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Cardiovascular Research 2001 49(1):152-160; doi:10.1016/S0008-6363(00)00238-8
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Copyright © 2000, European Society of Cardiology

Cysteinyl leukotrienes are involved in angiotensin II-induced contraction of aorta from spontaneously hypertensive rats

Françoise Stanke-Labesque*, Philippe Devillier1, Sophie Veitl, Françoise Caron, Jean-Luc Cracowski and Germain Bessard

Laboratory of Pharmacology, University of Medicine, LSCPA EA2937 F-38706, La Tronche Cedex, France

* Corresponding author. Tel.: +33-4-7663-7159; fax: +33-4-7651-8667 Francoise.Stanke{at}ujf-grenoble.fr

Received 13 April 2000; accepted 13 September 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Non specific lipoxygenase inhibitors have been reported to reduce the in vitro constrictor response and the in vivo pressor effect of angiotensin II in rats. The aim of this study was to assess the role of cysteinyl leukotrienes, in the vascular response to angiotensin II in spontaneously hypertensive rats (SHR). Methods: Rings of thoracic aorta from SHR and normotensive Wistar–Kyoto rats (WKY) were compared in terms of contractile responses and release of cysteinyl leukotrienes in response to angiotensin II. Results: Pretreatment with the specific 5-lipoxygenase inhibitor AA861 10 µM reduced the efficacy of angiotensin II in intact and endothelium-denuded aorta from SHR (% inhibition vs. control: 65±12.6% with endothelium (n = 6), P<0.05; 43±7.2% without endothelium (n = 6), P<0.05) but not in aorta from WKY. In addition, in aorta from SHR, the CysLT1 receptor antagonist MK571 1 µM reduced by 55±6.1% (n = 6, P<0.05) the contractile effects of angiotensin II in rings with endothelium but not in endothelium-denuded rings. Angiotensin II induced a 8.6±2.1-fold increase in cysteinyl leukotriene production in aorta rings from SHR with endothelium which was prevented by the AT1 receptor antagonist losartan 1 µM but not by the AT2 receptor antagonist PD123319 0.1 µM. In aorta rings from WKY, cysteinyl leukotriene production remained unchanged after exposition to angiotensin II. The cysteinyl leukotrienes (up to 0.1 µM) induced contractions in aorta rings from SHR but not from WKY. Conclusions: These data suggest that cysteinyl leukotrienes, acting at least in part on endothelial CysLT1 receptors, are involved in the contractile response to angiotensin II in isolated aorta from SHR but not from WKY.

KEYWORDS Angiotensin; Arteries; Contractile function; Hypertension; Lipid metabolism; Receptors


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Angiotensin II (Ang II)-mediated vasoconstriction is involved in both the physiological maintenance of arterial pressure and the pathogenesis of various forms of experimental and human hypertension. It is now well documented that Ang II stimulates synthesis and release of many different eicosanoids from a variety of cells and tissues [1]. This action is primarily the consequence of phospholipase activation resulting in the formation of free arachidonic acid available for metabolism by oxygenases.

Compelling evidence suggests that lipoxygenase-derived eicosanoids contribute to prohypertensive mechanisms in Ang II-dependent hypertension [1]. Lipoxygenases catalyze the formation of 5-,12- and 15-HPETEs which are subsequently transformed to the corresponding HETEs and in the case of 5-HETE to leukotrienes. Phenidone and baicalein which inhibit the three lipoxygenases have been shown to markedly reduce the in vitro constrictor response of rat femoral artery to Ang II [2], to attenuate the in vivo pressor response to Ang II in normotensive rats [2], and to decrease blood pressure in two-kidneys, one clip hypertensive rats [3]. In addition, the magnitude of the hypotensive effect of phenidone was considerably higher in SHR than in WKY [4]. These data suggest that the Ang II-mediated pressor effects may be subserved by lipoxygenase-derived metabolites of arachidonic acid and that these mediators are much more involved in the control of the vascular tone in SHR than in WKY.

The 5-lipoxygenase pathway leads to the formation of 5-HETE which can be converted to leukotrienes B4 (LTB4) and to the cysteinyl leukotrienes (LTC4, LTD4 and LTE4). The cysteinyl leukotrienes have been shown to exert relaxant and contractile effects on different vascular preparations [5–12]. In addition, intravenous injection of LTD4 induced a transient dose-dependent increase in mean blood pressure, which was greater in SHR than in WKY [13]. Moreover, in hypoxic rats [14], specific inhibition of leukotriene synthesis by blockade of 5-lipoxygenase activating protein (FLAP) decreased Ang II-mediated pulmonary pressor response, suggesting the enhancing effect of leukotrienes in the Ang II pressor effects. Furthermore, it has recently been shown that Ang II infusion induced an up regulation of leukotriene A4 hydrolase expression in the heart of rat [15]. Leukotrienes can be produced by the smooth muscle cells [9,16,17] or by the monocyte/macrophages found in the intima of SHR vessels [18]. In this context, the present study was designed to assess the involvement of the 5-lipoxygenase metabolites, particularly the cysteinyl leukotrienes, in the vasoconstrictor effects of Ang II on isolated aorta from spontaneously hypertensive rats (SHR) and control normotensive Wistar–Kyoto rats (WKY).


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Measurement of arterial blood pressure and rat aorta preparation
The care and use of animals in this work were in accordance 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). Experiments were conducted on adult male SHR and age- and weight-matched WKY (12–15 weeks old). The right carotid artery of rats, anesthetized with sodium pentobarbital (50 mg/kg intra peritoneal), was cannulated with polyethylene tubing (PE-50) connected to a pressure transducer (Statham, USA) for recording of arterial blood pressure on a polygraph (Windograph, Gould Instrument, USA). After mean arterial blood pressure (MABP) measurement, the thoracic aorta was excised, transferred to dish filled with Krebs bicarbonate buffer, cleared of periadventitial tissue, and cut into ring segments (3.0 mm in length). In certain rings, the endothelium was removed by gentle rubbing of the intimal surface with small forceps; in the remaining rings, care was taken not to touch the inner surface of the blood vessels.

2.2 Measurement of isometric tension in rings of aorta
Each aortic ring was placed inside a 10-ml organ bath filled with Krebs solution of the following composition (mM): NaCl (118.0), KCl (4.7), CaCl2 (25), MgSO4 (1.0), KH2PO4 (1.0), NaHCO3 (25) and glucose (11.0). The Krebs solution was continuously bubbled with a gas mixture (5% CO2, 95% O2) and thermostated at 37°C. The rings were mounted between two stainless steel wires, the lower wire was fixed to a micrometer (Mitutoyo, Japan) and the upper wire was attached to a force transducer (UF-1 Pioden, UK) through which changes in isometric forces were continuously displayed on recorder (Linseis 200, France). The rings of aorta were initially stretched to a given preload of 1.5 g and then were allowed to equilibrate for 60 min with change of the medium every 15 min. After the 60-min equilibration period, experiments were initiated by obtaining in each ring a reference contraction in response to KCl (90 mM). KCl 90 mM elicited similar contractions in aortic rings of normotensive (1.69±0.07 g, n = 92) and hypertensive rats (1.62±0.04 g, n = 114). The endothelial function was assessed by testing the relaxant effect of acetylcholine 1 µM to 0.1 mM on aortic rings precontracted with methoxamine 3 µM. The failure of acetylcholine to elicit relaxation of aortic rings previously subjected to rubbing of the intimal surface was taken as proof of endothelium removal. Subsequently, the rings were allowed to equilibrate for another hour, with the Krebs solution being changed every 15 min. Only one cumulative concentration–response curve was established for Ang II (0.5log increments, 0.1 nM–1 µM) in each ring. The contribution of cysteinyl leukotrienes was assessed with prior incubation of the preparations with either the specific 5-lipoxygenase inhibitor AA861 (10 µM for 30 min) [19] or with the specific CysLT1 receptor antagonist (MK571 1 µM for 30 min) [20,21]. Appropriate controls (incubation with solvents) were run under similar experimental conditions in rings obtained from the same aorta.

The contractile effects of the cysteinyl leukotrienes was also assessed in intact aortic rings from both SHR and WKY. Cumulative concentration response curves were constructed for either LTB4, LTC4, LTD4 or LTE4 (1-log increment, 1 pM–0.1 µM) on intact preparations pretreated or not with AA861 to avoid endogenous leukotriene effects. Pretreatment with AA861 10 µM or solvent was made for 30 min, and then the Krebs solution containing AA861 or vehicle was changed every 10 min for a second period of 30 min. The effect of MK571 on concentration–response curves for LTD4 (1-log increment, 1 pM–0.1 µM) were performed on intact or endothelium-denuded aortic rings of SHR pretreated with MK571 1 µM or solvent for 30 min in the presence of AA861. Lastly, in order to study the involvement of NO or endothelin-1 in LTD4-evoked contractions, cumulative concentration–response curves for LTD4 were performed on intact aortic rings after incubation for 30 min with either N-{omega}-nitro-L-arginine (NLA, 100 µM) or with the ETA receptor antagonist BQ123 (1 µM) [22,23] and the ETB receptor antagonist BQ788 (1 µM) [23].

2.3 Measurement of cysteinyl leukotriene release in rings of rat aorta
Rings of SHR and WKY were prepared as described for organ bath experiments. Rings with and without endothelium were placed in a siliconized tube containing 1 ml Krebs solution oxygenated with 95% O2/5% CO2. The rings were allowed to equilibrate for 60 min at 37°C, the Krebs solution being changed every 15 min. Intact or endothelium-denuded ring vessels were then incubated for 30 min with either Ang II (3 nM, 30 nM and 0.3 mM) or solvents (basal levels of eicosanoid release). In a different series of experiments aortic rings were preincubated with the AT1 receptor antagonist losartan 1 µM [24], or with the AT2 receptor antagonist PD123319 0.1 µM [24] for 30 min prior to challenge with the submaximal concentration of Ang II (0.3 µM) for another 30 min. In addition, the effect of the calcium ionophore (A23187 [GenBank] 10 µM for 30 min) on cysteinyl leukotriene production was also assessed in intact aortic rings. The Krebs solution was collected and samples were frozen at –80°C. The rings were dried in an oven for measurement of dry weight. Cysteinyl leukotrienes were measured by enzyme immunoassay on unextracted samples using reagents purchased from Cayman (Ann Arbor, USA). The detection limit of the assay was 3.2 pg/ml, the EC50 value (50% B/B0) was 36.6 pg/ml and the intra and inter-assay coefficients of variations were <10%.

2.4 Drugs
Drugs used and their source were: KCl (Prolabo Normapur, France), angiotensin II, acetylcholine, methoxamine, AA861 (2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone), N-{omega}-nitro-L-arginine, and the calcium ionophore A23187 [GenBank] from Sigma (L'Isle d'Abeau, France). Leukotriene B4, leukotriene C4, leukotriene D4, leukotriene E4, and the CysLT1 receptor antagonist MK571 (propanoic acid, 3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-(E)-, sodium salt) were purchased from Cayman (Ann Arbor, USA). Losartan (Dup 753) and PD123319 ((S)1-[[4-(dimethylamino)-3-methylphenyl]-methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]-pyridine-6-carboxylic acid, ditriflouroacetate, monohydrate), were kindly provided by Merck (Rahway, USA) and Research Biochemicals International (Natrick, USA), respectively. BQ123 (cyclo(D-{alpha}-aspartyl-L-propyl-D-valyl-L-leucyl-D-tryptophyl), and BQ788 (N-[N-[N-[(2,6-dimethyl-1-piperidinyl)carbonyl]-4-methyl-L-leucyl}-1-(methoxycarbonyl)-D-tryptophyl]-D-norleucine monosodium) were from RBI (Natick, USA).

2.5 Statistical analysis
The cysteinyl leukotriene data were expressed as pg/mg dry weight tissue. Contractile responses were expressed as percentage of the contraction induced by KCl 90 mM. The maximal effect (Emax) was the greatest response obtained with the agonist. The concentration of agonist producing 50% of the maximal effect (EC50) was determined from each curve by a logistic curve-fitting equation. The pD2 value is the negative logarithm of the EC50.

Results are expressed as mean±standard error of the mean (S.E.M.) for the specified number of preparations tested. Statistical analysis were performed using analysis of variance (ANOVA) for repeated measures followed by Bonferroni corrected t-test. Individual comparisons were made by Student's t-test for paired or unpaired data as appropriate. P values <0.05 were considered to be significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Blood pressure and body weight
MABP of SHR (189.8±56.1 mmHg; n = 36) was significantly higher (P<0.0001) than MABP of control WKY (107.3±2.4 mmHg; n = 26) whereas body weight was similar in both groups (SHR: 288.8±6.7 g (n = 36) vs. WKY: 292.7±2.7 g (n = 26) NS).

3.2 Organ chamber experiments
In terms of both potency and efficacy, Ang II elicited similar concentration-dependent contractions on intact aortic rings taken from WKY and SHR (Table 1). Removal of the endothelium enhanced (P<0.0001) the maximal contraction elicited by Ang II in both WKY and SHR preparations. On endothelium-denuded rings, constrictor responses to Ang II were greater (P<0.006) in aortic rings of WKY than of SHR (Table 1).


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  		Table 1 Potency (pD2) and efficacy (Emax, expressed as percentage of 90 mM KCl-induced contraction) of Ang II in aorta from SHR or WKY with or without endotheliuma

 
The 5-lipoxygenase inhibitor AA861 (10 µM) induced decreases of the resting tone of 0.03±0.04 g (n = 8) and 0.07±0.08 g (n = 8) in isolated aorta from WKY with and without endothelium, respectively, and of 0.12±0.08 g (n = 6) and 0.13±0.2 g (n = 6) in isolated aorta from SHR with and without endothelium, respectively. The 5-lipoxygenase inhibitor did not affect the contractions elicited by Ang II in aortic rings of WKY with or without endothelium (Fig. 1a, Table 1). In marked contrast, AA861 reduced the amplitude of the contraction to Ang II in aorta from SHR with or without endothelium by about 65% and 40%, respectively (Fig. 1b, Table 1).


Figure 1
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  		Fig. 1 Effect of AA861 10 µM on concentration–response curves to Ang II in aortic rings with (E+) or without (E–) endothelium from WKY (a) and SHR (b). Results are expressed as percentage of the contraction elicited by KCl 90 mM and are presented as mean±S.E.M. of six to eight experiments. *P<0.05 with respect to control.

 
The CysLT1 receptor antagonist MK571 (1 µM) for 30 min did not alter the resting tone in rings of WKY but induced a slight decrease of the resting tone of 0.04±0.02 g (n = 6) and 0.01±0.02 g (n = 10) in rings of SHR with and without endothelium, respectively. In intact or endothelium-denuded aortic rings from WKY, MK571 had no effect on Ang II-induced contractions (Fig. 2a, Table 1). In contrast, in rings of SHR with endothelium, MK571 significantly reduced by 54% Ang II-induced contractions whereas it did not alter contractions elicited by Ang II in endothelium-denuded rings (Fig. 2b, Table 1). LTC4, LTD4 or LTE4 induced no contraction of intact aorta from WKY and SHR (n = 4 experiments performed on different aortic rings for each leukotrienes). However, on preparations pretreated with AA861 and after the repeated washings to eliminate the cysteinyl leukotrienes spontaneously released by the preparations, LTD4 induced concentration-dependent contractions of the intact aorta from SHR (pD2=7.4±0.1, Emax: 6.4±2.1%, n = 11) whereas LTC4 and LTE4 induced weaker contractions (Emax=3.9±2.4% for LTC4 (n = 4) and 1.5±0.35% for LTE4 (n = 3), Fig. 3a) and LTB4 induced no contraction (n = 3). In contrast, the intact aorta from WKY remained unresponsive to each leukotriene (up to 0.1 µM) in these experimental conditions. In endothelium-denuded aorta from SHR, LTD4 and LTC4 at the maximal concentration of 0.1 µM induced weak contractions (Emax: 4.3±1.7% for LTD4 (n = 4) and 5.0±2.0% for LTC4 (n = 3)). Pretreatment with MK571 (1 µM) completely abolished the LTD4-induced contraction in intact aorta (Emax: 0.3±0.3% (n = 4), P<0.05 vs. control) (Fig. 3b) but not in endothelium-denuded aorta from SHR (Emax: 54.5±1.5% (n = 4)). Pretreatment with the ETA and ETB receptor antagonists (BQ123 (1 µM) and BQ788 (1 µM)) did not induced any significant change of LTD4-induced contractions in intact aortic ring from SHR. The maximal contractions elicited by LTD4 (0.1 µM) were 6.4±2.1% (n = 11) and 6.4±2.6% (n = 4) in the absence (control) or the presence of BQ123 and BQ788, respectively. Pretreatment with NLA which by itself induced an increase on the basal tone of 0.12±0.04 g (n = 4), enhanced LTD4-induced contractions in intact aortic rings from SHR (Emax: 6.4±2.1% (control, n = 11) vs. 14.8±4.7% (NLA, n = 4) P<0.01; PD2: 7.4±0.1 (control, n = 11) vs. 8.1±0.1 (NLA, n = 4) P<0.004).


Figure 2
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  		Fig. 2 Effect of MK571 1 µM on concentration–response curves to Ang II in aortic rings with (E+) or without (E–) endothelium from WKY (a) and SHR (b). Results are expressed as percentage of the contraction elicited by KCl 90 mM and are presented as mean±S.E.M. of six to ten experiments. *P<0.05 with respect to control.

 

Figure 3
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  		Fig. 3 (a) Contractile effect of LTB4 (n = 3), LTC4 (n = 4), LTD4 (n = 11) and LTE4 (n = 3) on the basal tone of intact aorta from SHR, (b) inhibition by MK571 (1 µM) on LTD4-induced contraction in aortic rings with (E+) or without endothelium (E–) from SHR. The vascular preparations were pretreated with AA861 10 µM. Results are expressed as percentage of the contraction elicited by KCl 90 mM and are presented as mean±S.E.M. of n experiments; n = 4 for experiments performed on intact aortic rings in the presence of MK571 1 µM, n = 4 for experiments performed in endothelium-denuded aortic rings in the presence or the absence (control) of MK571. *P<0.05 with respect to control.

 
3.3 Measurement of cysteinyl leukotriene production
The basal level of cysteinyl leukotriene production in aorta from SHR with or without endothelium was significantly lower than in aorta from WKY (Table 2). On intact and endothelium-denuded aortic rings from SHR, Ang II induced concentration-dependent increases in cysteinyl leukotriene production. For 0.3 µM Ang II, the mean increases in cysteinyl leukotriene production were 8.7±2.1-fold in intact rings and 4.8±1.2-fold in rings without endothelium (P<0.05 vs. controls) (Table 2). Pretreatment with the AT1 receptor antagonist, losartan (1 µM), prevented the increase in cysteinyl leukotriene production induced by 0.3 µM Ang II in SHR aortic rings with or without endothelium. Blockade of the AT2 receptors with PD123319 (0.1 µM) had no effect on Ang II-mediated cysteinyl leukotriene production on intact aortic rings. In contrast, in aorta from WKY, Ang II (3 nM, 30 nM, 0.3 µM) failed to modify cysteinyl leukotriene production, and neither losartan nor PD123319 had influence on cysteinyl leukotriene levels (Table 2).


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  		Table 2 Release of cysteinyl leukotrienes from SHR or WKY aorta rings with or without endothelium, in the absence (basal) or in the presence of Ang II (3 nM, 30 nM or 0.3 µM), losartan (1 µM) plus Ang II (0.3 µM), PD123319 (0.1 µM) plus Ang II (0.3 µM)a

 
The calcium ionophore A23187 [GenBank] (10 µM) induced 2.5- and 2.1-fold increases in cysteinyl leukotriene production in intact aorta from SHR and WKY, respectively (pg/mg dry weight tissue: SHR: (21.0±6.2 (basal, n = 6) vs. 48.9±10.1 (A23187 [GenBank] , n = 6), P<0.05) and WKY: (35.8±16.3 (basal, n = 6) vs. 116.0±43.5 (A23187 [GenBank] , n = 6), P<0.05)).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The results of this study provide the first evidence for the involvement of cysteinyl leukotrienes in the in vitro constriction induced by Ang II in aorta from SHR but not from WKY.

4.1 Contractile responses to Ang II in aorta from SHR and WKY
The cardiovascular system of SHR is characterized by structural and functional alterations compared to WKY. In terms of functional alterations, increases in contractile responses to Ang II have been reported in mesenteric [25,26], femoral [27] or renal arteries [28]. However, in the present study, the contractions elicited by Ang II were similar in intact aortic rings from both SHR and WKY and, after endothelium removal, were higher in WKY rings than in SHR rings. These results are in full agreement with a previous study conducted on rat aorta preparations with angiotensin II [29] and may reflect differences in the adaptive processes taking place as a consequence of elevated blood pressure within the arterial wall of different vascular beds. The enhanced contractile response to Ang II in SHR and WKY aorta rings without endothelium is in line with the inhibitory effect of endothelium on Ang II-induced response in aorta [30], carotid artery [31] and mesenteric artery [32] in both rat strains.

4.2 Inhibitory effect of a 5-lipoxygenase inhibitor on Ang II-induced contractions
A pivotal finding of the present study is the inhibitory effect of the specific 5-lipoxygenase inhibitor, AA861, on the Ang II-induced contraction in intact or endothelium-denuded aorta from SHR but not from WKY.

Phenidone and baicalein which inhibit the 5-, 12- and 15-lipoxygenases have been previously shown to markedly reduce the in vitro constrictor response of Sprague–Dawley rat femoral artery to Ang II [2]. Blockade of lipoxygenases with these two inhibitors also attenuated the in vivo pressor response to Ang II [2] and phenidone induced a greater decrease of intraarterial systolic pressure in SHR than WKY [4]. In this regard, specific inhibition of 12-lipoxygenase was subsequently reported to induce an hypotensive effect in SHR but not in WKY [33]. These data suggested that lipoxygenase products were much more involved in the control of the vascular tone in SHR than in WKY. The present study demonstrates that, in addition to 12-lipoxygenase products, 5-lipoxygenase products play a major role in Ang II constrictor effect in aorta from SHR since specific inhibition of 5-lipoxygenase inhibited the response to Ang II. In contrast, none of these effects occurred in aorta from WKY. These results are in line with the previous demonstration of a modulatory role of the 5-lipoxygenase pathway on Ang II-induced aldosterone production in rat glomerulosa cells [34].

4.3 Cysteinyl leukotriene production in SHR and WKY aorta
The unstimulated aorta preparations from WKY and SHR produced cysteinyl leukotrienes. An unexpected finding was that aorta from WKY produced higher basal levels of cysteinyl leukotrienes than aorta from SHR. Leukotrienes can be produced by the vascular smooth muscle cells [9,16,35], the endothelial cells [36,37] and the monocyte/macrophages infiltrating the vascular wall [9]. In contrast with the SHR, no monocyte/macrophage infiltration occurred in the intima of WKY aorta [18]. In addition, the production of cysteinyl leukotrienes was similar in WKY aorta with and without endothelium suggesting that cysteinyl leukotriene production in WKY could take place at the vascular smooth muscle level. An alteration of the production of eicosanoids between the two rat strains has been previously shown in several vascular beds of age-matched WKY and SHR [38–40], and may explain difference in the production of cysteinyl leukotrienes.

Although a higher basal production of cysteinyl leukotrienes was found in aorta from WKY, 5-lipoxygenase inhibition did not alter the contractile response to Ang II. In addition, no increase in the release of cysteinyl leukotrienes occurred in response to Ang II in aorta from WKY. Although Ang II did not cause production of cysteinyl leukotrienes in WKY aorta, a calcium ionophore (A23187 [GenBank] ) induced a similar increase in cysteinyl leukotriene production in aorta from WKY and SHR. These results provide evidence that Ang II-induced vasoconstriction is not coupled to cysteinyl leukotriene release in WKY.

In contrast to WKY aorta, Ang II induced a concentration-dependent release of cysteinyl leukotrienes in SHR aorta. Ang II mediates contraction of small mesenteric arteries of young SHR through stimulation of AT1 and AT2 receptors [41]. These two Ang II receptor subtypes are involved in arachidonic acid release from rabbit and porcine aortic smooth muscle cells [42,43]. In addition, the two subtypes are expressed by human monocytes and macrophages [44,45] and by rat coronary endothelial cells [46]. The possible contributions of the AT1 and AT2 receptors to the Ang II-induced release of cysteinyl leukotrienes has been therefore studied by using by the selective AT1 and AT2 receptor antagonists, losartan and PD123319, respectively. The increase in cysteinyl leukotriene production induced by Ang II in aorta rings from SHR was blocked by losartan but not by PD123319 indicating that Ang II was acting through activation of AT1 receptors. The release of cysteinyl leukotrienes in response to Ang II and the inhibitory effect of losartan on this response were not altered by endothelium removal supporting previous reports that an increase in lipoxygenase activity can be mediated through AT1 receptors both on vascular smooth muscle cells [47] and macrophages [48].

4.4 Contractile responses of SHR and WKY aorta to leukotrienes
Cysteinyl leukotrienes have been already reported to induce contraction of rat isolated vessels [11,12,49]. Constriction of mesenteric [12] and pulmonary vessels [49] has been shown in response to LTD4/E4 and LTE4, respectively. In rat aorta preparations, a weak contraction in response to LTD4 (0.2 µM) has been observed [11] whereas in another study, no contraction to LTC4 and LTD4 was found [5]. In the present study, LTC4, LTD4 and LTE4 caused no contraction of intact aorta rings from SHR and WKY. However, washings of isolated aorta and pretreatment with AA861 to eliminate endogenous leukotrienes unmasked reproducible and concentration-dependent contractions in response to LTC4 and LTD4 (up to 0.1 µM) in SHR but not in WKY preparations. These results suggest a better responsiveness of SHR aorta than WKY aorta to the contractile activity of cysteinyl leukotrienes. In this context, it is interesting to note that a contractile effect of LTC4 and LTD4 has been reported in human atherosclerotic coronary arteries but not in non atherosclerotic arteries, that was associated with the presence of specific leukotriene binding sites in atherosclerotic arteries [9]. In addition, in line with a previous report on human artery and vein [8], LTB4 failed to contract SHR or WKY aorta. These results suggest that the cysteinyl leukotrienes are the 5-lipoxygenase products involved in SHR aorta contraction.

4.5 Modulation of Ang II-induced contraction by endothelial cysteinyl leukotriene receptors in SHR aorta
The inhibitory effect of the selective CysLT1 receptor antagonist MK571 on Ang II-induced contraction in intact aorta from SHR but not WKY provided further evidence of the involvement of cysteinyl leukotrienes in Ang II-mediated contraction in aorta from hypertensive rat. Furthermore, the observation that MK571 inhibited the Ang II-induced contraction in intact rings but not in deendothelialized rings of SHR suggests that stimulation of endothelial CysLT1 receptors potentiates contractile response to Ang II. This hypothesis is reinforced by the blocking effect of MK-571 on LTD4-induced contraction in intact SHR aorta rings and the inefficacy of this antagonist in deendothelialized rings. Pertaining with this finding, it has been reported that endothelial CysLT1 receptors mediated a contractile response in human pulmonary veins through the release of a contractile factor which remains however to be characterized [50]. In aorta from SHR, this factor is not the endothelins since the LTD4 contractile response in intact aorta was not altered by the combination of ETA and ETB receptor antagonists (present study). In addition to endothelial CysLT1 receptor associated with contraction, endothelial CysLT2 receptors associated with relaxation have been suggested in rat aorta [10] and in human pulmonary vessels [50]. In intact SHR aorta precontracted with U46619 [GenBank] , LTD4-induced relaxation was potentiated by MK-571 (data not shown) suggesting, as previously reported in human pulmonary vein, that the full relaxation to LTD4 may have been overridden by the endothelial CysLT1 receptor-dependent contraction. The endothelium-dependent relaxation induced by LTD4 in rat aorta was inhibited by free hemoglobin suggesting the involvement of nitric oxide [10]. This data is in line with the increase in LTD4-induced contraction in the presence of nitric oxide synthase inhibitor (present study). Together, these data suggest that the inhibitory effect of MK571 on Ang II-induced contractions in intact SHR aorta resulted from both the direct blockade of the contraction mediated by endothelial CysLT1 receptors and the indirect potentiating effect of the relaxation mediated by endothelial CysLT2 receptors.

4.6 Modulation of Ang II-induced contraction by smooth muscle cysteinyl leukotriene receptors in SHR aorta
In addition to endothelial CysLT2 receptors, the existence of CysLT2 receptors on the smooth muscle of SHR aorta is supported by the contractile response to LTD4 in deendothelialized aorta which remains unchanged in presence of the CysLT1 antagonist MK571. This result is in line with the LTD4-induced contraction in human pulmonary vein which was resistant to the inhibitory effect of CysLT1 antagonists [51] suggesting the presence of a contractile CysLT2 receptor located on the smooth muscle. The presence of CysLT2 receptors on the smooth muscle of SHR aorta could explain the inhibitory effect of the 5-lipoxygenase inhibitor AA861 on Ang II-induced contraction in endothelium-denuded aorta preparations whereas the CysLT1 antagonist (MK571) was ineffective. However, the confirmation of the involvement of CysLT2 receptor-mediated mechanisms in the response to Ang II awaits the development of selective CysLT2 antagonists.

In conclusion, this study strongly supports the involvement of the cysteinyl leukotrienes in Ang II-induced vascular contraction in SHR. The modulatory role of cysteinyl leukotrienes is complex and involves both direct and indirect mechanisms mediated by a CysLT1 receptor located on the endothelium and at least a different type of receptor (CysLT2) located both on the endothelium and the smooth muscle. With regard to the involvement of proinflammatory mediators, and particularly lipoxygenase products, in the pathogenesis of hypertension [52,53], these data support the concept of the potential involvement of 5-lipoxygenase pathway in hypertension.

Time for primary reviews 27 days.


   Notes
 
1 Present address: Laboratory of Pharmacology, IFR 53, EA2070, Faculty of Medicine, F-51092 Reims, France. Back


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Nasjletti A. The role of eicosanoids in angiotensin II-dependent hypertension. Hypertension (1998) 31(2):194–200.[Abstract/Free Full Text]
  2. Stern N., Golub M., Nozawa K., et al. Selective inhibition of angiotensin II-mediated vasoconstriction by lipoxygenase blockade. Am J Physiol (1989) 257:H434–H443.[Web of Science][Medline]
  3. Nozawa K., Tuck M.L., Golub M., et al. Inhibition of lipoxygenase pathway reduces blood pressure in renovascular hypertensive rats. Am J Physiol (1990) 259:H1774–H1780.[Web of Science][Medline]
  4. Stern N., Nozawa K., Golub M., et al. The lipoxygenase inhibitor phenidone is a potent hypotensive agent in the spontaneously hypertensive rat. Am J Hypertens (1993) 6:52–58.[Web of Science][Medline]
  5. Berkowitz B.A., Zabko-Potapovich B., Valocik R., Gleason J.G. Effects of the leukotrienes on the vasculature and blood pressure of different species. J Pharmacol Exp Ther (1984) 229:105–112.[Abstract/Free Full Text]
  6. Labat C., Ortiz J.L., Norel X., et al. A second cysteinyl leukotriene receptor in human lung. J Pharmacol Exp Ther (1992) 263:800–805.[Abstract/Free Full Text]
  7. Allen S.P., Chester A.H., Piper P.J., et al. Effects of leukotrienes C4 and D4 on human isolated saphenous veins. Br J Clin Pharmacol (1992) 34:409–414.[Web of Science][Medline]
  8. Allen S.P., Chester A.H., Dashwood M.R., et al. Preferential vasoconstriction to cysteinyl leukotrienes in the human saphenous vein compared with the internal mammary artery. Implication for graft performance. Circulation (1994) 90:515–524.[Abstract/Free Full Text]
  9. Allen S., Dashwood M., Morrison K., Yacoub M. Differential leukotriene constrictor responses in human atherosclerotic coronary arteries. Circulation (1998) 97:2406–2413.[Abstract/Free Full Text]
  10. Lawson D.L., Mehta J.L., Mehta P., Nichols W.W. Endothelium-dependent relaxation of rat aortic rings by leukotriene D4: importance of the magnitude of preload. Eicosanoids (1989) 2:175–181.[Medline]
  11. Lawson D.L., Smith C., Mehta J.L., Mehta P., Nichols W.W. Leukotriene D4 potentiates the contractile effects of epinephrine and norepinephrine on rat aortic rings. J Pharmacol Exp Ther (1988) 247:953–957.[Abstract/Free Full Text]
  12. Siren A.L., Letts G., Feuerstein G. N-acetyl-leukotriene E4 is a potent constrictor of rat mesenteric vessels. Eur J Pharmacol (1988) 146:331–335.[CrossRef][Web of Science][Medline]
  13. Zukowska-Grojec Z., Bayorh M.A., Kopin I.J., Feuerstein G. Overall and regional hemodynamic effects of leukotriene D4 in spontaneously hypertensive rats. Hypertension (1985) 7:507–513.[Abstract/Free Full Text]
  14. Voelkel N.F., Tuder R.M., Wade K., et al. Inhibition of 5-lipoxygenase-activating protein (FLAP) reduces pulmonary vascular reactivity and pulmonary hypertension in hypoxic rats. J Clin Invest (1996) 97:2491–2498.[Web of Science][Medline]
  15. Ishizaka N., Nakao A., Ohishi N., et al. Increased leukotriene A4 hydrolase expression in the heart of angiotensin II-induced hypertensive rat. FEBS Lett (1999) 463:155–159.[CrossRef][Web of Science][Medline]
  16. Larrue J., Rigaud M., Razaka G., et al. Formation of monohydroxyeicosatetraenoic acids from arachidonic acid by cultured rabbit aortic smooth muscle cells. Biochem Biophys Res Commun (1983) 112:242–249.[CrossRef][Web of Science][Medline]
  17. Piomelli D., Feinmark S.J., Cannon P.J. Leukotriene biosynthesis by canine and human coronary arteries. J Pharmacol Exp Ther (1987) 241:763–770.[Abstract/Free Full Text]
  18. Clozel M., Kuhn H., Hefti F., Baumgartner H.R. Endothelial dysfunction and subendothelial monocyte macrophages in hypertension. Effect of angiotensin converting enzyme inhibition. Hypertension (1991) 18:132–141.[Abstract/Free Full Text]
  19. Yoshimoto T., Yokoyama C., Ochi K., et al. 2,3,5-Trimethyl-6-(12 hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA861), a selective inhibitor of the 5-lipoxygenase reaction and the biosynthesis of slow-reacting substance of anaphylaxis. Biochem Biophys Acta (1982) 713:470–473.[Medline]
  20. Lynch K.R., O'Neill G.P., Liu Q., et al. Characterization of the human cysteinyl leukotriene CysLT1 receptor. Nature (1999) 399:789–793.[CrossRef][Medline]
  21. Heise C.E., O'Dowd B.F., Figueroa D.J., et al. Characterization of the cysteinyl leukotriene 2 (CysLT2) receptor. J Biol Chem (2000) 275:30531–30536.[Abstract/Free Full Text]
  22. Taddei S., Vanhoutte P.M. Role of endothelium in endothelin-evoked contractions in the rat aorta. Hypertension (1993) 21:9–15.[Abstract/Free Full Text]
  23. Bax W., Saxena P.R. The current endothelin receptor classification: time for reconsideration? Trends Pharmacol Sci (1994) 15:379–386.[CrossRef][Medline]
  24. Griendling K.K., Lassègue B., Murphy T.J., Alexander R.W. Angiotensin II receptor pharmacology. Adv Pharmacol (1994) 28:269–306.[CrossRef][Medline]
  25. Cortes S.D., Andriantsitohaina R., Stoclet J.C. Alteration of cyclo-oxygenase products and NO in response to angiotensin II of resistance arteries from the spontaneously hypertensive rats. Br J Pharmacol (1996) 24:1635–1641.
  26. Endemann D., Touyz R.M., Li J.S., Deng L.Y., Schiffrin E.L. Altered angiotensin II-induced small artery contraction during the development of hypertension in spontaneously hypertensive rats. Am J Hypertens (1999) 12:716–723.[CrossRef][Web of Science][Medline]
  27. Urabe M., Su C., Lee T.J. Pre- and postsynaptic effects of angiotensins in the femoral artery of spontaneously hypertensive and Wistar–Kyoto rats. Blood Vessels (1987) 24:1–10.[Web of Science][Medline]
  28. Jackson E.K., Herzer W.A., Vyas S.J., Kost C.K. Jr. Angiotensin II-induced renal vasoconstriction in genetic hypertension. J Pharmacol Exp Ther (1999) 291:329–334.[Abstract/Free Full Text]
  29. Zerrouk A., Auguet M., Chabrier P.E. Augmented endothelium-dependent contraction to angiotensin II in the SHR aorta: role of an inducible cyclooxygenase metabolite. J Cardiovasc Pharmacol (1998) 31:525–533.[CrossRef][Web of Science][Medline]
  30. Gruetter C.A., Ryan E.T., Lemke S.M., et al. Endothelium-dependent modulation of angiotensin II-induced contraction in blood vessels. Eur J Pharmacol (1998) 146:85–95.[CrossRef]
  31. Boulanger C.M., Caputo L., Lévy B.I. Endothelial AT1-mediated release of nitric oxide decreases angiotensin II contractions in rat carotid artery. Hypertension (1995) 26:752–757.[Abstract/Free Full Text]
  32. Asano M., Oaki K., Matsuda T. Quantitative changes of maximum contractile response to norepinephrine in mesenteric arteries from spontaneously hypertensive rats during the development of hypertension. J Cardiovasc Pharmacol (1984) 6:727–731.[Web of Science][Medline]
  33. Stern N., Kisch E., Knoll E. Platelet lipoxygenases in spontaneously hypertensive rats. Hypertension (1996) 27:1149–1152.[Abstract/Free Full Text]
  34. Stern N., Natarajan R., Tuck M.L., Laird E., Nadler J.L. Selective inhibition of angiotensin II-mediated aldosterone secretion by 5-hydroxyeicosatetraenoic acid. Endocrinology (1989) 125:3090–3095.[Abstract/Free Full Text]
  35. Piper P.J., Letts L.G., Galton S.A. Generation of a leukotriene-like substance from porcine vascular and other tissues. Prostaglandins (1983) 25:591–599.[CrossRef][Web of Science][Medline]
  36. Cole O.F., Fan T.P., Lewis G.P. Release of eicosanoids from cultured rat aortic endothelial cells; studies with arachidonic acid and calcium ionophore A23187. Cell Biol Int Rep (1986) 10:407–413.[CrossRef][Web of Science][Medline]
  37. Kähler J., Conforto A., Dudek R., Termin A., Bing R.J. Synthesis of leukotrienes by freshly harvested endothelial cells. J Mol Cell Cardiol (1993) 25:647–653.[CrossRef][Web of Science][Medline]
  38. Gecse A., Sonkondi S., Mezei Z., Telegdy G. Arachidonate cascade in mesenteric blood vessels and platelets of spontaneously hypertensive rats. Agents Action (1987) 22(Suppl):43–48.[CrossRef][Web of Science][Medline]
  39. Kunimoto M., Soma M., Kanmatsuse K. Production of eicosanoids and angiotensin II in resistance vessels in spontaneously hypertensive rats. Clin Exp Pharmacol Physiol (1998) 15:430–434.
  40. Namaguchi Y., Harada M., Osanai H., et al. Altered gene expression of prostacyclin synthase and prostacyclin receptor in the thoracic aorta of spontaneously hypertensive rats. Cardiovasc Res (1999) 41:682–688.[Abstract/Free Full Text]
  41. Touyz R.M., Endemann D., He G., Li J.S., Schiffrin E.L. Role of AT2 receptors in angiotensin II-stimulated contraction of small mesenteric arteries in young SHR. Hypertension (1999) 33:366–372.[Abstract/Free Full Text]
  42. Muthalif M.M., Benter I.F., Uddin M.R., Harper J.L., Malik K.U. Signal transduction mechanisms involved in angiotensin-(1-7)-stimulated arachidonic acid release and prostanoid synthesis in rabbit aortic smooth muscle cells. J Pharmacol Exp Ther (1998) 284:388–398.[Abstract/Free Full Text]
  43. Jaiswal N., Tallant E.A., Jaiswal R.K., Diz D.I., Ferrario C.M. Differential regulation of prostaglandin synthesis by angiotensin peptides in porcine aortic smooth muscle cells: subtypes of angiotensin receptors involved. J Pharmacol Exp Ther (1993) 265:664–673.[Abstract/Free Full Text]
  44. Lijnen P., Fagard R., Petrov V. Cytosolic calcium changes induced by angiotensin II in human peripheral blood mononuclear cells are mediated via angiotensin II subtype 1 receptors. J Hypertens (1997) 15:871–876.[CrossRef][Web of Science][Medline]
  45. Okamura A., Rakugi H., Ohishi M., et al. Upregulation of renin-angiotensin system during differentiation of monocytes to macrophages. J Hypertens (1999) 17:537–545.[CrossRef][Web of Science][Medline]
  46. Stoll M., Steckelings U.M., Paul M., et al. The angiotensin AT2-receptor mediates inhibition of cell proliferation in coronary endothelial cells. J Clin Invest (1995) 95:651–657.[Web of Science][Medline]
  47. Natarajan R., Gonzales N., Lanting L., Nadler J. Role of the lipoxygenase pathway in angiotensin II-induced vascular smooth muscle cell hypertrophy. Hypertension (1994) 23(Suppl_I):I142–I147.[Web of Science][Medline]
  48. Scheidegger K.J., Butler S., Witztum J.L. Angiotensin II increases macrophage-mediated modification of low density lipoprotein via lipoxygenase-dependent pathway. J Biol Chem (1997) 272:21609–21615.[Abstract/Free Full Text]
  49. Feddersen C.O., Mathias M., Murphy R.C., Reeves J.T., Voelkel N.F. Leukotriene E4 causes pulmonary vasoconstriction, not inhibited by meclofenamate. Prostaglandins (1983) 26:869–883.[CrossRef][Web of Science][Medline]
  50. Ortiz J.L., Gorenne I., Cortijo J., et al. Leukotriene receptor on human pulmonary vascular endothelium. Br J Pharmacol (1995) 115:1382–1386.[Web of Science][Medline]
  51. Labat C., Ortiz J.L., Norel X., Gorenne I., et al. A second cysteinyl leukotriene receptor in human lung. J Pharmacol Exp Ther (1992) 263:800–805.[Abstract/Free Full Text]
  52. Natarajan R., Rosdahl J., Gonzales N., Bai W. Regulation of 12-lipoxygenase by cytokines in vascular smooth muscle cells. Hypertension (1997) 30:873–879.[Abstract/Free Full Text]
  53. Patricia M.K., Kim J.A., Harper C.M., et al. Lipoxygenase products increase monocyte adhesion to human aortic endothelial cells. Arterioscler Thromb Vasc Biol (1999) 19:2615–2622.[Abstract/Free Full Text]

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