughout the layers of smooth muscle cells (Figure 1). It appeared difficult to ascertain the presence of EP2 receptors on the monolayer of endothelial cells due to the weakness of the signal. EP3 receptors were found on the endothelial cells and throughout the smooth muscle layers (1A). There was no apparent difference in the abundance of EP3 receptors between WKY and SHR. Positive staining of EP4 receptors could not be obtained in aortas, kidney, or brain sections of the rat.


Figure 1
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  		Figure 1 (A) Immunohistochemical detection of EP1, EP2, and EP3 receptors in aortas from 36-weeks-old WKY and SHR. Western blots for (B) EP1, (C) EP2, (D) EP3, (E) EP4, (F) TP receptors and β-actin in WKY (n = 6) and SHR (n = 6) aortic homogenates. All protein expressions were normalized to the abundance of β-actin and then further expressed in relative changes compared to the preparations from WKY. *P < 0.05 vs. WKY; #Band disappeared after the incubation of the primary antibody with the respective blocking peptide. BP, blocking peptide. For colour version of Figure 1A, see Supplementary material online, Figure S1A.

 
3.3 Protein expression
The proteins of the four EP receptor subtypes were expressed in the aorta of both WKY and SHR (Figure 1B–E).

The EP1 polyclonal antibody detected one discrete protein band between 37 and 49 kDa. The expression of EP1 was statistically significantly higher in the WKY than in the SHR aorta (Figure 1B).

The EP2 polyclonal antibody detected one band between 49 and 64 kDa. There was no statistically significant difference in EP2 protein expression between the two strains (Figure 1C).

The EP3 polyclonal antibody detected five separate bands with molecular masses of approximately 120, 90, 65, 50, and 40 kDa (Figure 1D). All five bands were no longer evident in the absence of the primary antibody or when the primary antibody was pre-incubated with the appropriate antigenic peptide. There was no statistically significant difference in the expression level of the EP3 (~120 kDa) and EP3 (~65 kDa) proteins between WKY and SHR. However, significantly lower amount of the EP3 (~90 kDa) but higher amounts of the EP3 (~50 kDa) and EP3 (~40 kDa) proteins were expressed in the SHR compared to the WKY aorta (Figure 1D). The expression of the total EP3 receptor protein (five bands combined) did not differ between aortic samples of WKY and SHR (data not shown). The EP3 polyclonal antibody detected six discrete protein bands in homogenates of the brain. Five of the six bands mimicked those obtained from aortic samples, but there was an extra band at ~72 kDa (Figure 1D). In the kidney homogenates, the EP3 polyclonal antibody detected six discrete protein bands, three bands with molecular masses greater than 115 kDa and the other bands corresponded to molecular masses of approximately 90, 65, and 50 kDa (Figure 1D).

The EP4 polyclonal antibody detected three bands. One band corresponded to approximately 52 kDa, the second band lied between the 35 and 47 kDa marker and the third band corresponded to approximately 25 kDa (Figure 1E). The band between 37 and 47 kDa disappeared, but the bands at ~52 and ~25 kDa remained when the primary antibody was pre-incubated the appropriate antigenic peptide (Figure 1E). There was no statistically significant difference in the expression level of the EP4 receptor protein between WKY and SHR (Figure 1E).

The TP polyclonal antibody detected a band between 82 and 115 kDa. There was no statistically significant difference in the expression level of the TP receptor protein between WKY and SHR (Figure 1F).

3.4 Concentration–responses to prostaglandin E2 in contracted rings
Prostaglandin E2 induced cumulative concentration- and endothelium-dependent relaxations of phenylephrine-contracted aortic rings of WKY, but not of SHR (Figure 2). The maximal relaxation approximated to 20% of the phenylephrine-induced contraction and this was achieved at a prostaglandin E2 concentration as low as 1 nmol/L (Figure 2). Higher concentrations (>0.1 µmol/L) of prostaglandin E2 induced contractions in aortas of both WKY and SHR (Figure 2).


Figure 2
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  		Figure 2 Response to increasing concentrations of prostaglandin E2 in phenylephrine (PE)-contracted aortic rings of WKY and SHR with and without endothelium. Data are means±SEM; n = 5. (+EC = with endothelium; –EC = without endothelium).

 
3.5 Concentration–responses to prostaglandin E2 and sulprostone in quiescent rings
Prostaglandin E2 and sulprostone caused cumulative concentration-dependent contractions in quiescent aortic rings with and without endothelium of WKY and SHR (Figure 3). The presence of the endothelium significantly reduced the contractions to prostaglandin E2 (Figure 3A and C) and sulprostone (Figure 3B and D) in both rat strains. A significantly greater reduction was observed in aortas of WKY during contractions to prostaglandin E2 (Figure 3A vs. C). Pre-treatment of the aortic rings with L-NAME (10–4 mol/L) abolished the inhibitory effects of the endothelium against the contractions induced by prostaglandin E2 and sulprostrone (Figure 3A–D). In aortic rings without endothelium, the contractions to prostaglandin E2 and sulprostone were not significantly different between preparations of WKY and SHR (Figure 3E and F).


Figure 3
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  		Figure 3 Effects of the endothelium and L-NAME treatment (100 µmol/L) on concentration–response curve to (A and C) prostaglandin E2 and (B and D) sulprostone in aortic rings of WKY and SHR. Comparison of the concentration–response curves to (E) prostaglandin E2 and (F) sulprostone in aortic rings without endothelium of WKY and SHR. Data are means±SEM and expressed as percentage of a reference contraction to KCl (60 mmol/L). n = 12 for WKY(–EC) and SHR(–EC); n = 5 for all other data sets. *P < 0.05 vs. +EC. (+EC = with endothelium; –EC = without endothelium; L-NAME = nitric oxide synthase inhibitor; sulprostone = preferential EP1 and EP3 receptor agonist).

 
3.6 Effect of inhibitors in rings without endothelium
In rings without endothelium of WKY, SQ29,548 (10 µmol/L), BAYu3405 (1 µmol/L), and Terutroban (0.1 µmol/L) nearly abolished the contractions induced to prostaglandin E2 (Figure 4A) and sulprostone (Figure 4B). In contrast, in rings without endothelium of SHR, SQ29,548 (10 µmol/L), BAYu3405 (1 µmol/L), and Terutroban (0.1 µmol/L) equally, but only partly reduced the contractions to prostaglandin E2 (Figure 4C) and sulprostone (Figure 4D). In rings without endothelium of SHR, full inhibition of the prostaglandin E2 or sulprostrone-induced contraction was achieved only when rings were treated with AH6809 (non-selective EP receptor antagonist; 30 µmol/L) in combination of Terutroban (0.1 µmol/L) or SC19220 (selective EP1 receptor antagonist; 100 µmol/L) in combination with Terutroban (0.1 µmol/L) (Figure 4C and D).


Figure 4
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  		Figure 4 Effect of Terutroban, BAYu3405, SQ29,548, AH6809 plus Terutroban or SC19220 plus Terutroban on concentration–response curves to (A and C) prostaglandin E2 and (B and D) sulprostone in aortic rings without endothelium of WKY and SHR. Data are means±SEM and expressed as percentage of a reference contraction to KCl (60 mmol/L); n = 12 for WKY (–EC) and SHR (–EC); n = 5 for all other data sets. *P < 0.05 vs. control. (AH6809 = non-selective EP receptor antagonist; SC19220 = selective EP1 receptor antagonist; SQ29,548, BAYu3405, Terutroban = TP receptor antagonist; sulprostone = preferential EP1 and EP3 receptor agonist).

 
3.7 Endothelium-dependent contractions
Acetylcholine caused concentration-dependent contractions in aortic rings with endothelium of 36-week-old SHR. The contraction was prevented when the endothelial layer was removed (data not shown) and was inhibited by indomethacin [10 µmol/L; a non-selective cyclooxygenase inhibitor (data not shown)], terutroban (0.1 µmol/L), AH6809 (30 µmol/L), SQ29,548 (10 µmol/L), and BAYu3405 (1 µmol/L) (Figure 5A and B). The preferential EP1 receptor antagonist, SC19220 (100 µmol/L) did not significantly reduce endothelium-dependent contractions to acetylcholine (Figure 5C). Endothelium-dependent contractions were not observed in age-matched WKY (data not shown).


Figure 5
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  		Figure 5 (A) Effect of Terutroban and AH6809; (B) SQ29,548 and BAYu3405; and (C) SC19220 on concentration–response curves to acetylcholine in aortic rings with endothelium of 36-weeks old SHR. Experiments were carried out in the presence of L-NAME (100 µmol/L). (D) Effect of Terutroban, AH6809, and SC19220 on concentration–response curves to U46619 in aortic rings with endothelium of 36-week-old SHR. Data are means±SEM and expressed as percentage of a reference contraction to KCl (60 mmol/L); n = 5. *P < 0.05 vs. control. (AH6809 = non-selective EP receptor antagonist; SC19220 = selective EP1 receptor antagonist; SQ29,548, BAYu3405, Terutroban = TP receptor antagonist; U46619 = TP receptor agonist).

 
3.8 Concentration–responses to U46619
U46619, the synthetic TP-receptor agonist25 caused potent concentration-dependent contractions in SHR rings with endothelium. SC19220 (100 µmol/L) significantly shifted the concentration–response curve to U46619 [GenBank] to the right. AH6809 (30 µmol/L) also caused a rightward shift of the concentration–response curve to U46619 [GenBank] ; its inhibitory effects was statistically significantly more pronounced than that of SC19220. Terutroban (0.1 µmol/L) abolished U46619 [GenBank] -mediated contractions (Figure 5D).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusions
 Supplementary material
 Funding
 References
 
4.1 Presence of EP receptors
Both the immunohistochemical and protein expression data demonstrated the abundant presence of EP receptors throughout the rat aorta (Figure 6). Using western blotting, the four EP receptor subtypes were detected in the aorta of both WKY and SHR. The molecular mass of EP1 and EP2 receptors detected in the present study was 42 and 53 kDa, respectively, which is in line with previously reported data in other organs and in different species.31,32


Figure 6
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  		Figure 6 Proposed role of EP and TP receptors in the vascular effects of prostaglandin E2 in the aorta of the SHR. TP and all four subtypes of EP receptors are expressed in the SHR aorta. The protein expression of EP1 is down-regulated in the SHR aorta, but the expression of EP2, EP4, and TP receptors does not differ between preparations of WKY and SHR. The EP3 polyclonal antibody detected five discrete EP3 immunoreactive bands. The protein expression of EP3 (~90 kDa) is decreased, but EP3 (~50 kDa) and EP3 (~40 kDa) are increased, and there are no changes in the expression of EP3 (~120 kDa) and EP3 (~65 kDa) in the SHR aorta when compared with the WKY. In the SHR aorta, prostaglandin E2 fails to induce relaxation but evokes contraction which is dependent on the dual activation of EP1 and TP receptors. In the WKY, prostaglandin E2-mediated contraction is largely dependent on the latter only. Endothelium-dependent contraction by acetylcholine produces cyclooxygenase-dependent vasoconstrictor prostanoids that activate TP receptors but not EP1 receptors. The involvement of EP3 receptors in endothelium-dependent contractions cannot be excluded. SC19220, the preferential inhibitor of EP1 receptor, and AH6809, the non-selective EP receptors antagonist inhibit TP receptors via competitive antagonism. (ACh, acetylcholine; COX, cyclooxygenase; EDCF, endothelium-derived contracting factor; m, muscarinic receptor; NO, nitric oxide).

 
The EP3 antibody identified five protein bands with molecular masses corresponding to approximately 120, 90, 65, 50, and 40 kDa, representing five different protein products. All five bands disappeared when the primary antibody was co-incubated with the EP3 blocking peptide, indicating that the bands are indeed immuno-specific for the EP3 receptor antigen. The five proteins of varying molecular masses were also detected in the brain homogenates. The bands of approximately 120, 90, 65, and 50 kDa were also found in the kidney homogenates. The exact molecular mass of the EP3 receptor protein is controversial, presumably due to the existence of multiple variants of EP3 receptors arising from alternative splicing which differs in the length of their cytoplasmic tails.33 The molecular masses of EP3 receptors in different tissues of different species have been reported to vary from 33 to >100 kDa.19,33–41 Alternatively, the differences in protein masses could be due to the different extent of post-translational modifications of the EP3 protein. Blotting of the EP3 receptor protein was carried out under reducing condition, thus the higher masses of the EP3 receptor proteins detected in the current study is unlikely to reflect dimerization or oligomerization of the EP3 receptors through disulfide bonds. However, dimers of the β2-adrenergic receptor42 and oligomers of the dopamine D3 receptors43 in transfected cells were resistant to reducing agents. Likewise, elimination of the disulfide linkage does not perturb the dimer formation and ligand signalling of the metabotropic glutamate receptor, since the dimerization of this receptor is dependent on the cryptic dimer interface.44 Thus, in the present study, the higher molecular mass proteins detected by the EP3 antibody may represent dimers or oligomers which are insensitive to reducing agents.

Previous studies have described EP4 receptors with molecular masses of 38, 52, 53, 55, or 62 kDa.34,45–47 In the present study, the EP4 polyclonal antibody detected three discrete bands; however, only the band between the 37 and 47 kDa marker was specific to the antigen of the EP4 antibody, as judged by the disappearance of this band when the EP4 antibody was co-incubated with its blocking peptide. The poor specificity of the EP4 receptor antibody as evidenced by the generation of multiple non-specific bands may explain the absence of staining in the aortic samples or the positive control tissues during the histochemical studies.

4.2 Responses to prostaglandin E2
The present study, using the rat aorta, confirms that lower concentrations of prostaglandin E2 causes endothelium-dependent relaxation.18 It does so by EP4 receptor-mediated release of nitric oxide, at least in the aorta of the mouse.18 The relaxation was observed in the aorta of WKY but was absent in the SHR. The impaired relaxation in the hypertensive rats is not due to altered protein level of EP2 or EP4 receptors, as these receptors were expressed at comparable levels in preparations of WKY and SHR.

At higher concentration, prostaglandin E2 and sulprostone (a preferential EP1 and EP3 agonist) caused concentration-dependent contractions in aorta with or without endothelium of WKY and SHR. The contractions were significantly lowered by the presence of the endothelium, but the inhibitory effect of the endothelium against prostaglandin E2-mediated contractions was greater in rings of WKY. The generalized reduction in prostaglandin E2 or sulprostone-induced contractions observed in all preparations with endothelium in rings from both WKY and SHR is explained best by the basal release of nitric oxide. However, the larger inhibitory effect of the endothelium against prostaglandin E2-induced contraction in the WKY aorta is presumably due to prostaglandin E2 causing EP2 or EP4-mediated release of EDRF. Such conclusion is supported by the following observations: (i) treatment of the rings with L-NAME abolished the inhibitory effects of the endothelium and (ii) the greater inhibitory effect of the endothelium against prostaglandin E2-mediated contraction was absent in the SHR aortas, where no endothelium-dependent relaxations to prostaglandin E2 were observed.

The expression of EP1 receptor was down-regulated in the aorta of the SHR. However, the vascular smooth muscle responsiveness to prostaglandin E2 did not differ between preparations of WKY and SHR. The down-regulation of the constrictive EP1 receptor may serve as a self-regulatory response to maintain a controlled smooth muscle responsiveness to prostaglandin E2. The total EP3 receptor protein expression did not differ between preparations of WKY and SHR, but when comparisons were analysed band by band, the EP3 (~90 KDa) was down-regulated, but the EP3 (~50 kDa) and EP3 (~40 kDa) were up-regulated in SHR aorta. The differential expression and the biological relevance of each EP3 receptor proteins are currently unknown.

The relative roles of EP and TP receptors in the contraction-producing effect of prostaglandin E2 was studied using different EP and TP receptor antagonists. In aortic ring without endothelium of WKY, contractions were induced prominently by high concentrations (≥1 µmol/L) of prostaglandin E2 (and sulprostone) and such contractions were virtually prevented by three chemically distinct TP receptor antagonists (Terutroban, SQ29,548, BAYu3405), implying that in the aorta of the WKY, prostaglandin E2 (and sulprostone) mediates contraction mainly through activation of TP rather than EP receptors, despite the abundant expression of the latter. The ability of prostaglandin E2 to cross activate vascular TP receptors has been described.48 In the aortic rings without endothelium of the SHR, prostaglandin E2 (and sulprostone) initiated contractions at much lower concentrations. The contractions induced by higher concentrations (≥1 µmol/L) of prostaglandin E2 (and sulprostone) were partially reduced by three different TP receptor antagonists, while the combined treated of Terutroban with AH6809 or Terutroban with SC19220 was required to fully inhibit the contraction. This implies that in the aorta of the SHR, prostaglandin E2 (and sulprostone) at high concentrations is likely to mediate contractions by the activation of both TP and EP receptors.

4.3 Endothelium-dependent contractions
In the present study, Terutroban, SQ29,548 and BAYu3405 prevented the endothelium-dependent contractions to acetylcholine, confirming that TP receptors are the main prostanoid receptor responsible for the contraction.3,8 Western blotting demonstrated that TP receptors are expressed in the rat aorta. However, since there is no difference in its expression between WKY and SHR, the larger occurrence of EDCF-mediated contractions in the SHR cannot be explained by a greater presence of this receptor subtype. The involvement of EP receptors in endothelium-dependent contraction has been suggested in small arteries from diabetic rats.11 However, activation of EP1 receptors is not involved in the development of acetylcholine-induced endothelium-dependent contractions in the rat aorta, as judged by the ineffectiveness of SC19220 to reduce such contractions. The non-selective EP antagonist, AH6809 which has equal affinity for EP1, EP2, and EP3 receptors27 reduced endothelium-dependent contraction to acetylcholine. The lack of effect by SC19220 and the fact that EP2 receptors are primarily involved in cAMP-dependent relaxation12,13 suggest that AH6809 does not inhibit endothelium-dependent contractions by preventing EP1 or EP2 receptor-mediated responses. However, it remains possible that AH6809 may have suppressed endothelium-dependent contractions through inhibition of the EP3 receptors, as this EP receptor subtype is both expressed and present in the SHR aorta. The possibility that the sequential or synergistic activation of EP3 and TP receptors are required to produce endothelium-dependent contractions cannot be ruled out. The most logical way to rule out the involvement of EP3 receptor in endothelium-dependent contractions is to use selective EP3 receptor antagonists that carry no antagonizing effect for the TP receptor. At the present time, we have no access to such high-quality selective antagonists. The ability of AH6809 to prevent endothelium-dependent contractions to acetylcholine may also be attributed to its partial antagonism at TP receptor, as supported by its ability to inhibit U46619 [GenBank] -mediated contractions. SC19220, the putative EP1 antagonist is also a partial TP receptor antagonist, but its effect was weaker than that of AH6809. This explains why SC19220 failed to significantly reduce EDCF-mediated responses.

The occurrence of endothelium-dependent contraction is not limited to the aorta, but it has been reported in many other arteries, including the femoral,11 mesenteric,49 carotid,50 renal,51 and basilar artery.52 The present study utilized the aorta to study the role of EP and TP receptors involvement in endothelium-dependent contractions, since this conduit aorta produces endothelium-dependent contractions that mimic those that occur in smaller arteries, but offers the benefit such as of greater retrieval of protein content.


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
Supplementary material
 Funding
 References
 
The identification of the exact nature of EDCF and their possible sites of action is important in order to reveal potential therapeutic targets that possess minimal side-effects to treat the imbalanced release of vasoactive substances that occurs with aging and hypertension. The current study revealed that high concentrations of prostaglandin E2 evoke contractions through the synergistic activation of both EP1 and TP receptor in the SHR aorta (Figure 6). However, the activation of TP but not EP1 receptors is required to develop endothelium-dependent contractions, implying that despite the fact that prostaglandin E2 is released by various endothelium-dependent vasoconstrictors including acetylcholine, the calcium ionophore A23187 [GenBank] and ATP,6,9,10 its contribution to endothelium-dependent contraction must be negligible in the aorta of the SHR. The ability of AH6809, the non-selective EP receptor antagonist to inhibit endothelium-dependent contraction is explained best by to its partial antagonism at TP receptor. Low concentrations of prostaglandin E2 induce endothelium-dependent relaxations in the aorta of the WKY but not in that of the SHR. This impaired EP receptor-dependent relaxation may contribute to endothelial dysfunction and indirectly permits the emergence of endothelium-dependent contractions in the aorta of the hypertensive rat.


   Supplementary material
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 Supplementary material
Funding
 References
 
Supplementary material is available at Cardiovascular Research online.

Conflict of interest: none declared.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 Supplementary material
 Funding
References
 
Hong Kong Research Grant Council (University of Hong Kong-777507M).


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 Supplementary material
 Funding
 References
 

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