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Am J Physiol Heart Circ Physiol 291: H2255-H2264, 2006. First published June 23, 2006; doi:10.1152/ajpheart.01115.2005
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In SHR aorta, calcium ionophore A-23187 releases prostacyclin and thromboxane A2 as endothelium-derived contracting factors

Pascale Gluais,1 Jerôme Paysant,1 Cécile Badier-Commander,1 Tony Verbeuren,1 Paul M. Vanhoutte,2 and Michel Félétou1

1Institut de Recherches Servier, Suresnes, France; and 2Department of Pharmacology, Faculty of Medicine, Hong Kong, China

Submitted 21 October 2005 ; accepted in final form 2 June 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In mature spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY), acetylcholine and the calcium ionophore A-23187 release endothelium-derived contracting factors (EDCFs), cyclooxygenase derivatives that activate thromboxane-endoperoxide (TP) receptors on vascular smooth muscle. The EDCFs released by acetylcholine are most likely prostacyclin and prostaglandin (PG)H2, whereas those released by A-23187 remain to be identified. Isometric tension and the release of PGs were measured in rings of isolated aortas of WKY and SHR. A-23187 evoked the endothelium-dependent release of prostacyclin, thromboxane A2, PGF2{alpha}, PGE2, and possibly PGH2 (PGI2 >> thromboxane A2 = PGF2{alpha} = PGE2). In SHR aortas, the release of prostacyclin and thromboxane A2 was significantly larger in response to A-23187 than to acetylcholine. In response to the calcium ionophore, the release of thromboxane A2 was significantly larger in aortas of SHR than in those of WKY. In both strains of rat, the inhibition of cyclooxygenase-1 prevented the release of PGs and the occurrence of endothelium-dependent contractions. Dazoxiben, the thromboxane synthase inhibitor, abolished the A-23187-dependent production of thromboxane A2 and inhibited by approximately one-half the endothelium-dependent contractions. U-51605, an inhibitor of PGI synthase, reduced the release of prostacyclin elicited by A-23187 but induced a parallel increase in the production of PGE2 and PGF2{alpha}, suggestive of a PGH2 spillover, which was associated with the enhancement of the endothelium-dependent contractions. These results indicate that in the aorta of SHR and WKY, the endothelium-dependent contractions elicited by A-23187 involve the release of thromboxane A2 and prostacyclin with a most likely concomitant contribution of PGH2.

arteries; endothelial factors; hypertension; TP receptors; spontaneously hypertensive rats; Wistar-Kyoto rats

THE IMPAIRMENT of endothelium-dependent relaxations in the aorta of hypertensive rats (27, 47) was among the first examples of endothelial dysfunctions ever reported. Endothelial dysfunction is a generic term that encompasses many different disorders (11). For instance, in the hypertensive salt-sensitive Dahl rat, the production of endothelial NO is reduced and the endothelin (ET) system is activated (8, 28). In contrast, in the genetic model of spontaneously hypertensive rats (SHR), endothelial dysfunction is attributed to the release of an endothelium-derived contracting factor (EDCF) that counterbalances the effect of NO with no or only minor alteration in the production of the latter (29). In response to acetylcholine, the endothelium-dependent contraction involves the production of reactive oxygen species, the activation of cyclooxygenase (COX)-1, the diffusion of EDCF, and the subsequent stimulation of thromboxane-endoperoxide (TP) receptors on vascular smooth muscle. Because inhibitors of thromboxane synthase do not affect or only poorly affect the endothelium-dependent contraction to acetylcholine, thromboxane A2 is not the EDCF released by acetylcholine (14, 29). As a matter of fact, in the aorta of SHR the EDCFs released by acetylcholine have been identified as prostaglandin (PG)H2 and, paradoxically, prostacyclin (13, 14, 35).

Interestingly, different forms of endothelial dysfunction are also observed in hypertensive patients. In patients with hypertension secondary to primary aldosteronism or to renovascular disease, who exhibit curtailed endothelium-dependent vasodilatations, the inhibition of COX does not improve the response to acetylcholine, suggesting that EDCF plays a minimal role in this endothelial dysfunction. In contrast, in essential hypertensive patients, indomethacin increases, and indeed almost normalizes, the vasodilator response to acetylcholine (45). These findings demonstrate that in essential human hypertension vasoconstrictor products of COX are mainly responsible for the abnormal reaction to endothelium-dependent vasodilators.

Endothelium-dependent contractions are elicited not only by acetylcholine but also in response to other agonists that stimulate G protein-coupled receptors as well as to substances that increase endothelial intracellular Ca2+ concentration in a receptor-independent manner (calcium ionophore, thapsigargin, cyclopiazonic acid). These responses are observed in rat arteries and in blood vessels from other species including the mouse and the human (34, 43, 45). In the aorta of SHR, the characteristics of the endothelium-dependent contractions in response to the calcium ionophore A-23187 are similar to those elicited by acetylcholine. They also rely on the activation of endothelial COX and the stimulation of TP receptors (51). However, despite these similarities, the EDCF(s) released by A-23187 may differ from those produced in response to acetylcholine. This would explain why the amplitude of the endothelium-dependent contractions elicited by A-23187 in arteries from rats and mice is consistently larger than that produced by acetylcholine (43, 51).

The parallelism of the endothelial dysfunction as observed in SHR and in humans with essential hypertension is rather striking, and it warrants the conclusion that EDCFs must play a key role in the resulting endothelial dysfunction and that the nature of these contractile factors must be identified. Therefore, the purpose of the present study was to determine whether or not the endothelium-dependent contractions elicited by A-23187 in the aorta of the SHR can be explained, like those produced by acetylcholine, by the sole release of prostacyclin and PGH2.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
This study was performed in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals as well as with the guidelines established by the ethical committee of the Institut de Recherches Servier. The principal investigator of the present work has been granted a license from the French government to conduct animal research (License No. 03661 delivered by the Ministère de l’Agriculture et de la Forêt, Service Vétérinaire de la Santé et de la Protection Animale).

Experiments were performed on thoracic aortas from 1-yr-old male SHR (400 g, n = 108) and normotensive Wistar-Kyoto rats (WKY; 430 g, n = 58; Charles River, l'Arbresle, France). The rats were anesthetized with pentobarbital sodium (50 mg/kg ip), and blood pressure was measured from the carotid artery (systolic blood pressure 188 ± 5 and 108 ± 4 mmHg in SHR and WKY, respectively; P < 0.05). The aorta was then dissected free, excised, and placed in cold modified Krebs-Ringer bicarbonate solution of the following composition (mM): 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2SO4, 25.0 NaHCO3, 0.026 edetate calcium disodium, and 11.1 glucose (control solution). In some aortas, the endothelium was removed from segments of various lengths by infusing a saponin solution (1 mg/ml for 20 s) that was subsequently flushed with control solution (10). The aorta was then cut into rings (4–5 mm in length). In some rings the isometric tension was recorded, whereas in others the release of prostanoids was studied.

Isometric tension recording. The rings were suspended in organ chambers (20 ml) that contained control solution (37°C) aerated with 95% O2 and 5% CO2. They were connected to a force transducer to record isometric contraction. They were stretched progressively to reach the optimal point of their length-active tension relationship (~2 g). Drug incubation time was at least 30 min for most of the experiments. Concentration-response curves were obtained in a cumulative manner. Each ring was exposed to only one set of cumulative concentrations of a given agonist. Contractile responses were expressed as a percentage of the reference contraction to KCl (60 mM), obtained in each ring at the beginning of the experiment (52).

Release of PGs. These experiments were performed independently from the isometric tension experiments. To measure the release of prostanoids, rings were placed in thermostated minichambers containing 1 ml of control solution (37°C) aerated with 95% O2 and 5% CO2. These aortic rings devoted to PG measurement were not subjected to passive tension. Although flow and dynamic changes such as acute stretch or pulsatile flow evoke the release of PGs (4, 5, 32), in steady-state conditions and in the absence of flow the level of stretch or the level of transluminal pressure, per se, do not appear to affect PG release (4, 5, 7, 9). The equilibration time was 1 h, during which the solution was changed every 15 min. The incubation period with drugs was 20 min, and agonists were applied for 10 min in presence of the drugs. Each ring was exposed once and to a single concentration of agonist. The aortic rings were then removed, and the minichambers were freeze clamped in liquid nitrogen and stored at –80°C for further analysis. The rings were placed in a dry hot box (60°C for 48 h), and the dry weight was measured (14).

PGs were measured with the following enzyme immunoassay kits from Cayman Chemical (Ann Arbor, MI): 6-keto-PGF1{alpha}, thromboxane B2, PGE2, and PGF2{alpha}. Undiluted 50-µl samples were used, with the exception of the 6-keto-PGF1{alpha} measurement, which required a systematic 50x dilution in control solution, and some samples that were subjected to a 2x dilution for the assessment of PGE2 and PGF2{alpha}. The various assays were performed as indicated by the manufacturer's procedure booklet.

Drugs. Acetylcholine hydrochloride, calcium ionophore A-23187, indomethacin, and NG-nitro-L-arginine were obtained from Sigma (La Verpillère, France). NS-398, SC-560, prostacyclin, PGH2, PGF2{alpha}, PGE2, 9{alpha},11{alpha}-azoprosta-5Z,13E-dien-1-oic acid (U-51605) and 9,11-dideoxy-9{alpha},11{alpha}-methanoepoxy PGF2{alpha} (U-46619) were purchased from Cayman Chemical (Ann Arbor, MI). 3-[(6-amino-(4-chlorobenzensulfonyl)-2-methyl-5,6,7,8-tetrahydronapht)-1-yl]propionic acid (S-18886) and dazoxiben were synthesized at Institut de Recherches Servier (Suresnes, France). Drug concentrations are expressed as final molar concentrations in the bath solution.

Data analysis. Data are expressed as means ± SE; n refers to the number of rats from which aortas were taken. Statistical analysis was performed by two-tailed Student's t-test for control and treatment comparisons and by one-way or two-way ANOVA for multiple comparisons followed by a Newman-Keuls or Bonferroni post hoc test, respectively, where appropriate. Differences were considered to be statistically significant when P was <0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Endothelium-dependent and -independent contractions. The calcium ionophore A-23187 (1 nM–1 µM) produced a concentration-dependent and endothelium-dependent contraction in aortas of SHR. The concentration-response curve to A-23187 was significantly shifted to the left in the presence of NG-nitro-L-arginine (100 µM). The TP receptor antagonist S-18886 (100 nM) abolished the endothelium-dependent contraction (Fig. 1). In the presence of NG-nitro-L-arginine (100 µM), the endothelium-dependent contractions in response to A-23187 were significantly larger in aortas of SHR than in those of WKY (Fig. 1).


Figure 1
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  		Fig. 1. Endothelium-dependent contractions produced by the calcium ionophore A-23187 in isolated aortic rings. Top: rings from spontaneously hypertensive rat (SHR) with endothelium. Effect of the NO synthase inhibitor NG-nitro-L-arginine (L-NNA; 100 µM; n = 11) and the thromboxane-endoperoxide (TP) receptor antagonist S-18886 (100 nM; n = 3). Data are means ± SE. Bottom: rings from SHR and Wistar-Kyoto rat (WKY) with and without endothelium (n = 11–35).

 
In the SHR, the maximal amplitude of the endothelium-dependent contractions produced by the calcium ionophore were significantly larger than those evoked by acetylcholine [maximal contraction in % of reference contraction to KCl: 46.7 ± 2.7 (n = 52) and 106.5 ± 3.2 (n = 40) for 10 µM acetylcholine and 1 µM A-23187, respectively; P < 0.05]. Furthermore, the contractions in response to A-23187 were sustained, whereas those observed in response to acetylcholine were transient (data not shown).

In SHR aortas with endothelium, the stable analog of thromboxane A2, U-46619, PGF2{alpha}, PGE2, and the endoperoxide PGH2 produced concentration-dependent contractions, whereas prostacyclin produced only a small increase in tone at the highest concentration tested (30 µM). However, the presence of NG-nitro-L-arginine (100 µM) or the denudation of the endothelium significantly and similarly shifted the concentration curves to the left in response to these various prostanoids. Under these conditions, the order of potency of the agonists was U-46619 >> PGF2{alpha} = PGH2 > PGE2 > PGI2. The prostanoid-induced contractions were virtually abolished by S-18886 (100 nM; Fig. 2).


Figure 2
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  		Fig. 2. Concentration-response curves in response to various prostanoids in isolated rings of SHR. Effects of endothelium removal, L-NNA (100 µM), and TP receptor antagonist S-18886 (100 nM) are shown. Top: U-46619 (1 nM–1 µM; n = 4), prostaglandin (PG)F2{alpha} (10 nM–30 µM; n = 5), and PGE2 (10 nM–30 µM; n = 5). Bottom: prostacyclin (100 nM–30 µM; n = 8) and PGH2 (1 nM–1 µM; n = 5).

 
Release of PGs. In aortas of both SHR and WKY, A-23187 (1 µM) evoked the release of 6-keto-PGF1{alpha} (stable metabolite of prostacyclin), thromboxane B2 (stable metabolite of thromboxane A2), PGE2, and PGF2{alpha}. In both strains, this release was endothelium dependent. The release of prostacyclin was 15–20 times larger than that of the other PGs. The release of thromboxane A2 was significantly larger in preparations of SHR than in those of WKY (Fig. 3).


Figure 3
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  		Fig. 3. A-23187(1 µM)-dependent release of PGs in rat aorta. Accumulation of PGs [prostacyclin and thromboxane A2 (top) and PGE2 and PGF2{alpha} (bottom)] in the incubation medium of isolated aortic rings with or without endothelium taken from WKY and SHR rats in the absence (control) or presence of A-23187 is shown. In response to A-23187, the production of prostaglandins is totally (PGI2 and thromboxane A2) or principally (PGE2 and PGF2{alpha}) endothelium dependent. Data are means ± SE of at least 4 different experiments. *Significant effect of A-23187; #significant difference between WKY and SHR.

 
The maximally effective concentration of A-23187 (1 µM) evoked a significantly larger production and release of prostacyclin (~1.5-fold increase) and, especially, thromboxane A2 (6-fold increase) than the maximally effective concentration of acetylcholine (10 µM). However, the production of PGE2 and PGF2{alpha} was similar in response to the two agents (Fig. 4).


Figure 4
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  		Fig. 4. Acetylcholine (10 µM)- and A-23187 (1 µM)-dependent release of PGs (prostacyclin, thromboxane A2, PGE2, and PGF2{alpha}) in SHR aortas with and without endothelium. Data are means ± SE of at least 4 different experiments. *Statistically significant difference between acetylcholine and A-23187.

 
Cyclooxygenases. In both SHR and WKY aortas, the release of PGs (prostacyclin, thromboxane A2, PGE2, and PGF2{alpha}) induced by A-23187 (1 µM) was not affected significantly by the presence of the preferential COX-2 inhibitor NS-398 (1 µM). In contrast, the selective COX-1 inhibitor SC-560 (0.3 µM) significantly inhibited the production of these various PGs, virtually abolishing that of prostacyclin and thromboxane A2 (Fig. 5).


Figure 5
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  		Fig. 5. Effect of the preferential cyclooxygenase-2 inhibitor NS-398 (1 µM) and the selective cyclooxygenase-1 inhibitor SC-560 (0.3 µM) on the production of PGs (prostacyclin, thromboxane A2, PGE2, and PGF2{alpha}) induced by A-23187 (1 µM) in both SHR (left) and WKY (right) aortas with endothelium. Data are means ± SE of at least 4 different experiments. *Statistically significant effect produced by a cyclooxygenase inhibitor.

 
In both WKY and SHR, the endothelium-dependent contractions induced by A-23187 were partially but significantly inhibited by NS-398 and prevented by the presence of SC-560. The combination of SC-560 plus NS-398 was not more effective than the presence of SC-560 alone. In contrast, the presence of NS-398, SC-560, or their combination did not significantly influence the contraction produced by U-46619 (Fig. 6). The nonselective inhibitor of cyclooxygenase indomethacin (5 µM) abolished both the endothelium-dependent contractions and the release of PGs (data not shown).


Figure 6
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  		Fig. 6. Effect of the preferential cyclooxygenase-2 inhibitor NS-398 (1 µM), the selective cyclooxygenase-1 inhibitor, SC-560 (0.3 µM), and their combination on the endothelium-dependent contraction induced by A-23187 (top; in the presence of 100 µM NG-nitro-L-arginine) and the endothelium-independent contraction induced by the TP receptor agonist U-46619 (bottom) in both SHR (left) and WKY (right) aortas with endothelium. Data are means ± SE of 6 different experiments.

 
Thromboxane A2. In SHR aorta, the thromboxane synthase inhibitor dazoxiben (10 µM) abolished the A-23187-dependent release of thromboxane A2 (Fig. 7), without affecting that of prostacyclin or PGE2 (data not shown), and produced a partial but statistically significant inhibition of endothelium-dependent contractions. In the presence of dazoxiben, these contractions were no longer sustained but transient (Fig. 7). Similar results were obtained in rings of WKY (data not shown).


Figure 7
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  		Fig. 7. Effects of dazoxiben (10 µM) on A-23187-induced endothelium-dependent contractions in isolated aortas of SHR with endothelium (in the presence of 100 µM L-NNA) as well as the release of thromboxane A2 and prostacyclin in the SHR aorta. Top: original traces of A-23187-induced endothelium-dependent contractions. Bottom left: concentration-response curves of the endothelium-dependent contraction. Bottom right: A-23187-induced thromboxane A2 release. Data are means ± SE of 6 different experiments. *Significant inhibitory effect.

 
Prostacyclin. The nonspecific prostacyclin synthase inhibitor U-51605 up to 1 µM produced a concentration-dependent and partial but significant inhibition of A-23187-induced prostacyclin release without significantly affecting that of thromboxane A2. In contrast, 0.5 and 1 µM U-51605 produced a statistically significant increase in A-23187-induced PGE2 and PGF2{alpha} release (Fig. 8). A-23187-induced endothelium-dependent contractions were significantly potentiated by 0.5 µM U-51605 but were significantly inhibited by the compound at 1 µM (Fig. 9).


Figure 8
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  		Fig. 8. Effects of U-51605 (0.5 and 1 µM) on A-23187 (1 µM)-stimulated release of prostacyclin, thromboxane A2, PGE2, and PGF2{alpha} in SHR aortas with endothelium. Data are means ± SE of at least 4 different experiments. *Significant effect of U-51605.

 

Figure 9
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  		Fig. 9. Effects of U-51605 (0.5 and 1 µM) on the endothelium-dependent contractions in response to A-23187 in SHR aortas with endothelium (in the presence of 100 µM NG-nitro-L-arginine). Data are means ± SE of 5 different experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrates that in the SHR aorta endothelium-dependent contractions in response to the calcium ionophore A-23187, in contrast to those evoked by acetylcholine, involve the production and release of thromboxane A2.

The endothelium-dependent contractions elicited by A-23187 and acetylcholine share many similarities. Those evoked by A-23187 are abolished by S-18886, a selective TP receptor antagonist (36), and SC-560, a potent and selective inhibitor of COX-1, but only partially inhibited by NS-398, a moderately selective inhibitor of COX-2 (21). The inhibitory effect of SC-560 is paralleled by a profound reduction of PG release. In contrast, the partial inhibition of the endothelium-dependent contractions by NS-398 is not associated with a significant decrease in PG production. Although NS-398 does not affect the responses to U-46619, indicating that this compound does not interact directly with TP receptors, a yet-undetermined nonspecific effect of NS-398 may explain the partial inhibition of the endothelium-dependent contraction. Therefore, the endothelium-dependent contractions produced by A-23187, like those elicited by acetylcholine (29, 49, 50), involve COX-1 activation and TP receptor stimulation.

However, the release of thromboxane A2 is six times larger in response to A-23187 than in response to acetylcholine. This release is fully endothelium dependent and is abolished by the thromboxane synthase inhibitor dazoxiben (14). The effect of dazoxiben appears specific, because the production of prostacyclin and PGE2 is not affected. Acetylcholine-induced contractions are not affected by dazoxiben (3, 14, 22, 24, 29), whereas those induced by A-23187 are reduced by half and over the whole concentration range of A-23187. These observations indicate that in the SHR aorta thromboxane A2 is an EDCF released by A-23187. Although various PGs contract the smooth muscle cells by activating TP receptors, thromboxane A2 is by far the most potent agonist (14). The enhanced release of thromboxane A2 in response to A-23187 certainly explains the larger amplitude and the difference in the kinetics (sustained vs. transient contractions) of the endothelium-dependent contractions produced by the calcium ionophore compared with those elicited by the muscarinic agonist. Indeed, in the presence of dazoxiben, the maximal amplitude and the transient nature of the time course of the contractions produced by the two mediators became similar.

The involvement of thromboxane A2 has been previously reported in both SHR and WKY arteries, for instance, in the endothelium-dependent and -independent contractions in response to free radical generation (18) or in the endothelium-dependent component of the contractions evoked by ET-1 (41, 42). The expression of TP receptors in vascular smooth muscle cells of the rat aorta is well documented (46), and hypertension does not appear to affect its expression (23). However, in response to A-23187, the increased production of thromboxane A2, in the SHR aorta compared with that of WKY is sufficient to explain the enhanced EDCF-mediated responses in these hypertensive animals.

As dazoxiben abolished thromboxane A2 synthesis but only produced a 50% inhibition of the endothelium-dependent contractions, another mediator(s) must be involved in the responses to A-23187. The endothelium-dependent contractions elicited by acetylcholine in the aorta of mature SHR and WKY most likely involve the release of prostacyclin (14). This earlier conclusion was based on the following observations: 1) In isolated WKY and SHR aortic rings, prostacyclin is a contracting but not a relaxing factor. Indeed, prostacyclin is generally described as an endothelium-derived vasodilator, which, by stimulating its receptor [prostacyclin (IP) receptors] and activating adenylate cyclase, elevates intracellular cAMP concentration and produces relaxation of the vascular smooth muscle (48). However, in WKY and SHR older than 30 wk, neither prostacyclin nor its stable analog iloprost produces a relaxation (14, 25, 35). This is a phenotypic peculiarity of these two strains, because aortic rings from Fischer rats up to 23 mo of age relax in response to IP receptor agonists (35). In both WKY and SHR, IP receptor gene expression decreases with age and, at any given age, is systematically less expressed in SHR than in WKY (33). 2) Prostacyclin is a more potent contracting agent in SHR than in WKY. 3) Prostacyclin- and endothelium-dependent contractions both involve activation of TP receptors. 4) Prostacyclin is the most abundant PG released by acetylcholine and is of endothelial origin. 5) The time course of the release of prostacyclin is compatible with the time course of the observed endothelium-dependent contractions. 6) The endothelium-dependent contractions and the release of prostacyclin are blocked by COX-1 inhibitors (14, 25, 29, 35, 45, 51, 53). All the arguments developed above for a contribution of prostacyclin in acetylcholine-induced endothelium-dependent contractions also apply to A-23187. Because the calcium ionophore releases even more prostacyclin than acetylcholine does, it is legitimate to assume that prostacyclin contributes to the contraction produced by A-23187. In the rat aorta, prostacyclin is a weak agonist of the TP receptor and induces a contractile response, whereas its stable metabolite 6-keto-PGF1{alpha} is virtually ineffective (14). The rapid metabolism of prostacyclin may explain the transient nature of the endothelium-dependent contractions observed in response to either acetylcholine or A-23187 in presence of dazoxiben.

The involvement of prostacyclin in endothelium-dependent contractions to A-23187 is reinforced by the effects of the inhibitor of PGI2 synthase U-51605. U-51605 is a stable analog of PGH2 that inhibits both prostacyclin and thromboxane synthases, with a slight selectivity toward the former (14–16). It is also a partial agonist at TP receptors (14, 19, 30). In the present study, U-51605 at a dose of 0.5 µM produced a partial inhibition of prostacyclin release but did not affect that of thromboxane A2. However, at this concentration the compound already produces a substantial inhibition of TP receptors (14). Paradoxically, the inhibition of the release of prostacyclin, one of the putative EDCFs, and the concomitant blockade of TP receptors were associated with the enhancement of the endothelium-dependent contractions to A-23187. This is only an apparent paradox, because the inhibition of prostacyclin release was compensated by a major increase in PGE2 and PGF2{alpha} production. PGH2 is an unstable PG that undergoes spontaneous or enzymatic transformation toward PGF2{alpha} and PGE2 (2, 6, 14). In endothelial cells, if the constitutive presence of the soluble PGE synthase associated with COX-1 is debatable, the parallel induction of the membrane-bound form of PGE-synthase with COX-2 is well documented (31, 37). Because the induction of COX-2 has been suggested in the aorta of SHR and old WKY (1, 17), the production of PGE2 might be of enzymatic origin. However, it cannot be excluded, especially when PGI synthase is inhibited, that the rate of formation of PGH2 exceeds its metabolism. Therefore, the production of PGE2 and PGF2{alpha} can partially represent spillover of PGH2. Functionally, in the rat aorta PGH2, PGE2, and PGF2{alpha} all activate TP receptors and have similar potency and affinity (14). A higher concentration of U-51605 (1 µM) did not produce any further potentiation and even caused a small inhibition of the A-23187-induced endothelium-dependent contractions, most likely because of the overwhelming antagonistic properties of this compound toward TP receptors (14). Tranylcypromine is often considered to be a nonspecific inhibitor of this enzyme (12). However, under the present experimental conditions, the monoamine oxidase inhibitor did not affect the production of prostacyclin, therefore precluding its utilization (14).

The amplitude of endothelium-dependent contraction in the SHR and WKY aorta is positively correlated with the level of arterial blood pressure (20). However, chronic treatment with either a cyclooxygenase inhibitor or a TP receptor antagonist does not prevent the establishment of hypertension and does not decrease arterial blood pressure in SHR (26, 38, 54) but restores the endothelium-dependent relaxation in the aorta (44). These results suggest that in SHR the production of EDCF is secondary to chronic hypertension and exacerbates the endothelial dysfunction. Similarly, in patients with essential hypertension, the endothelial dysfunction must precede the onset of high arterial blood pressure because in young genetically predisposed normotensive offspring of essential hypertensive patients the response to acetylcholine is already reduced (40). Furthermore, antihypertensive agents do not have the same impact on endothelial function despite similar reduction of arterial blood pressure (39). Altogether the available data suggest that the endothelial dysfunction in SHR and in patients with essential hypertension is not directly related to blood pressure values and is probably genetically determined. The identification of the EDCFs released may help to design better treatments to restore proper endothelial function in hypertension.

In conclusion, the endothelium-dependent contractions elicited by A-23187 and acetylcholine in the aorta of SHR involve the release of different PGs: thromboxane A2, prostacyclin, and most likely PGH2 for the former but only prostacyclin and PGH2 for the latter.


   ACKNOWLEDGMENTS
 
The authors thank E. Bonhomme, M. Gaudin, G. Gautier, and M. Germain for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Félétou, Département Angiologie, Institut de Recherches Servier, 11 rue des Moulineaux, 92150 Suresnes, France (e-mail: michel.feletou{at}fr.netgrs.com)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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