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(Circulation Research. 1995;76:1003-1010.)
© 1995 American Heart Association, Inc.

Articles

Endothelium-Dependent Contractions Are Associated With Both Augmented Expressionof Prostaglandin H Synthase-1 and Hypersensitivity to Prostaglandin H₂in the SHR Aorta

Tong Ge, Helen Hughes, Didier C. Junquero, Kenneth K. Wu, Paul M. Vanhoutte, Chantal M. Boulanger

From the Center for Experimental Therapeutics, Baylor College of Medicine, Houston, Tex.

Correspondence to Chantal M. Boulanger, PhD, Baylor College of Medicine, Center for Experimental Therapeutics, One Baylor Plaza, Room 826E, Houston, TX 77030.

	Abstract

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Abstract Prostaglandin H₂ (PGH₂ [endoperoxide]) is an immediate product of prostaglandin H (PGH) synthase activity (cyclooxygenase) and a likely candidate to mediate endothelium-dependent contractions evoked by acetylcholine in the aorta of the spontaneously hypertensive rat (SHR). Experiments were designed to investigate whether or not endothelium-dependent contractions were associated with an increased expression of PGH synthase, an augmented acetylcholine-induced release of PGH₂, and/or a hypersensitivity of the smooth muscle to endoperoxides in SHR aorta compared with normotensive Wistar-Kyoto (WKY) aorta. In SHR aorta, endothelium-dependent contractions to acetylcholine were abolished by tenidap (10^-8 mol/L), a preferential PGH synthase-1 inhibitor, but slightly impaired by NS-398 (10^-6 mol/L), a preferential PGH synthase-2 inhibitor. PGH synthase-1 expression, which was evaluated by both reverse transcriptase–polymerase chain reaction and Western blotting, was about twofold greater in preparations with endothelium from SHR than from WKY rats. There was no difference in PGH synthase-1 expression between preparations with and those without endothelium in both strains. In SHR but not WKY aortas, acetylcholine (10^-5 mol/L, 5 minutes) caused a significant endothelium-dependent release of PGH₂, as measured by gas chromatography/mass spectrometry. PGH₂ evoked more potent contractions in rings without endothelium from SHR than from WKY rats, whereas the thromboxane analogue U46619 and prostaglandin F₂ caused a comparable response in both preparations. These results show that endothelium-dependent contractions to acetylcholine in SHR aorta are associated with a greater expression of PGH synthase-1, a significant release of PGH₂, and a hypersensitivity of the smooth muscle to the endoperoxide.

Key Words: thromboxane • tenidap • NS-398 • acetylcholine • cyclooxygenase

	Introduction

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Endothelial cells modulate the response of vascular smooth muscle to hormones, neuromediators, and aggregating platelets by releasing relaxing and/or contracting factors.^{1 2 3 4} In the aorta of the normotensive Wistar-Kyoto (WKY) rat, acetylcholine evokes endothelium-dependent relaxations, which are mediated by the release of nitric oxide and of endothelium-derived hyperpolarizing factor; this response is insensitive to inhibitors of cyclooxygenase.^{5 6 7} However, in the aorta of spontaneously hypertensive rats (SHR), the endothelium-dependent relaxations evoked by acetylcholine are blunted because of the concomitant release of a cyclooxygenase-dependent contracting factor.^{5 8} This contracting factor likely contributes to the impaired vasodilatation to acetylcholine observed in the forearm of patients with essential hypertension, since a normal response is restored after treatment with an inhibitor of cyclooxygenase.⁹ The endothelium-derived contracting factor is distinct from thromboxane but activates thromboxane-endoperoxide receptors to cause contraction of the smooth muscles. Therefore, it is likely to be prostaglandin H₂ (PGH₂), an endoperoxide precursor of other prostaglandins.^{5 8 10} PGH₂ is the immediate product of the activity of cyclooxygenase (prostaglandin H [PGH] synthase), which is the rate-limiting enzyme in the synthesis of prostaglandins.¹¹ PGH synthase exists under two isoforms, PGH synthase-1 and PGH synthase-2, a cytokine-inducible form.^{12 13 14 15 16 17}

The presence of endothelium-dependent contractions to acetylcholine in SHR but not WKY aortas suggests either an augmented production (associated with an increased expression of PGH synthase) or an increased reactivity to PGH₂ in the vessel wall of the SHR. The first hypothesis is supported by the fact that prolonged exposure of WKY aorta to interleukin-2, a cytokine augmenting cyclooxygenase expression in endothelial cells, induces endothelium-dependent contractions to arachidonic acid that are comparable to those observed in control SHR aorta.^{18 19 20} The purpose of the present study was to examine (1) the sensitivity of endothelium-dependent contractions to acetylcholine to inhibitors of either PGH synthase-1 or PGH synthase-2 activity in SHR aorta, (2) the expression of cyclooxygenase (PGH synthase) in the vascular wall of SHR and WKY rats, (3) the subsequent production of PGH₂ upon acetylcholine stimulation, and (4) the response to PGH₂ of the vascular smooth muscle from both strains.

	Materials and Methods

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Animals
Experiments were performed on thoracic aortas from male SHR and WKY rats (35 weeks old, Harlan Sprague Dawley, Indianapolis, Ind). All procedures using animals were in accordance with the guidelines of the Animal Protocol Review Committee of Baylor College of Medicine. Systolic arterial blood pressure was measured by the tail-cuff method and averaged 157.2±3.2 (n=31) and 239.6±6.0 (n=37) mm Hg in WKY rats and SHR, respectively. The rats were anesthetized with pentobarbital sodium (50 mg/kg IP). The thoracic aorta was dissected free, excised, and placed into cold modified Krebs-Ringer bicarbonate solution of the following composition (mmol/L): NaCl 118.3, KCl 4.7, MgSO₄ 1.2, KH₂PO₄ 1.2, CaCl₂ 2.5, NaHCO₃ 25.0, EDTA 0.026, and glucose 11.1 (control solution, pH 7.4). The blood vessels were cleaned of adherent connective tissue. For organ-chamber experiments, the preparations were immersed in control solution and used immediately. For RNA extraction and Western blotting, they were either frozen immediately in liquid N₂ (preparations with endothelium) or opened longitudinally with the intimal layer being scraped away mechanically before freezing (preparations without endothelium). Then the preparations were kept in a freezer (-70°C) until assays were performed.

Organ-Chamber Experiments
Thoracic aortas from SHR and WKY rats were cut into rings (4 to 5 mm long). In some preparations, the endothelium was removed by gently rubbing the intimal surface with a small forceps. In the remaining rings, care was taken not to touch the inner surface of the blood vessel. The presence or absence of endothelial cells was confirmed by the presence or absence of relaxation in response to thrombin (1 U/mL), respectively.⁵ The rings were suspended horizontally between two stainless steel wires in organ chambers, which contained 15 or 25 mL of control solution (37°C) aerated with 95% O₂/5% CO₂. The preparations were connected to force transducers (Statham Universal UC2 or Grass FT 03C) for recording of changes in isometric force. Before experimentation, the preparations were stretched progressively and exposed to KCl (45 mmol/L) at each level of force until the optimal point of the length–active force relation was reached. After this procedure, the rings were allowed to equilibrate for 40 minutes. All rings were then exposed to KCl (60 mmol/L) to determine their maximal responsiveness.

When endothelium-dependent contractions to acetylcholine were investigated, aortic rings with endothelium from SHR were incubated with N^G-nitro-L-arginine (10^-4 mol/L, 20 minutes, Aldrich) to prevent the formation of nitric oxide⁶ and to optimize endothelium-dependent contractions.^{21 22} The endothelium-dependent contractions to acetylcholine were examined in parallel rings with endothelium of SHR aorta in control solution (control) or in the presence of tenidap (10^-8 mol/L, a preferential inhibitor of PGH synthase-1; Reference 23²³ and personal communication, Drs J. Bonnet and C. Tordjman, 1994) or NS-398 (10^-6 mol/L, a preferential inhibitor of PGH synthase-2)^{24 25 26} (both a kind gift from Institut de Recherche Servier). The concentration of NS-398 used in the present study was approximately three times smaller than the reported IC₅₀ for PGH synthase-2 (3.6x10^-6 mol/L),²⁶ because pilot experiments revealed that it was the highest concentration that did not cause a significant decrease of the basal tension of SHR aorta during the incubation period (data not shown).

Contractions to increasing concentrations of the thromboxane analogue U46619 (Cayman Chemical Co), prostaglandin F₂ (PGF₂, Upjohn), or PGH₂ (Biomol) were obtained in parallel rings without endothelium from WKY and SHR aortas.

All drugs were prepared daily (stock solution, 10^-2 mol/L) and further diluted in distilled water, except for PGH₂, which was obtained as a solution in acetone (100 µg/mL, -70°C) and further diluted in acetone, and NS-398, which was dissolved first in pure ethanol (10^-2 mol/L) and further diluted in water. The highest concentration of acetone used was 1.2% of the final chamber volume (to reach 10^-7 mol/L PGH₂ in the bath solution), because at higher concentrations, it decreased vascular tone to a comparable extent in both strains (data not shown). Ethanol (final concentration, 0.01% to 0.1%) did not affect the subsequent response to acetylcholine in SHR aortas with endothelium (data not shown).

Amplification of PGH Synthase-1 mRNA by Reverse Transcriptase–Polymerase Chain Reaction
RNA Preparation
Total RNA was extracted by using an RNA STAT-30 kit (Tel-Test B, Inc). The frozen thoracic aortas were ground individually into a fine powder in a marble mortar and suspended in the homogenization buffer until the tissue was dispersed thoroughly. The RNA was isolated according to the protocol given by the supplier, and the concentration was measured two times for each sample in a spectrophotometer (model UV160U, Shimadzu). Aliquots of RNA were loaded on a denatured formamide agarose gel to check the RNA quality.

Reverse Transcriptase–Polymerase Chain Reaction
The relative expression of PGH synthase-1 (Cox-1) mRNA in WKY and SHR aortas was evaluated by reverse transcriptase (RT)–polymerase chain reaction (PCR) with appropriate internal controls.^{27 28} Samples with and without endothelium from SHR and WKY rats were studied in parallel. Total RNA of 2 µg from each rat aorta was reverse-transcribed into cDNA by using the SuperScript preamplification system (GIBCO BRL). The random hexamer (1 ng/µL, GIBCO BRL) was used as primer for the first-strand cDNA synthesis. The reverse transcription reactions were performed according to the protocol given by the supplier and were stopped by heating at 90°C for 5 minutes. cDNA aliquots (10 µL) were amplified by PCR with primers specific for Cox-1, GAPDH, or ß-actin in a DNA thermal cycler (Perkin Elmer). Amplification of the GAPDH (or ß-actin) cDNA was used as an internal standard for each preparation. The primers for Cox-1 were obtained from Baylor College of Medicine, Molecular Core Facility. The primers for GAPDH and ß-actin were purchased from Clontech. The PCR mixture included 200 µmol/L of each dNTP plus 10 µCi of [-³²P]dCTP (3000 Ci/mmol), 1 mmol/L of each primer, and 2 U of Taq polymerase (Perkin Elmer) in PCR buffer (20 mmol/L Tris-HCl [pH 8.4], 2.5 mmol/L MgCl₂, 50 mmol/L KCl, and 100 ng/mL bovine serum albumin), with a final volume of 50 µL. For quantitative purposes, the cDNAs were amplified by 28 cycles (each cycle consisting of DNA denaturation at 94°C for 40 seconds), primer annealing at 54°C for 40 seconds, and primer extension at 72°C for 90 seconds. The amplified cDNAs (212, 452, and 764 bp for Cox-1, GAPDH, and ß-actin, respectively) were resolved on 8% polyacrylamide gels and quantified by a Beta-Scope 603 blot analyzer (Betagen).

Western Blots
Frozen thoracic aortas were ground as described for RNA extraction and then homogenized in PBS buffer (mmol/L: NaCl 135, KCl 2.7, Na₂HPO₄ 10, and KH₂PO₄ 1.8) with 0.1% Triton X-100, 1 mmol/L phenylmethylsulfonyl fluoride, 0.01% EDTA, and 0.03% leupeptin (400 µL, pH 7.4), sonicated (2 times 30 seconds), boiled for 5 minutes, and centrifuged for 15 minutes at 13 000 rpm. The protein concentration was measured in the supernatant by using the Micro BCA protein assay (Pierce). Aliquots (50 µg protein) were frozen at -70°C until use. Samples were electrophoresed on a 10% polyacrylamide gel under reducing and denaturing conditions and transferred onto a nitrocellulose-ECL membrane (Amersham) by using a transfer buffer (25 mmol/L Tris and 192 mmol/L glycine, 85 V, 70 minutes).²⁸ Nonspecific binding sites were blocked overnight (4°C) in a 0.05% Tween 20–PBS buffer (T-PBS, pH 7.4) containing 5% nonfat milk (Bio-Rad). The blots were then incubated for 3 hours (room temperature) with the primary antibodies in T-PBS with 5% nonfat milk. Monoclonal anti–PGH synthase-1 antibody (Oxford Biomedical Research) and monoclonal anti–-smooth muscle actin (as an internal standard, Sigma Chemical Co) were used at a 1:200 and 1:2000 dilution, respectively. All washes were performed in T-PBS. The second antibody was an anti-mouse horseradish peroxidase–conjugated whole antibody (from sheep; dilution, 1:1000; Amersham). Both PGH synthase-1 and -smooth muscle actin were revealed with ECL Western blotting detection reagents and after a 15-second to 2-minute exposure of the blots to Hyperfilm-ECL (Amersham) by following instructions given by the manufacturer. Films were analyzed by densitometry (Biomed Instruments, Inc), and for each preparation with or without endothelium, data are expressed as the ratio of the peak absorbance for PGH synthase-1 and -smooth muscle actin.

Measurement of PGF₂ and PGH₂
PGH₂ is the unstable precursor of prostaglandins and thromboxanes, and when its rate of formation exceeds its rate of metabolism, measurable concentrations are released from aortic segments. PGH₂ isomerizes to prostaglandin E₂ (PGE₂) under nonreducing conditions (ethanol) but is converted to PGF₂ under reducing conditions (SnCl₂). Therefore, by measuring the difference in PGF₂, in a sample of aortic medium placed in ethanol versus that reduced with SnCl₂, it is possible to estimate the amount of PGH₂ released into the medium.^{29 30} Paired segments of WKY and SHR aortas (1 cm in length) were equilibrated for 1 hour at 37°C in 3 mL of control solution gassed with 5% CO₂/95% O₂ (pH 7.4). Then the buffer was changed, and baseline samples were taken 5 minutes later. The aortic segments were then stimulated with acetylcholine (10^-5 mol/L); samples were taken after 5 minutes, since in pilot experiments the rate of prostaglandin production was close to maximal at this time. Two samples of incubation media (0.3 mL) were removed simultaneously: one was placed into ethanol (6 mL); the other, into SnCl₂ (6 mL, 0.5% in ethanol). After 2 minutes at room temperature, sodium hydroxide (80 µL, 10%) was added to the SnCl₂ samples. The internal standard ²H₄-PGF₂ (250 pg) was added to all samples, the SnCl₂ samples were centrifuged, and the supernatants were transferred to clean tubes. All samples were stored at -20°C before extraction and analysis.

PGF₂ was extracted by using solid-phase extraction with C-18 Bond-Elute extraction columns (Varian) that were preconditioned with methanol (10 mL) and water (10 mL). The ethanolic samples were poured into 35 mL water, and the pH was adjusted to between 2.5 and 3.0 with HCl (2 mol/L). These solutions were loaded on the preconditioned columns, which were subsequently washed with water and hexane (10 mL of each) before elution of the prostaglandins with 2.5 mL ethyl acetate. This eluate was evaporated to dryness, derivatized to the pentafluorobenzyl (PFB) ester,³¹ and purified further by thin-layer chromatography. Samples were redissolved in ethyl acetate (0.1 mL), spotted on prescored silica gel plates (LK6D, Whatman International Ltd), and developed in hexane/isopropanol (80:20 [vol/vol]). PGE₂–PFB ester (10 µg) was run on one lane of the plates, and the lipids were visualized under long-wave UV light after spraying with a solution of 0.1% diphenylhexatriene in hexane.³² A 0.5-cm-wide band just below the PGE₂-PFB standard was scraped, and the prostaglandins were eluted with ethyl acetate (2 mL). The samples were then evaporated to dryness and derivatized with bis-(trimethylsilyl)-trifluoroacetamide containing 1% trimethylchlorosilane (Sylon BFT, Supelco, Inc) for 30 minutes at 60°C.

Gas chromatography/mass spectrometry was accomplished on a Fisons (VG) QUATTRO tandem quadrupole instrument by using the first quadrupole for the analyses. The instrument was operated in the negative ion mode with methane as the reagent gas. The gas chromatograph was a Hewlett-Packard 5890 equipped with an all-glass falling needle injector system (Allen Scientific) and a DB-1 capillary column (internal diameter, 30 mx0.32 mm; J&W Scientific) that was heated from 200°C to 300°C at 10°C/min. Ions were monitored at mass-to-charge ratios of 569 and 573, corresponding to the loss of the PFB group from the molecular ion of endogenous PGF₂ and the internal standard, respectively. Quantification was accomplished from peak area ratios with reference to standard curves, the linear regression of which gave a correlation coefficient >.999. The coefficient of intra-assay variation was 3.9% for 10 pg PGF₂.

Statistics
All experiments were performed in parallel on preparations with or without endothelium from age-matched SHR and WKY rats; n represents the number of preparations from different rats (WKY or SHR), either with or without endothelium.

Results are given as mean±SEM. For organ-chamber experiments and measures of PGH synthase expression, statistical analysis was performed by using Student's t test for paired and unpaired observations or a two-way ANOVA followed by a Bonferroni test when more than two means were compared.³³ For the measurement of PGH₂ and PGF₂, statistical analysis was performed by using a Kruskal-Wallis nonparametric ANOVA followed by Dunn's test.³⁴ All data were analyzed with INSTAT software (GraphPad). Means were considered significantly different at P<.05.

	Results

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Response to Acetylcholine in SHR Aorta
Acetylcholine (10^-8 to 10^-5 mol/L) induced endothelium-dependent contractions in the SHR aorta (Fig 1). The endothelium-dependent component of the response to acetylcholine was abolished by tenidap (a preferential PGH synthase-1 inhibitor, 10^-8 mol/L). In preparations with endothelium treated with tenidap, the response to acetylcholine was no longer different from that observed in control rings without endothelium (Fig 1). NS-398 (a preferential PGH synthase-2 inhibitor, 10^-6 mol/L) reduced the contraction evoked by high concentrations of acetylcholine to a significantly lesser extent than did tenidap (Fig 1).

View larger version (16K): [in this window] [in a new window]   Figure 1. Graph showing the effect of prostaglandin H (PGH) synthase-1 and PGH synthase-2 inhibitors on the contractions evoked by acetylcholine in rings with endothelium from spontaneously hypertensive rat aorta. The experiments were performed under control conditions () or in the presence of either tenidap (10-8 mol/L, ) or NS-938 (10-6 mol/L, ). The response evoked by acetylcholine in preparations without endothelium is represented by the open symbol (). The contractions evoked by KCl (60 mmol/L) averaged 3.7±0.1 g in control rings (with endothelium), 3.8±0.2 g in rings treated with NS-398, 3.7±0.2 g in rings exposed to tenidap, and 3.4±0.4 g in preparations without endothelium (n=5 or 6). The asterisks denote a significant difference (P<.05) from the response observed in control rings with endothelium.

Expression of PGH Synthase-1 in WKY and SHR Aortas
Cox-1 and GAPDH mRNAs were reverse-transcribed and amplified during 28 cycles by PCR; under these conditions, the rate of amplification was comparable for Cox-1 and GAPDH (Fig 2). In vessels with endothelium, the ratio of Cox-1 to GAPDH cDNAs was significantly greater in SHR (2.4±0.2) than in WKY rats (1.1±0.1) (n=5, Fig 3). However, there was no difference in the ratio of Cox-1 to GAPDH cDNAs in preparations with and without endothelium in both WKY (1.1±0.1 and 1.2±0.2) and SHR (2.4±0.2 and 2.2±0.2) aortas, respectively (n=5, Fig 3). Comparable results were observed when ß-actin instead of GAPDH was used as an internal control; indeed, in preparations with endothelium, the ratio of Cox-1 to ß-actin cDNAs was significantly greater in SHR (2.2±0.4) than in WKY rats (1.0±0.1) (n=4).

View larger version (10K): [in this window] [in a new window]   Figure 2. Graph showing the results of reverse transcriptase–polymerase chain reaction (PCR) analysis of prostaglandin H synthase-1 (Cox-1) and GAPDH mRNA expression in Wistar-Kyoto rat aorta. Total RNA (2 µg) was reverse-transcribed into cDNA, and cDNA aliquots (10 µL) were amplified with either Cox-1 or GAPDH sense and anti-sense primers (, Cox-1; , GAPDH). At indicated cycles of PCR amplification, 10-µL aliquots were taken, separated on a polyacrylamide gel, and stained with ethidium bromide. Incorporation of 32P into the PCR products (in counts per minute [cpm]) was quantified by using a blot analyzer. Representative experiments out of three others (24 to 32 cycles).

View larger version (51K): [in this window] [in a new window]   Figure 3. Autoradiograms showing the amplification by reverse transcriptase–polymerase chain reaction (PCR) of prostaglandin H synthase-1 (Cox-1, 212 bp, top) and GAPDH (452 bp, bottom) cDNAs in Wistar-Kyoto (WKY, n=6, left) and spontaneously hypertensive rat (SHR, n=6, right) aortas with (+) or without (-) endothelium. Experiments were performed in parallel; one lane represents the PCR product obtained from one rat aorta. The autoradiograms were exposed for 30 minutes (Cox-1) and 40 minutes (GAPDH); the size of the PCR products is indicated by the DNA markers (in base pairs).

When WKY and SHR aortas were compared, the expression of PGH synthase-1 as detected by Western blotting was also greater in preparations with and without endothelium obtained from SHR than from WKY rats (Fig 4). The densitometric analysis showed that the ratio of PGH synthase-1 to -smooth muscle actin was 42±9 (n=3) and 76±1 (n=3) in preparations with endothelium and 32±7 (n=3) and 54±11 (n=3) in preparations without endothelium from WKY and SHR aortas, respectively. In both WKY and SHR aortas, there was no statistically significant difference in PGH synthase-1 expression in preparations with and without endothelium.

View larger version (30K): [in this window] [in a new window]   Figure 4. Densitometric analysis showing detection of prostaglandin H (PGH) synthase-1 (top) and -smooth muscle actin (bottom) in protein homogenates from Wistar-Kyoto (WKY) and spontaneously hypertensive rat (SHR) aortas with and without endothelium (50 µg per lane). Migration of the standards is indicated on the left of the figure. The estimated molecular mass (MW) values are 70 and 45 kD for the protein detected with the PGH synthase-1 antibody and that detected with the -smooth muscle actin monoclonal antibody, respectively. The same results were obtained with two other sets of WKY and SHR preparations with and without endothelium.

Release of PGH₂ and PGF₂
The release of PGH₂ from WKY and SHR aortas with endothelium was determined under basal conditions and after stimulation with acetylcholine (10^-5 mol/L, 5 minutes, Fig 5). In WKY aortas with endothelium, acetylcholine did not significantly augment the release of PGH₂ or PGF₂ above basal values (n=5 or 6, Fig 5). Removal of the endothelium or incubation with indomethacin did not affect the production of PGH₂ or PGF₂ from WKY aortas exposed to acetylcholine (P>.05, Fig 5).

View larger version (15K): [in this window] [in a new window]   Figure 5. Bar graphs showing release of prostaglandin H2 (PGH2, left) and prostaglandin F2 (PGF2, right) from segments with or without endothelium from Wistar-Kyoto (WKY) and spontaneously hypertensive rat (SHR) aortas. Experiments were performed in parallel on segments obtained from one pair of WKY and SHR aortas, either under basal conditions (open bars, n=5 pairs) or during stimulation with acetylcholine (10-5 mol/L, 5 minutes, hatched bars, n=6 pairs). In some experiments, the effect of acetylcholine was examined in denuded segments (solid bars, n=3 pairs) or in preparations with endothelium exposed for 45 minutes with indomethacin (10-5 mol/L, bars with horizontal lines, n=3 pairs). There was no significant difference in the dry weight (expressed in milligrams) of the different aortic segments used for these experiments (with endothelium: WKY, 8.5±0.7 [n=6]; SHR, 9.1±0.5 [n=6]; without endothelium: WKY, 6.5±0.6 [n=3]; SHR, 10.1±0.7 [n=3]). *Statistically significant (P<.05) effect of acetylcholine compared with basal values. #Statistically significant (P<.05) effect of endothelium removal. $Statistically significant (P<.05) effect of indomethacin in preparations exposed to acetylcholine. Statistically significant difference (P<.05) between WKY and SHR preparations.

In SHR aortas with endothelium, the release of both PGH₂ and PGF₂ was augmented significantly upon stimulation with acetylcholine (n=5 or 6, P<.05, Fig 5). Removal of the endothelium significantly decreased the acetylcholine-induced release of PGH₂ and PGF₂ (n=3, P<.05, Fig 5). In preparations with endothelium of SHR aorta exposed to acetylcholine, indomethacin caused a significant decrease in the production of PGH₂ and PGF₂ (n=3, P<.05, Fig 5). The acetylcholine-induced release of PGF₂ was greater in preparations with endothelium from SHR than from WKY rats (n=6 pairs, P<.05), whereas that of PGH₂ did not reach statistical significance (Fig 5).

Contractions to PGH₂, U46619, and PGF₂
Both the thromboxane analogue U46619 and PGF₂ evoked comparable responses in rings without endothelium from SHR and WKY aortas (Fig 6). However, PGH₂ (3x10^-8 to 10^-7 mol/L) induced significantly greater contractions in rings from SHR than from WKY rats (Fig 6). In preparations without endothelium, there was no difference in the response evoked by PGH₂ (10^-8 mol/L) in SHR compared with the response evoked by PGH₂ (3x10^-8 and 10^-7 mol/L) in WKY rats (Fig 6).

View larger version (13K): [in this window] [in a new window]   Figure 6. Graphs showing contraction of rings without endothelium from Wistar-Kyoto (WKY, ) and spontaneously hypertensive rat (SHR, ) aortas to increasing concentrations of U46619 (left), prostaglandin F2 (PGF2, middle), and prostaglandin H2 (right). Experiments were performed on parallel rings without endothelium; results are given in grams of tension developed and expressed as mean±SEM. When responses to U46619 were examined, the contraction evoked by KCl (60 mmol/L) averaged 1.62±0.15 and 1.62±0.18 g in preparations from WKY rats (n=5) and SHR (n=6), respectively. When the response to PGF2 was studied, the contraction evoked by KCl (60 mmol/L) reached 2.77±0.24 and 3.13±0.36 g in WKY and SHR preparations (n=6 each), respectively. When contractions to prostaglandin H2 were investigated, the contractions evoked by KCl (60 mmol/L) were 1.52±0.32 and 1.72±0.14 g in rings from WKY rats (n=6) and SHR (n=5), respectively. The responses to prostaglandin H2 and U46619 were obtained with different groups of rats and on different experimental days than that of PGF2. *Significant difference (P<.05) between preparations from SHR and WKY rats.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present experiments demonstrate that endothelium-dependent contractions to acetylcholine in the SHR aorta are associated with a twofold increase in expression of PGH synthase-1, a significant release of PGH₂, and an augmented sensitivity of the vascular smooth muscle to the endoperoxide.

In the SHR aorta, endothelium-dependent contractions to acetylcholine require activation of PGH synthase, since they are abolished by PGH synthase inhibitors such as indomethacin.⁵ The present results suggest that the response to acetylcholine in SHR aorta is caused by the activation of PGH synthase-1, since tenidap, a preferential PGH synthase-1 inhibitor, abolished the endothelium-dependent contraction to acetylcholine at concentrations below those reported to inhibit 5-lipoxygenase activity (Reference 23²³ and Drs J. Bonnet and C. Tordjman, personal communication, 1994). This was also supported by the fact that high concentrations of NS-398 (10^-6 mol/L), a preferential inhibitor of PGH synthase-2 (References 24²⁴ to 26 and Drs J Bonnet and C. Tordjman, personal communication, 1994), only minimally impaired the maximal response to acetylcholine. The small inhibitory effect of this concentration of NS-398 may be due to a partial inhibition of PGH synthase-1 or to nonspecific effect(s) of the compound under the present experimental conditions.

Two hypotheses could explain the existence of endothelium-dependent contractions to acetylcholine in the SHR aorta: (1) There was an increased production of the contracting factor caused by a greater expression of PGH synthase in these preparations. (2) There was an augmented sensitivity of the SHR smooth muscle to the contracting factor. The contracting factor released by acetylcholine in the SHR aorta is a metabolite of the PGH synthase activity that is different from thromboxane, since thromboxane synthase inhibitors do not impair the response.^{5 8 10 18} It has been proposed that the contracting factor released by acetylcholine in hypertensive rats is PGH₂, an endoperoxide precursor of thromboxanes and other prostaglandins; this is based on the following facts: (1) The contractions are prevented by antagonists of thromboxane/endoperoxide receptors.^{8 10} (2) Acetylcholine induced a larger release of PGE₂ and 6-ketoprostaglandin F₁ (but not thromboxane A₂) in SHR than in WKY rats. (3) Exogenous PGH₂ and PGF₂ are more potent than PGE₂, prostaglandin D₂, or prostaglandin I₂ in inducing contraction of the smooth muscle from SHR.¹⁰

The present results suggest that endothelium-dependent contractions to acetylcholine in the SHR aorta are associated with an increased expression of cyclooxygenase. Indeed, both the ratio of Cox-1 to GAPDH cDNAs and the PGH synthase-1 protein level were approximately twofold greater in these blood vessels compared with blood vessels from age-matched WKY rats; this difference in PGH synthase-1 expression could account for the significant acetylcholine-induced release of PGH₂ and PGF₂ from SHR aorta. However, the present results do not allow us to conclude whether this difference in PGH synthase-1 mRNA expression between SHR and WKY is acquired genetically, whether it results from the presence of an endogenous inducer of PGH synthase-1 expression in SHR (and not in WKY rats), or whether it is due to a different stability of the mRNA between the two strains. A surprising finding was that there was no difference in the expression of PGH synthase-1 between preparations with and without endothelium from both strains, despite the obligatory role of the endothelium in the occurrence of cyclooxygenase-dependent contractions to acetylcholine (the present study and References 5 and 35^{5 35} ). One likely explanation is that although vascular endothelial cells contain 20 times more PGH synthase than do smooth muscle cells,^{36 37} the ratio of endothelial to smooth muscle cells may be very small in the rat aorta; therefore, the level of endothelial PGH synthase-1 expression would be undetectable under the present experimental conditions. However, if PGH synthase is expressed to a comparable level in WKY and SHR endothelial cells, one could envisage the release of an unknown "endothelium-derived indirect contractile factor" by acetylcholine; this factor would activate smooth muscle PGH synthase, thus causing contraction in the SHR aorta. Although further experiments would be required to strengthen this hypothesis, it is supported by the fact that the biological half-life of PGH₂ (5 minutes at 37°C and pH 7.5)^{38 39} is hard to reconcile with the difficulty of bioassaying the endothelium-derived contracting factor under experimental conditions in which endothelium-derived nitric oxide, a substance with a much shorter half-life (6 to 60 seconds), can be detected.^{40 41 42 43}

In addition to the significant acetylcholine-induced release of PGH₂ in the SHR aorta, another cause of the occurrence of endothelium-dependent contractions could be the hypersensitivity of SHR smooth muscle to PGH₂. This result is in agreement with the increased response to the endoperoxide (10^-7 mol/L) observed in the aortas of rats with aortic coarctation-induced hypertension, where a lipoxygenase product impairs the metabolism of PGH₂ into prostacyclin, the major arachidonic acid metabolite in this preparation.⁴⁴ Thus, the impaired metabolism of PGH₂ into prostacyclin could contribute to the augmented local concentration and effect of PGH₂ in blood vessels from these hypertensive rats. Although the present experiments do not exclude the possibility that a similar mechanism could cause the augmented response of SHR smooth muscle to the endoperoxide, this hypothesis may be hard to reconcile with the fact that after stimulation with acetylcholine, a greater amount of prostacyclin (approximately twofold) is formed in rings with endothelium in SHR aorta compared with WKY aorta.⁸

The responses to the thromboxane analogue U46619 and PGF₂ were also investigated in rings without endothelium from WKY and SHR aortas, since U46619, like the endothelium-derived contracting factor, activates thromboxane-endoperoxide receptors and PGF₂, together with PGH₂, causes potent contractions of rat aortic smooth muscle. The present results show that the responses to U46619 and PGF₂ are comparable in WKY and SHR aortas. PGF₂ could contribute to some extent to endothelium-dependent contractions in aortas from SHR, since it has a low affinity for thromboxane-endoperoxide receptors.⁴⁵ The different response of WKY and SHR smooth muscles to PGH₂, but not to U46619, suggests that either the metabolism of PGH₂ is affected in the SHR smooth muscle or that PGH₂ and the thromboxane analogue U46619 activate different subtypes of thromboxane-endoperoxide receptors on SHR and WKY smooth muscle cells. However, further experiments would be required to investigate these possibilities.

Both an increased expression of PGH synthase-1 leading to a significant release of endoperoxides and an augmented contraction to PGH₂ could contribute to the impairment of endothelium-dependent responses associated with the release of a cyclooxygenase-dependent contracting factor in SHR resistance blood vessels and could play a role in the increase of vascular resistance.^{46 47 48 49} Interestingly, an increased PGH synthase expression has been associated with the release of a cyclooxygenase-dependent vasoconstrictor altering endothelial function in blood vessels from vitamin E–deprived rats.⁵⁰ The present results could also explain the dysfunction observed in essential hypertension; indeed, in this group of patients the impaired vasodilatation to acetylcholine is restored after inhibition of PGH synthase.⁹ In addition, the augmented release and effect of endoperoxides may contribute to the increased proliferation of the hypertensive smooth muscle cells, since activation of thromboxane-endoperoxide receptors generates growth-promoting effects.^{51 52 53 54 55}

	Acknowledgments

 
This study was supported by National Institutes of Health grant HL-35614 and a grant-in-aid from the Institut de Recherches Internationales Servier (Paris, France). The authors wish to thank Drs Teresa Zyglewska, Stavros Topouzis, Tom Scott-Burden, and Nancy S. Day for their advice, Barnabas Desta and Nusret Didzik for superb technical assistance, and Melody Whitus for secretarial help.

Received June 29, 1994; accepted February 17, 1995.

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