Cardiovascular Research

Cardiovascular Research 2000 47(2):376-383; doi:10.1016/S0008-6363(00)00112-7
© 2000 by European Society of Cardiology

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Copyright © 2000, European Society of Cardiology

Angiotensin II-induced contractions in human internal mammary artery: effects of cyclooxygenase and lipoxygenase inhibition

Françoise Stanke-Labesque^a,*, Philippe Devillier^b, Pierrick Bedouch^a, Jean Luc Cracowski^a, Olivier Chavanon^c and Germain Bessard^a

^aLaboratory of Pharmacology, University of Medicine, LSCPA EA2937, F-38706 La Tronche, Cedex, France
^bLaboratory of Pharmacology, Maison Blanche Hospital, F-51092 Reims, France
^cDepartment of Cardiac Surgery, Albert Michallon Hospital, CHU Grenoble, BP217, F-38043 Grenoble, Cedex 9, France

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

Received 16 January 2000; accepted 19 April 2000

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: This study investigated, in isolated human internal mammary artery, the involvement of the cyclooxygenase and the lipoxygenase pathways of arachidonic acid metabolism in the contraction induced by angiotensin II. Methods: Rings of human internal mammary arteries were suspended in organ baths for recording of isometric tension. In addition, the release of eicosanoids in response to angiotensin II (0.3 µM) was measured by enzyme immunoassay. Results: In human arterial rings without endothelial dependent relaxation in response to substance P or acetylcholine, the angiotensin II-induced contractions were significantly (P<0.05) reduced by 27% in the presence of GR32191 0.3 µM (thromboxane A₂ (TXA₂) receptor antagonist) but remained unchanged in the presence of dazoxiben 100 µM (thromboxane synthase inhibitor). In addition, angiotensin II failed to modify TXB₂ and 6-keto-PGF₁ production. These results suggest the contribution of a TXA₂/PGH₂ agonist other than TXA₂ in angiotensin II-induced contractions. However, indomethacin increased (P<0.05) angiotensin II-mediated contractile response and cysteinyl leukotriene production, suggesting a redirection of arachidonic acid metabolism from the cyclooxygenase pathway to the lipoxygenase pathway. Indeed, the contractions induced by angiotensin II were inhibited (P<0.05) by phenidone 100 µM (cyclooxygenase and lipoxygenase inhibitor), baicalein 100 µM (5-, 12- and 15-lipoxygenases inhibitor), AA861 10 µM (5-lipoxygenase inhibitor) and MK571 1 µM (CysLT₁ receptor antagonist). Cysteinyl leukotrienes were released in response to angiotensin II (pg/mg dry weight tissue: 32±9 (basal, n=6) vs. 49±9 (angiotensin II 0.3 µM, n=6), P<0.05). LTD₄, and at a lesser degree LTC₄, induced contractions of internal mammary artery and MK571 1 µM abolished the contraction to LTD₄. Conclusions: This study suggests that the in vitro vasoconstrictor effects of angiotensin II in human internal mammary artery are enhanced at least in part by eicosanoids produced by the cyclooxygenase pathway, probably PGH₂, acting on TXA₂/PGH₂ receptors, and by lipoxygenase-derived products, particularly cysteinyl leukotrienes acting on CysLT₁ receptors.

KEYWORDS Angiotensin; Arteries; Lipid metabolism; Prostaglandins; Receptors

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Angiotensin-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 angiotensin-II stimulates synthesis and release of many different eicosanoids from a variety of cells and tissues. This action is primarily the consequence of phospholipase activation resulting in the formation of free arachidonic acid available for metabolism by oxygenases.

The modulation of angiotensin-II-induced contractions by cyclooxygenase products has been well documented in various animal models. Angiotensin II stimulated PGI₂ and PGE₂ release from vascular smooth muscle cells [1–3] and from aortic rings [4,5] as well as thromboxane A₂ (TXA₂) release from aortic rings [5–7]. In normotensive rats, infusion of angiotensin II stimulated the production of PGI₂, PGE₂ and TXA₂ [4,8] and administration of a TXA₂ receptor antagonist markedly attenuated the short-term pressor response to angiotensin II [8]. In rats with angiotensin II–salt-induced-hypertension, TXA₂ production was increased [5] and administration of a thromboxane A₂/prostaglandin H₂ (TXA₂/PGH₂) receptor antagonist decreased blood pressure.

In addition to the cyclooxygenase products, lipoxygenase metabolites contribute to the vasoconstrictor action of angiotensin II [9–11]. Lipoxygenases catalyze the formation of 5-,12- or 15-HPETEs, which are subsequently transformed to the corresponding HETEs and in the case of 5-HETE to leukotrienes. 5- and 12-Lipoxygenase inhibitors have been reported to reduce the constrictor effect of angiotensin II on isolated vessels [9–12], the pressor effects of angiotensin II in normotensive rats [12] and the blood pressure in rats with spontaneous hypertension [13] as well as in rats with renovascular hypertension [14].

Most of the data on the functional involvement of eicosanoids in angiotensin-II-mediated vascular effects are derived from in vivo and in vitro studies in animal models. Therefore, the aim of the present study was to assess the involvement of the cyclooxygenase and the lipoxygenase pathways of arachidonic acid metabolism in the vasoconstrictor response to angiotensin II in human internal mammary artery (IMA).

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Tissue preparation
IMA segments were harvested from 58 patients undergoing IMA coronary artery graft surgery. The investigation conforms with the principles outlined in the Declaration of Helsinki and was approved by the Medical Ethics Committee of the Academic Medical Hospital. Among the patients, 33% had hypertension, 70% had hypercholesterolemia and 20% had diabetes mellitus. The preoperative drug therapy in these patients was as follows: 25% were on calcium blockers, 70% on β-adrenoceptor blockers, 15% on potassium channels openers, 25% on angiotensin converting enzyme inhibitors, 70% on nitrates and 37% on hydroxyl-methyl CoA reductase inhibitors.

The IMAs were removed together with their pedicle including two satellite veins and the surrounding fat tissue and the discarded distal IMA segments were obtained prior to anastomosia. The vessel samples were immediately placed in oxygenated Belzer solution maintained at +4°C, and transported to the laboratory where they were stored at 4°C and used within 24 h. The composition of the Belzer solution was: hydroxyethyl starch (5.0 mM), potassium lactobionate (100.0 mM), KHPO₃ (28.5 mM), raffinose pentahydrate (30.0 mM), adenosine (5.0 mM), allopurinol (1.0 mM), total glutathione (3.0 mM). The IMA were carefully dissected free from the surrounding connective tissue and cut into 3-mm length rings. The rings were then used in experiments to examine the effects of angiotensin II on isometric tension development and to evaluate the vascular release of eicosanoids.

2.2 Measurement of isometric tension in rings of human IMAs
The rings were mounted on parallel wires and placed in a 10-ml organ bath filled with Krebs solution maintained at 37°C and gassed with a mixture of 5% CO₂, 95% O₂. The Krebs solution had the following composition: NaCl (118.0 mM), KCl (4.7 mM), CaCl₂ (2.5 mM), MgSO₄ (1.0 mM), KH₂PO₄ (1.0 mM), glucose (11.0 mM), NaHCO₃ (25.0 mM). The lower wire was fixed to a micrometer (Mitutoyo, Japan) and the upper wire was attached to a force transducer (UF-1, Pioden, UK) to allow changes in tension to be recorded isometrically. Changes in tension were monitored on Linseis recorder (Bioblock, France). Each ring of IMA was set up under an initial tension of 5 g as previously described [15] and allowed to equilibrate for 60 min, the Krebs solution being changed every 15 min. The rings were then challenged twice by the addition of 90 mM KCl. These initial maximal contractions were performed to stabilize the preparations and ensure reproducible contractile responsiveness. Absolute values of the second contractions elicited by 90 mM KCl were 2.5±0.1 g (n=109). The arterial rings were then submaximally precontracted with 3 µM of norepinephrine. After contraction had stabilized, the preparations were exposed to either acetylcholine (1 µM) or substance P (1 µM) to assess endothelial function. None of the preparation tested relaxed to acetylcholine or substance P. However, we have recently performed histological examination which revealed an intact endothelium on the IMA [16]. Therefore, we conclude that the lack of clear-cut relaxation to acetylcholine or substance P was related to endothelial dysfunction. The ring was then washed four times with Krebs solution to restore the baseline tension and during the following h, the Krebs solution was changed every 15 min.

Cumulative concentration–response curves were constructed for angiotensin II (0.5 log increments, 10 pM–0.3 µM). The contribution of prostanoids was assessed by prior incubations of the preparations with either the cyclooxygenase inhibitor indomethacin (1 µM for 30 min), the potent and selective TXA₂/PGH₂ receptor antagonist GR32191 (0.3 µM for 45 min) [17] or the selective thromboxane synthase inhibitor dazoxiben (100 µM for 60 min) [18].

The contribution of lipoxygenase-derived eicosanoids was assessed by prior incubation with either the dual cyclooxygenase and lipoxygenase inhibitor phenidone (10 µM for 30 min) [19], the inhibitor of the 5-, 12- and 15-lipoxygenases, baicalein (100 µM for 30 min) [20,21], the selective 5-lipoxygenase inhibitor AA861 (10 µM for 30 min) [22] and the CysLT₁ antagonist MK571 (1 µM for 30 min) [23,24]. In addition, cumulative concentration–response curves were constructed for either leukotriene B₄ (LTB₄), the cysteinyl leukotrienes LTC₄, LTD₄ and LTE₄, the hydroxyeicosatetraenoic acids 12(S)-HETE and 15(S)-HETE (1-log increments, 1 pM to 0.1 µM) to assess their contractile effects in IMAs.

Only a single cumulative–response curve for angiotensin II was obtained in each preparation since the second cumulative–response curve was inhibited indicating major tachyphylaxis (data not shown). Appropriate controls (incubation with solvents) were run under similar experimental conditions in rings of IMA obtained from the same patient. In addition, all experiments were conducted with aluminum foil-covered organ baths to prevent light-induced degradation of the drugs.

2.3 Measurement of eicosanoid release in rings of human IMAs
Rings of IMA (3 mm length) 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. The ring vessels were then incubated with either angiotensin II (0.3 µM, 30 min), indomethacin (1 µM, 30 min), indomethacin (1 µM) plus angiotensin II (0.3 µM) (indomethacin added 30 min before angiotensin II) or solvents (basal release of eicosanoids). The Krebs solution was collected and samples were frozen at –80°C. The rings were dried in an oven for measurement of dry weight. The levels of TXA₂ (measured as TXB₂), PGI₂ (measured as 6-keto-PGF₁) and cysteinyl leukotrienes were determined by enzyme immunoassay on unextracted samples using reagents purchased from Cayman (Ann Arbor, USA). The detection limits of the assays were 3.2, 13.3 and 15.0 pg/ml and the EC₅₀ values (50% B/B0) were 36.6, 50.6 and 39.0 pg/ml for the cysteinyl leukotrienes, TXB₂ and 6-keto PGF₁ immunoassays, respectively. In addition, the intra-and inter-assay coefficients of variation were <10%. The results are expressed as picograms of eicosanoids per milligram of tissue dry weight.

2.4 Analysis of results
Concentrations referred to final organ bath concentrations. Contractile responses were expressed as a percentage of the contraction induced by the second KCl (90 mM) challenge. The concentration of agonist producing 50% of the maximal effect (EC₅₀) was determined from each curve by a logistic curve-fitting equation. The pD₂ represents the negative logarithm of the EC₅₀. Results are expressed as mean±S.E.M. for the specified number of preparations tested. Analysis of variance (ANOVA) for repeated measures was performed, followed by Bonferroni corrected t-test. Individual comparisons were made by Student's t-test for paired data. P values <0.05 were considered to be significant.

2.5 Chemicals
Drugs used and their sources were: KCl Normapur (Prolabo, France), angiotensin II, acetylcholine chloride, phenidone (1-phenyl-3 pyrazolidone), AA861 (2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone), and baicalein (5,6,7-trihydroxyflavone) from Sigma (L’Isle d’Abeau, France). The thromboxane synthase inhibitor dazoxiben {4-[-2-(1H-imidazol-1-yl)ethoxy]benzoic acid hydrochlolide} and the thromboxane A₂ receptor antagonist GR32191 {[1R-[1(Z),2β,3β,5]]-(+)-7-[5-[[1,1'-biphenyl)-4-yl]methoxy]-3-hydroxy-2-(1-piperidinyl)cyclopentyl]-4-heptanoic acid], hydrochloride} were kindly provided by Pfizer (Sandwich, UK) and GlaxoWellcome (Stevenage, UK), respectively. The leukotrienes (B₄, C₄, D₄ and E₄), 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), the 12(S)-HETE [12(S)-hydroxyeicosa-5Z,8Z,10E,14Z-tetraenoic acid] and the 15(S)-HETE [15(S)-hydroxyeicosa-5Z,8Z,13E-tetraenoic acid] were purchased from Cayman. All drugs were dissolved in distilled water excepted indomethacin, baicalein and phenidone. Stock solutions of indomethacin (0.1 mM) were prepared in ethanol and stock solutions of baicalein (1 mM) and phenidone (10 mM) were prepared in dimethylsulfoxide. The subsequent dilutions were performed in distilled water for indomethacin and in dimethylsulfoxide for baicalein and phenidone. The final concentrations of the solvents were 0.001% ethanol in the indomethacin experiments and 1% dimethylsulfoxide in the phenidone and baicalein experiments. These final concentrations of solvents did not alter the response to angiotensin II.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Measurement of isometric tension in rings of IMA
Angiotensin II-induced concentration-dependent contractions of IMA. In terms of efficacy and potency, the responses to angiotensin II displayed a variability, which was not significantly different among the control groups (Table 1). Incubation of IMA rings with either indomethacin, GR32191 and dazoxiben resulted in slow decreases of the basal tone which at the end of the incubation period were, respectively 0.4±0.1 g (n=11), 0.31±0.07 g (n=6) and 0.14±0.06 g (n=6). The contractile response to angiotensin II was potentiated by indomethacin, inhibited by GR32191 and was not modified by dazoxiben (Fig. 1, Table 1). Since dazoxiben did not alter angiotensin II-induced contractions whereas GR32191 inhibited the response to angiotensin II, the effect of GR32191 was assessed in presence of indomethacin to check whether its inhibitory effect was due to the blockade of a cyclooxygenase product acting on the TXA₂/PGH₂ receptors. In presence of indomethacin, GR32191 did not inhibit angiotensin II-induced contraction, the pD₂ and E_max values being, respectively 9.1±0.7 and 98.3±22.3% for the control rings (n=5) and 9.1±0.4 and 87.1±15.6% for the rings pretreated by indomethacin plus GR32191 (n=5).

View this table: [in this window] [in a new window]   Table 1 Potency (pD2) and efficacy (Emax, expressed as percentage of 90 mM KCl-induced contraction) of angiotensin II in the presence or absence of different drugs acting on eicosanoids biosynthesis or activitya

 

View larger version (10K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Effects of 1 µM indomethacin (a), 0.3 µM GR32191 (b) and 100 µM dazoxiben (c) or vehicle (solvent) on the contractions induced by angiotensin II in human IMA rings. Data are expressed as percentages of the contraction elicited by KCl (90 mM). Results are presented as mean±S.E.M. of n paired experiments: indomethacin, n=11; GR32191, n=6, dazoxiben, n=6. *, P<0.05 with respect to control.

 
Incubation of IMA rings with either phenidone, baicalein, AA861 and MK571 resulted in slow decreases of the basal tone which at the end of the incubation period were, respectively 0.20±0.06 g (n=6), 0.41±0.22 g (n=6), 0.14±0.06 g (n=5) and 0.03±0.06 g (n=8).

The dual cyclooxygenase and lipoxygenase inhibitor, phenidone, decreased the maximal response to angiotensin II but did not alter its potency (pD₂) (Fig. 2a, Table 1). Baicalein, at a concentration which has been reported to inhibit 5-, 12- and 15-lipoxygenases [20,21] also decreased the maximal response to angiotensin II without alteration of the pD₂ (Fig. 2b, Table 1). Pretreatment with indomethacin did not modify the inhibitory effect of baicalein on angiotensin II-induced contractions (baicalein: pD₂: 9.3±0.3; E_max: 62.4±13.4% (n=4) vs. indomethacin plus baicalein: pD₂=9.1±0.5, E_max: 52.0±9.8% (n=4)). In addition, 12(S)-HETE and 15(S)-HETE induced no change on the basal tone of the IMA (n=3 for each HETE).

View larger version (11K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Effects of 100 µM phenidone (a) and 100 µM baicalein (b) or vehicle (solvent) on the contractions induced by angiotensin II in human IMA rings. Data are expressed as percentages of the contraction elicited by KCl (90 mM). Results are presented as mean±S.E.M. of n paired experiments: phenidone, n=10; baicalein, n=8. *, P<0.05 with respect to control.

 
The involvement of 5-lipoxygenase metabolites in the contractile response to angiotensin II was assessed by selective inhibition of the 5-lipoxygenase with AA861. AA861 induced a decrease in the maximal response to angiotensin II (Fig. 3a, Table 1). In addition, the potent and specific CysLT₁ receptor antagonist MK571 inhibited the maximal response to angiotensin II (Fig. 3b, Table 1). The contractile effects of the four leukotrienes were tested in different preparations (n=4–5 for each leukotriene). LTB₄ and LTE₄ (up to 0.1 µM) did not cause contraction of the IMA. LTC₄ elicited a weak and variable contraction (E_max: 5.5±1.9%) and LTD₄ induced a concentration-dependent contraction with a pD₂ of 7.6±0.4 and a maximal response at 0.1 µM of 38.8±15.5%, respectively (n=5) (Fig. 4). MK571 totally abolished the response to LTD₄ (n=3).

View larger version (10K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Effects of 10 µM AA861 (a) and 1 µM MK571 (b) or vehicle (solvent) on the contractions elicited by angiotensin II in human IMA rings. Data are expressed as percentages of the contraction elicited by KCl (90 mM). Results are presented as mean±S.E.M. of n paired experiments: AA861, n=6; MK571, n=9. *, P<0.05 with respect to control.

 

View larger version (13K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Contractile effect of LTC4, LTD4 and LTE4 on the basal tone of IMA rings. Data are expressed as percentages of the contraction elicited by KCl (90 mM). Results are presented as mean±S.E.M. of n experiments: LTC4, n=6, LTD4, n=5, LTE4, n=4.

 
3.2 Measurement of eicosanoid release in rings of IMA
Angiotensin II did not change the production of 6-keto-PGF₁ and TXB₂ (Table 2) on human IMA rings. In contrast, angiotensin II induced a 1.5-fold significant increase in cysteinyl-leukotriene release from the IMA rings (Table 2). In the presence of indomethacin, the release of cysteinyl leukotrienes after angiotensin II challenge was potentiated whereas the levels of 6-keto-PGF₁ and TXB₂ remained unchanged (Table 2).

View this table: [in this window] [in a new window]   Table 2 Release of eicosanoids by IMA rings in presence of either 0.3 µM angiotensin II (solvent/Ang II), 1 µM indomethacin (Indo/basal), 0.3 µM angiotensin II and 1 µM indomethacin (Indo+Ang II) or vehicle (solvent/basal)a

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The major findings of the present study indicate that the contractile response of human IMA to angiotensin II involves cyclooxygenase metabolites, probably PGH₂, acting on TXA₂/PGH₂ (TP) receptors, and 5-lipoxygenase metabolites acting, at least in part, on CysLT₁ receptors.

The human IMA used in this study were devoid of functional endothelium inasmuch as no relaxation was observed in response to either acetylcholine or substance P. However, since the contractile response to angiotensin II was not altered by endothelium removal in human IMA [25], the absence of functional endothelium may not interfere with the conclusions and the clinical relevance of the study.

The involvement of arachidonic acid metabolites acting on TXA₂/PGH₂ receptors in the contractile response to angiotensin II is suggested by the inhibitory effect of GR32191. However, TXA₂ may not be involved in the contractile responses to angiotensin II since thromboxane synthase inhibition with dazoxiben did not modify angiotensin II-induced contractions. Furthermore, angiotensin II, at a concentration which induced a maximal contractile response in organ bath, did not produce TXA₂. These results allowed the conclusion that a TP receptor agonist, other than TXA₂, is involved in angiotensin II-induced contractions. The other agonists of the TP receptors include cyclooxygenase metabolites (PGH₂, PGF₂ and PGD₂) [26] and 15-lipoxygenase metabolites (15-HETE, 15-HPETE) [27]. However, the observation that indomethacin abolished the inhibitory effect of GR32191 on the response to angiotensin II suggests that the TP receptor agonist involved in angiotensin II-induced contractions is a metabolite of the cyclooxygenase pathway, although a non specific functional antagonism between GR32191 and indomethacin can not be ruled out. In addition, the possible contribution of 15-HETE appears unlikely given the lack of contractile effect of 15-HETE. Evidence in the literature supported the involvement of PGH₂ in the response to angiotensin II in animal models [6,7]. Moreover, it has recently been shown on rat vascular smooth muscle cells that angiotensin II induced the expression of cyclooxygenase-2 within 1 h [28]. With respect to the timing of our experiments, (angiotensin II concentration–response curves lasting 30–40 min), the involvement of prostanoids (mainly PGH₂) in angiotensin II contractile response may reflect the stimulation of either cyclooxygenase type 1 or 2 by angiotensin II.

In our experimental conditions, angiotensin II did not induce an increase in the in vitro release of 6-keto PGF₁, the stable metabolite of PGI₂, from human IMA rings. This finding may not be related to the cold storage of arterial preparations since storage at +4°C in Belzer solution for 24 h did not significantly alter the release of PGI₂ in response to arachidonate, acetylcholine and calcium ionophore on rabbit aorta [29]. In fact, angiotensin II-mediated production of PGI₂ appears variable among different animal vascular preparations [1,3,30,31]. In our preparations of human IMA, the absence of release of PGI₂ in response to angiotensin II suggests that the enhanced angiotensin II-induced contraction in the presence of indomethacin is not due to inhibition of the release of this relaxant prostaglandin. This potentiating effect of indomethacin is in line with the rise in pressure in response to angiotensin II in vitro on human placental cotyledon [11]. In human IMA, indomethacin has been reported to enhance the maximal contractile response to angiotensin II by 60% although this increase did not reach significance, probably since the low number of arterial rings tested [32]. In addition, indomethacin induced an increase in the release of cysteinyl leukotrienes from IMA rings in response to angiotensin II, suggesting that the enhanced response to angiotensin II in presence of indomethacin may have been due to a redirection of arachidonic acid metabolism from the cyclooxygenase pathway to the lipoxygenase pathway.

Phenidone is a dual cyclooxygenase and lipoxygenase inhibitor and has previously been shown to inhibit the contractile response to angiotensin II in rat femoral artery rings [12]. Since selective cyclooxygenase blockade with indomethacin enhanced the angiotensin II contractile response in the present study, the attenuation of the contractile response by phenidone may be ascribed to the lipoxygenase inhibitory activity of this agent and not to its cyclooxygenase blocking property. Baicalein, a potent inhibitor of the 5-, 12- and 15-lipoxygenases [20,21] was also effective in attenuating the in vitro contractile response of IMA to angiotensin II. Such an inhibitory effect of baicalein has been previously reported in rat femoral artery [12]. The involvement of 15-lipoxygenase derivatives acting by direct activation of TP receptors appears less likely in the human IMA preparation since the results with indomethacin and GR32191 and the absence of contractile effect of 15-HETE. Collectively, these data support the involvement of 5- or 12-lipoxygenase metabolites in the contraction of IMA in response to angiotensin II. 12-Lipoxygenase is expressed in human aortic smooth muscle cells [33] and angiotensin II increases 12-HETE release from human perfused placental cotyledon as well as from cultured umbilical artery smooth muscle cells [11]. 12-HETE is generally produced as stereoisomers by 12-lipoxygenase and cytochrome P-450 which produce 12(S)-HETE and 12(R)-HETE, respectively. 12(S)-HETE did not affect the tone of either IMA (present study), aortic rings from normotensive rats and rats with aortic coarctation-induced hypertension [34] or hamster aorta rings [35]. However, 12(S)-HETE potentiated the response to angiotensin II in hamster aorta rings [35]. It has been reported that the procontractile or vasopressor effect of 12(S)-HETE may be related to the inhibitory influence of this HETE on prostacyclin synthase and therefore on the release of the relaxant PGI₂ [3,36]. In the present study on human IMA, the inhibitory effect of baicalein on angiotensin-II-induced contractions was assessed in presence of indomethacin to determine whether or not baicalein acted by removal of an inhibitory influence of lipoxygenase-derived eicosanoids on prostacyclin synthase. Since angiotensin II did not induce production of PGI₂ and since 12(S)-HETE has no contractile effect, the participation of 12-lipoxygenase derivatives in the contractile response to angiotensin II appears unlikely. All these data support an inhibitory effect of baicalein on angiotensin-II-induced contraction through inhibition of 5-lipoxygenase.

Selective inhibition of the 5-lipoxygenase pathway also caused a decrease of the response to angiotensin II in IMA. Previous reports have shown that a blocker of the 5-lipoxygenase-activating protein (MK866) decreased the angiotensin II pulmonary pressor response in hypoxic rat [9]. Among the four leukotrienes, LTD₄ and, at a much lesser degree, LTC₄ elicited contraction of the IMA rings. These data are similar to previous results obtained on the same vascular preparation [37]. In addition, angiotensin II induced the release of cysteinyl leukotrienes from IMA rings supporting the direct involvement of these 5-lipoxygenase metabolites in the contractile response to angiotensin II. These data demonstrate the participation of the cysteinyl leukotrienes in the vascular response to angiotensin II. In addition, MK571, a selective CysLT₁ receptor antagonist, abolished the contraction induced by LTD₄ and reduced the response to angiotensin II. Since the contractile response to angiotensin II in IMA is not modulated by the endothelium [25] and since binding sites to LTC₄ or to LTD₄ have been localized by high resolution autoradiography on the tunica media of human IMA but not on the endothelium [37], our results suggest that the cysteinyl leukotrienes (particularly LTD₄), released in response to angiotensin II, participated in the vasoconstriction to this vasoactive peptide by acting mainly on CysLT₁ receptors located on the smooth muscle.

In conclusion, this study suggests that the in vitro vasoconstrictor effects of angiotensin II in human IMA is mediated at least in part by eicosanoids produced by the cyclooxygenase pathway, probably PGH₂ acting on TP receptors, and by the lipoxygenase pathway, notably LTD₄ acting on CysLT₁ receptors.

Time for primary review 16 days.

	Acknowledgements

 
The authors thank Mr. Charles Brink, Ph.D., for his help with improving the manuscript. This study was supported by the University of Medicine of Grenoble.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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